2ND SYMPOSIUM ON THE
ALPHA-AMYLASE FAMILY
SMOLENICE CASTLE, SLOVAKIA, OCT 3-7, 2004
PROGRAM AND ABSTRACTS
Program and abstracts of the
nd
2 Symposium on the Alpha-Amylase Family
held in Smolenice Castle, Slovakia, 3-7 Oct, 2004
Edited by
Štefan Janeček
ISBN 80-88820-29-4
© 2004 ASCO Art & Science Bratislava
All rights reserved
No part of this book may be reproduced, stored in a retrieval system, or transmitted in
any form or by any means, electronic, mechanical, photocopying, microfilming,
recording, or otherwise, without the permission of the publisher.
Printed in Slovakia
2
2nd Symposium on the Alpha-Amylase Family
Scientific Program Committee:
Štefan Janeček
Richard Haser
Bernard Henrissat
Takashi Kuriki
E. Ann MacGregor
Pierre Monsan
Kwan-Hwa Park
John F. Robyt
Birte Svensson
Marc J.E.C. van der Maarel
Bratislava, Slovakia
Lyon, France
Marseille, France
Osaka, Japan
West Lothian, U.K.
Toulouse, France
Suwon, Korea
Ames, IA, USA
Kgs. Lyngby, Denmark
Groningen, The Netherlands
Major Sponsors:
Sponsor:
Local Organising Committee:
Štefan Janeček
Anna Varcholová
Katarína Fabriciová
Alžbeta Janečková
Martin Machovič
Anna Medlenová
Karol Ondrovič
Richard Zona
3
Supporter:
4
2nd Symposium on the Alpha-Amylase Family
Foreword
Smolenice Castle (Slovakia), the Congress Centre of the Slovak Academy of Sciences,
welcomes all the participants of ALAMY_2!
The objective of its predecessor, the ALAMY_1, held also in Smolenice in 2001, was to
establish a forum for the presentation and discussion of research on enzymes and
proteins belonging to the α-amylase family. According to the response from the
participants, the ALAMY_1 served as an excellent platform for informal discussion
about the most recent results and international collaborations. There was, in fact,
unambiguously expressed agreement to organise the ALAMY_2 at the same place.
Now in the post-genome era, the ALAMY_2 scientific program has been broadened to
cover not only the α-amylase family, i.e. the clan of glycoside hydrolases GH-H
(families GH-13, GH-70 and GH-77), but also the “cousin” families containing amylolytic
enzymes or, in a wider sense, related glucosidases, mainly GH-31, GH-57 and GH-15.
Special attention has been paid to starch-binding domains currently classified in several
families of carbohydrate-binding modules, especially the CBM-20 and CBM-21, and
others.
The lectures consist of 13 Invited Lectures and 19 Oral Talks. The latter were carefully
selected from the submitted abstracts by the members of Scientific Program
Committee. The results of the rest of abstracts, also about 30, will be presented in the
form of posters that will be on display during the entire symposium.
The Local Organising Committee made every effort to prepare as interesting as
possible social program for all the participants. It includes the Opening Reception, trip
by boat to the water dam in Gabčíkovo, small trip to the cave “Driny” as well as
Banquet.
We hope to have a nice autumn weather that together with beautiful natural
surrounding and attractive interior of the Castle will create a really special atmosphere
for scientific discussions, relaxation and meeting friends.
I, on behalf of the organizers, wish all of you a pleasant stay and a fruitful meeting!
Štefan Janeček
5
6
2nd Symposium on the Alpha-Amylase Family
PROGRAM
7
______________________________________________________________
SUNDAY – 03 October 2004
______________________________________________________________
15:00
Registration Desk Open
Mounting of Posters
16:00 – 17:00
Conference buses from Bratislava and Vienna arrive at
Smolenice Castle
18:00 – 19:15
Chairman: Štefan Janeček
Keynote Lecture - L1
Pedro M. Coutinho (Marseille, France): Starch-active enzymes and related
proteins: data integration for evolutionary and functional analysis.
19:30 – 22:00
Opening Reception
______________________________________________________________
8
______________________________________________________________
MONDAY – 04 October 2004
______________________________________________________________
07:30 – 08:30
Breakfast
08:25
Announcements
08:30 – 12:10
Chairmen: Torben V. Borchert and Birte Svensson
Clan GH-H: Structure-function relationships I.
08:30 – 09:15
L2
Nushin Aghajari (Lyon, France): Carbohydrate recognition, binding and
processing in barley α-amylase isozymes.
09:15 – 09:40
L3
Stephanie Ravaud (Lyon, France): Crystallographic study of the sucrose
isomerase MutB from Pseudomonas mesoacidophila.
09:40 – 10:05
L4
Thomas R.M. Barends (Groningen, The Netherlands): The crystal structure
of Aquifex aeolicus amylomaltase explains the ring size of its
cycloamylose product.
10:05 – 10:35
Coffee/Tea Break and Posters
10:35 – 11:00
L5
Kazutoshii Fujii (Osaka, Japan): Protein engineering of amylomaltase
from Thermus aquaticus with random and saturation mutageneses.
11:00 – 11:25
L6
Slavko Kralj (Haren, The Netherlands): Molecular and biochemical
characterization of glucansucrases from lactobacilli.
11:25 – 12:10
L7
Narayanan Ramasubbu (Newark, NJ, USA): Structure-function
relationships in human salivary α-amylase: role of aromatic
residues.
12:10 – 13:30
Lunch
13:30 – 15:00
Poster Session 1
9
15:00 – 18:40
Chairmen: Takashi Kuriki and Pierre Monsan
Clan GH-H: Structure-function relationships II.
15:00 – 15:45
L8
Lubbert Dijkhuizen (Haren, The Netherlands): Structure/function
relationships determining product and reaction specificities in
cyclodextrin glycosyltransferase.
15:45 – 16:10
L9
Hossein Naderi-Manesh (Tehran, Iran): Structural investigation and
homology modeling studies of a Ca-independent α-amylase
purified from Bacillus sp. KR-8104: active and stable at low pH.
16:10 – 16:35
L10
Ge-xin Zhang (Jiangsu, China): The α-amylase amino acid feature in
different kingdoms.
16:35 – 17:05
Coffee/Tea Break and Posters
17:05 – 17:30
L11
Nivetha Ramachandran (Stellenbosch, South Africa): Domain architecture
of Lipomyces kononenkoae α-amylases: possible role of
N-terminal domain.
17:30 – 17:55
L12
Romina Rodriguez-Sanoja (Mexico, Mexico): Functional characteristics of
the Lactobacillus amylovorus α-amylase starch binding domain.
17:55 – 18:40
L13
Birte Svensson (Kgs. Lyngby, Denmark): Binding of carbohydrates and
protein inhibitors at the surface of α-amylases.
18:40 – 20:00
Supper
______________________________________________________________
10
______________________________________________________________
TUESDAY – 05 October 2004
______________________________________________________________
07:30 – 08:30
Breakfast
08:25
Announcements
08:30 – 12:10
Chairmen: Bauke W. Dijkstra and E. Ann MacGregor
“Cousin” families.
08:30 – 09:15
L14
Atsuo Kimura (Sapporo, Japan): Molecular analysis of α-glucosidase
(GH-family 31).
09:15 – 09:45
L15
Masahiro Mizuno (Tokyo, Japan): Three-dimensional structure of
glucodextranase, glycoside hydrolase family 15 enzyme.
09:45 – 10:15
L16
Jozef Ševčík (Bratislava, Slovakia): Structure of the complex of
S. fibuligera glucoamylase with acarbose indicates the presence
of the starch-binding site in the catalytic domain.
10:15 – 10:45
Coffee/Tea Break and Posters
10:45 – 11:15
L17
Heidi A. Ernst (Copenhagen, Denmark): Probing the structure of glucan
lyases - the lytic members of GH-31 - by sequence analysis,
circular dichroism and proteolysis.
11:15 – 12:00
L18
Michael J. O’Donohue (Reims, France): Characterization of a GH57
amylopullulanase: a survey of functionality and active site
organisation.
12:00 – 13:30
Lunch
14:00
Trip by boat to water dam Gabčíkovo
(conference buses leave for Bratislava)
19:00 – 21:00
Dinner
24:00
Arrival at the Smolenice Castle
______________________________________________________________
11
______________________________________________________________
WEDNESDAY – 06 October 2004
______________________________________________________________
07:30 – 08:30
Breakfast
08:25
Announcements
08:30 – 12:10
Chairmen: Richard Haser and John F. Robyt
Clan GH-H: Protein Engineering and
Laboratory Evolution.
08:30 – 09:15
L19
Bart A. van der Veen (Toulouse, France): CEASE: Combinatorial
Engineering to enhance Amylosucrase performance.
09:15 – 09:40
L20
Kwan-Hwa Park (Seoul, Korea): Dissociation/association properties of
cyclodextrin-hydrolyzing enzyme: an oligomeric state analysis by
pH and salt.
09:40 – 10:05
L21
Sophie Bozonnet (Kgs. Lyngby, Denmark): Molecular evolution techniques
as tools to improve barley α-amylase AMY2 expression.
10:05 – 10:35
Coffee/Tea Break and Posters
10:35 – 11:00
L22
Thijs Kaper (Groningen, The Netherlands): Engineering the reaction
specificity of GH77 amylomaltase from Thermus thermophilus HB8
by laboratory evolution.
11:00 – 11:25
L23
Lars Beier (Bagsvaerd, Denmark): Specificity engineering of a maltogenic
α-amylase.
11:25 – 12:10
L24
Lars Skov (Copenhagen, Denmark): Structural studies of sucrose utilizing
enzymes from glycoside hydrolase family 13.
12:10 – 13:30
Lunch
13:00 – 17:00
Trip to Cave “Driny” (optional; otherwise free time)
12
17:00 – 17:45
Chairman: Štefan Janeček
Special Lecture - L25
Hannes Lohi (Toronto, Canada): Laforin preferentially binds the
neurotoxic starch-like polyglucosans, which form in its absence in
progressive myoclonus epilepsy.
17:45 – 19:15
Poster Session 2 (including Refreshment)
19:15 – 20:00
Chairman: Birte Svensson
Banquet Lecture - L26
E. Ann MacGregor (West Lothian, U.K.): An overview of clan GH-H and
distantly-related families.
20:00 – 24:00
Symposium Banquet (+ Poster Prizes)
______________________________________________________________
13
______________________________________________________________
THURSDAY – 7 October 2004
______________________________________________________________
07:00 – 08:00
Breakfast
Vacation of the rooms; Poster removing
08:00 – 08:10
Announcements
08:10 – 11:55
Chairmen: Marc van der Maarel and Kwan-Hwa Park
Family GH-13 Enzymes and Inhibitors.
08:10 – 08:55
L27
Bharat Patel (Brisbane, Queensland, Australia): The thermohalophilic
amylases of Halothermothrix orenii.
08:55 – 09:20
L28
Lili Kandra (Debrecen, Hungary): Transglycosylations catalysed by
α-amylases.
09:20 – 09:45
L29
Pernilla Turner (Lund, Sweden): Two novel cyclodextrin-degrading
enzymes isolated from thermophilic bacteria, have similar domain
structures but differ in oligomeric state and activity profiles.
09:45 – 10:05
Coffee/Tea Break
10:05 – 10:30
L30
Elspeth A. MacRae (Auckland, New Zealand): Characterisation of putative
α-amylases from apple (Malus domestica) and Arabidopsis
thaliana.
10:30 – 11:15
L31
John F. Robyt (Ames, IA, USA): Synthesis and study of potent α-amylase
inhibitors.
11:15 – 11:55
Closing Lecture - L32
Takashi Kuriki (Osaka, Japan): The conclusive proof that supports the
concept of the α-amylase family: structural similarity and common
catalytic mechanism.
11:55 – 12:00
Closure of ALAMY_2
12:00
The 1st Conference Bus leaves for Bratislava and Vienna
12:00 – 13:00
Lunch
13:00
The 2nd Conference Bus leaves for Bratislava and Vienna
______________________________________________________________
______________________________________________________________
14
2nd Symposium on the Alpha-Amylase Family
LECTURE ABSTRACTS
15
KEYNOTE LECTURE - L1
Starch-active enzymes and related proteins: data
integration for evolutionary and functional analysis
Pedro M. COUTINHO
UMR6098, AFMB / CNRS / Universités Aix-Marseille I et II, 31 chemin Joseph Aiguier, 13402 Marseille
cedex 20, France; e-mail: [email protected]
Starch/glycogen-related polysaccharides play a central role in the metabolism of most organisms
as a source of energy and energy storage. Enzyme families involved in the degradation,
biosynthesis and modification of starch-related polysaccharides are present in the CarbohydrateActive Enzymes’ classification (CAZy found at URL: http://afmb.cnrs-mrs.fr/CAZY), and many
are used for commercial applications. Structurally, the over 3,200 starch-active enzymes and
related proteins presently found in CAZy belong to 10 families (GH13, GH14, GH15, GH31,
GH57, GH70, GH77, GT3, GT5, GT35), which include different structural classes,
complemented by at least 5 families of ancillary starch-binding modules (CBM20, CBM21,
CBM25, CBM26, CBM34). The description of the modular organisation combined with
phylogenetic analysis within each family helps in understanding both function and evolution.
The integration of sequence and modular information with available genomic, taxonomical and
biochemical data, provides means to measure the degree of knowledge within each family and to
orient research.
16
INVITED LECTURE - L2
Carbohydrate recognition, binding and processing
in barley α-amylase isozymes
1
1
1
2,3
Samuel TRANIER , Xavier ROBERT , Richard HASER , Sophie BOZONNET ,
2
2,3
1
Morten T. JENSEN , Birte SVENSSON & Nushin AGHAJARI
1
Institut de Biologie et Chimie des Protéines UMR 5086, Laboratoire de BioCristallographie, CNRS and
Université Claude Bernard Lyon I, 7 Passage du Vercors, 69367 Lyon cedex 07, France;
e-mail: [email protected]
2
Carlsberg Laboratory, Department of Chemistry, Gamle Carlsberg Vej 10, DK-2500 Valby, Denmark
3
Biochemistry and Nutrition Group, BioCentrum-DTU, Technical University of Denmark, Søltofts Plads,
Building 224, DK-2800, Kgs. Lyngby, Denmark
The barley α-amylase isozymes, AMY1 and AMY2, share nearly 80% sequence identity but
display remarkable differences in physico-chemical properties. Crystal structures of AMY2 are
known in the native state (KADZIOLA et al., 1994), in complex with the inhibitor acarbose
(KADZIOLA et al., 1998), and with the endogenous bifunctional barley α-amylase/subtilisin
protein inhibitor, BASI (VALLÉE et al., 1998), the latter only inhibiting AMY2. The 3D structure
of AMY1 has been solved to 1.5Å resolution (ROBERT et al., 2003). Furthermore a vast number
of enzyme/substrate and enzyme/inhibitor complexes, and mutant structures exist which allow
us to perform an extended analysis of the enzyme/carbohydrate interactions within these
systems (ROBERT et al., 2003; ROBERT et al., in prep.; TRANIER et al., in prep.). On the basis of
these data, differences in substrate recognition and specificity of the two isozymes have been
discerned. Though the fact that the organization of both isozymes is virtually identical and that
the local changes are very small, intriguingly, these isozymes behave very distinct when
interacting with substrates and inhibitors. As concerns the third and last identified substratebinding site in the C-terminal domain of AMY1, “the pair of sugar tongs”, (not present in
AMY2) a subtle but very significant conformational change occurs upon sugar binding.
Examination of the various enzyme/carbohydrate structures points out the crucial role of a
tyrosine and its conformational flexibility in AMY1 contributing to polysaccharide capture.
Interestingly, the presence of this extra carbohydrate site in AMY1 is fully consistent with an
increased affinity and activity (compared to AMY2) on starch granules. The C-terminal domain
in AMY1 with “the pair of sugar tongs”, proposed to be involved in starch granule binding,
seems to be a major determinant for barley α-amylase isozyme specificity.
Details of the structural and isozyme-specific differences in terms of substrate
recognition and processing will be presented.
KADZIOLA, A., ABE, J., SVENSSON, B. & HASER, R. 1994. J. Mol. Biol. 239: 104-121.
KADZIOLA, A., SØGAARD, M., S VENSSON, B. & HASER, R. 1998. J. Mol. Biol. 278: 205-217.
ROBERT, X., HASER, R., GOTTSCHALK, T.E., RATAJCZAK, F., DRIGUEZ, H., SVENSSON, B. & AGHAJARI, N. 2003. Structure
11: 973-984.
ROBERT, X., HASER, R, MORI, H., S VENSSON, B. & AGHAJARI, N. In preparation.
TRANIER, S., ROBERT , X., HASER, R, BOZONNET, S., JENSEN, M.T., S VENSSON, B. & AGHAJARI, N. In preparation.
VALLÉE, F., KADZIOLA, A., JUY, M., BOURNE, Y., RODENBURG, K., SVENSSON, B. & HASER, R. 1998. Structure 6: 649-659.
17
ORAL T ALK - L3
Crystallographic study of the sucrose isomerase
MutB from Pseudomonas mesoacidophila
1
2
2
Stéphanie RAVAUD , Hildegard W ATZLAWICK , Ralph MATTES , Richard HASER
1
& Nushin AGHAJARI
1
1
Laboratoire de BioCristallographie, IBCP UMR 5086, CNRS UCBL, IFR128 “Biosciences LyonGerland”, 7 passage du Vercors, 69367 Lyon Cedex 07, France; e-mail: [email protected]
Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany
2
Sucrose isomerase (MutB) from Pseudomonas mesoacidophila belonging to GH family 13, is an
enzyme consisting of 584 amino acids which catalyses the isomerization of sucrose into
isomaltulose (α-D-glucosylpyranosyl-1,6-D-fructofuranose), and trehalulose (α-D-glucosylpyranosyl-1,1-D-fructofuranose) as main products and glucose and fructose in residual amounts
from the hydrolytic reaction. As concerns the enzyme activity based on sucrose decomposition,
the optimum pH and the optimum temperature were pH 5.8 and 40 oC, respectively (NAGAI et
al., 1994). The MutB protein has been crystallized at 295 K using the hanging drop vapourdiffusion method. Two different crystal forms have been obtained: the first one diffracts X-rays
to 1.6 Å using synchrotron radiation and belongs to the space group P1, with unit cell
parameters a=64 Å, b=72 Å, c=82 Å, α=67,5°, β=73° and γ=70°, the second form diffracts to
1.8 Å using synchrotron radiation and belongs to the space group P21, with unitcell parameters
a=64 Å, b=86 Å, c=120 Å and β=98°. The structure of MutB has been determined by the
molecular replacement method using isomaltulose synthase (PalI) from Klebsiella sp. LX3 as a
search model (LI et al., 2002; ZHANG et al. 2003).
The MutB structure consists of three domains: an N-terminal catalytic (β/α)8 domain, a
subdomain, and a C-terminal domain displaying seven β-strands. The active site architecture of
MutB is identical to that of other glycoside hydrolase family 13 members, suggesting a similar
mechanism in substrate binding and hydrolysis. The refinement of the two structures is in
progress, and co-crystallization experiments with inhibitors and/or substrate analogues are
planned. Analysis of these structures, when completed, should contribute to explain the
molecular basis of sucrose decomposition, isomerization and the selectivity of this enzyme
leading to formation of different products.
NAGAI, Y., S UGITANI, T. & TSUYUKI, K. 1994. Biosci. Biotechnol. Biochem. 58: 1789-1793.
LI, N., ZHANG, D., ZHANG, L.H. & SWAMINATHAN, K. 2002. Acta Cryst. D59: 150-151.
ZHANG, D., LI, N., LOK, S.M., ZHANG, L.H. & SWAMINATHAN, K. 2003. J. Biol. Chem. 278: 35428-35434.
18
ORAL T ALK - L4
The crystal structure of Aquifex aeolicus
amylomaltase explains the ring size of its
cycloamylose product
1
1
2
Hilda KORF , Thomas R. M. BARENDS , Thijs KAPER , Marc J. E. C. VAN DER
3
2
1
MAAREL , Lubbert DIJKHUIZEN & Bauke W. DIJKSTRA
Centre for Carbohydrate Bioengineering TNO-University of Groningen
1
Laboratory of Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The
Netherlands; e-mail: [email protected]
2
Laboratory of Microbiology, University of Groningen, Kerklaan 30, 9751 NN, Haren, The Netherlands
3
Innovative Ingredients and Products Department, TNO Nutrition and Food Research, Rouaanstraat 27,
9723 CC, Groningen, The Netherlands
Amylomaltases can be used to produce large cyclic glucans, which have several applications as
complexing agents, like the smaller cyclodextrins. Control of the ring size of the cycloamyloses
produced by amylomaltase is therefore of great interest. The crystal structure of Aquifex
aeolicus amylomaltase has been detemined to 2.15 Å resolution (Fig. 1). From a previously
determined structure of another amylomaltase, it was proposed that the ring size is determined
by the structural elements the oligosaccharide substrate wraps around, to bring both its ends
together for cyclization (PRZYLAS et al., 2000). The current Aquifex amylomaltase structure
allows a comparison of the two structures, correlating these structural features with the ring size
of the cycloamyloses produced by both enzymes.
Figure 1. Crystal structure of the
amylomaltase from Aquifex aeolicus.
The catalytic residues are shown in
ball-and-stick representation.
PRZYLAS, I., TERADA, Y., F UJII, K., TAKAHA, T.,
SAENGER, W. & STRAETER, N. 2000. Eur. J.
Biochem. 267: 6903-6913.
19
ORAL T ALK - L5
Protein engineering of amylomaltase from Thermus
aquaticus with random and saturation mutageneses
1
2
1
1
Kazutoshi FUJII , Hirotaka MINAGAWA , Yoshinobu TERADA , Takeshi TAKAHA ,
Takashi KURIKI1, Jiro SHIMADA2 & Hiroki KANEKO3
1
Biochemical Research Laboratory, Ezaki Glico Co., Ltd., 4-6-5 Utajima, Nishiyodogawa-ku, Osaka 5558502, Japan; e-mail: [email protected]
2
Biomaterials Processing Fundamental Research Laboratories, NEC Co., Ltd., 34 Miyukigaoka, Tsukuba,
Ibaraki 305-8501, Japan
3
Department of Applied Physics, College of Humanities and Sciences, Nihon University, 3-25-40,
Sakurajousui, Setagaya-ku, Tokyo 156-8550, Japan
Cycloamylose is a large cyclic α-1,4-glucan with DP ranging from 17 to several hundreds. It is
highly soluble in cold water, and can form inclusion complex with various molecules, including
alcohols or fatty acids. Cycloamylose is going to be applied to pharmaceutical, cosmetic, food,
toiletry, agricultural, and chemical industries. It was recently reported that cycloamylose
functions as an artificial chaperone to refold denatured protein into active form. (MACHIDA et
al., 2000)
We have reported that cycloamylose is produced by the intra-molecular
transglycosylation reaction of various 4-α-glucanotransferases (EC 2.4.1.25, 4αGTase)
(TAKAHA & SMITH, 1999). Among 4αGTases, amylomaltase from Thermus aquaticus is
particularly useful for the production of cycloamylose, since it exhibits high thermal stability
and higher productivity. However, this enzyme has weak but significant hydrolytic activity
together with its major transglycosylation activity. Therefore, the yield of cycloamylose at the
end point of the reaction was decreased to about 50~60% of the maximum yield.
In order to obtain mutated amylomaltase with lower hydrolytic activity, we introduced
random mutations into the gene coding for this enzyme. From these results, we found amino
acids, which are involved in the hydrolytic activity. We also performed saturation mutagenesis
to these sites and finally obtained a mutated amylomaltase with dramatically decreased
hydrolytic activity.
This work was supported by a grant for the development of the next generation of bioreactor systems from the Society for
Techno-Innovation of Agriculture, Forestry and Fisheries (STAFF), Tokyo, Japan, to Ezaki Glico Co., Ltd.
MACHIDA, S., OGAWA, S., XIAOHUA, S., TAKAHA, T., FUJII, K. & HYASHI, K. 2000. FEBS Lett. 486: 131-135.
TAKAHA, T. & SMITH, S.M. 1999. Biotechnol. Genet. Eng. Rev. 16: 257-280.
20
ORAL T ALK - L6
Molecular and biochemical characterization of
glucansucrases from lactobacilli
1,4
1,2
Slavko KRALJ , Ineke G.H. VAN GEEL-SCHUTTEN , Marc J.E.C. VAN DER
MAAREL1,3 & Lubbert DIJKHUIZEN1,4
1
Centre for Carbohydrate Bioengineering TNO-University of Groningen
Innovative Ingredients and Products Department, TNO nutrition and food research, Utrechtseweg 48,
3704 HE Zeist, The Netherlands
3
\ Innovative Ingredients and Products Department, TNO nutrition and food research, Rouaanstraat 27,
9723 CC Groningen, The Netherlands
4
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB),
University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands; e-mail: [email protected]
2
Glucansucrases or glucosyltransferases (GTFs, EC 2.4.1.5) of lactic acid bacteria are large
extracellular enzymes responsible for the synthesis of alpha-glucans from sucrose. Much
research is performed on GTFs from oral streptococci and Leuconostoc species, whereas little is
known about GTFs of lactobacilli.
A large collection of Lactobacillus strains was screened for the production of
exopolysaccharides (VAN GEEL-SCHUTTEN et al., 1998). One of the positive strains,
Lactobacillus reuteri 121, produced two different soluble homopolysaccharides during growth
on sucrose, a fructan and glucan. Structural analysis of the polysaccharides produced by L.
reuteri 121 revealed that the fructan is a linear levan with β-(2,6) linked fructosyl units. The
glucan possessed a unique highly branched structure with α-(1,4) and α-(1,6) linkages together
with (4,6) branching points (a reuteran) (VAN GEEL-SCHUTTEN et al., 1999). Furthermore,
different dextran- [α-(1,6)] and mutan [α-(1,3)] synthesizing lactobacilli have been identified
(KRALJ et al., 2004a).
The L. reuteri 121 glucansucrase gene has been cloned, expressed in Escherichia coli
and the GTFA enzyme was purified. Analysis of the glucans (reuterans) produced by the
recombinant enzyme and of glucans isolated from supernatants of L. reuteri revealed that both
glucans were virtually identical (KRALJ et al., 2002). The purified recombinant GTFA was
molecularly and biochemically characterized, including the investigation of its acceptor reaction
(KRALJ et al., 2004b).
The variations in linkage specificity observed between different glucansucrase enzymes
is most likely based on differences in the acceptor binding sites. To investigate this in detail the
(putative) acceptor binding sites of the reuteransucrase (GTFA) from L. reuteri 121 were
subjected to site-directed mutagenesis. This resulted in marked changes in linkage specificity in
the glucans and oligosaccharides synthesized by the mutant enzymes. Largest differences were
obtained when mutations in different putative acceptor sites were combined.
VAN GEEL-SCHUTTEN, G.H., F LESCH, F., TEN BRINK, B., S MITH, M.R. & DIJKHUIZEN, L. 1998. Appl. Microbiol. Biotechnol.
50: 697-703.
VAN GEEL-SCHUTTEN, G.H., FABER, E.J., S MIT, E., ... & DIJKHUIZEN, L. 1999. Appl. Environ. Microbiol. 65: 3008-3014.
KRALJ, S., VAN GEEL-S CHUTTEN, G.H., RAHAOUI, H., ... & DIJKHUIZEN, L. 2002. Appl. Environ. Microbiol. 68: 4283-4291.
KRALJ, S., VAN GEEL-S CHUTTEN, G.H., DONDORFF, M.M.G., ... & DIJKHUIZEN, L. 2004a. submitted.
KRALJ, S., VAN GEEL-S CHUTTEN, G.H., V AN DER MAAREL, M.J. & DIJKHUIZEN, L. 2004b. Microbiology 150: 2099-2112.
21
INVITED LECTURE - L7
Structure-function relationships in human salivary
α-amylase: role of aromatic residues
1
1
1
Narayanan RAMASUBBU , Chandran RAGUNATH , Prasunkumar J. MISHRA ,
2
2
Gyöngyi GYÉMÁNT & Lili KANDRA
1
Department of Oral Biology, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103 USA;
e-mail: [email protected]
Department of Biochemistry, Faculty of Sciences, University of Debrecen, 4010 Debrecen, Hungary
2
Human salivary α-amylase (HSAmy) has three distinct functions relevant to oral health: 1)
hydrolysis of starch; 2) binding to hydroxyapatite; and 3) binding to bacteria (e.g. viridans
streptococci). Oral bacteria utilize the starch hydrolyzing activity to derive their nutrients from
dietary starch. Localized acid production by bacteria, through the metabolism of maltose
generated by HSAmy, can lead to the dissolution of tooth enamel, a critical step in dental caries
formation.
The structure of HSAmy consists of 496 residues, one calcium ion and one chloride ion
and folds into three domains A (residues 1-99+169-404), B (residues 100-167) and C (residues
405-496). The central N-terminal domain A serves as a scaffold on which two flexible domains
B (complex loop) and C (independent domain) are placed. The starch-binding site of HSAmy, a
deep cleft at the interface between domains A and B, consists of six subsites (-4 through +2).
The interactions between the substrate and HSAmy at subsites -1 and +1 (wherein the cleavage
occurs) are very well conserved among many α-amylases. The residues at other subsites are not
well conserved. Nonetheless, among chloride-containing α-amylases, aromatic residues are
prominently located in the active site (immediately next to the subsites where cleavage occurs)
as well as at the chloride-binding site.
We focused on determining the role of these aromatic residues in the structure-function
relationships of HSAmy. 1) We hypothesized that aromatic residues at the active site provide a
scaffold on to which the substrate positions itself first before additional stabilizing interactions
can occur. We tested this hypothesis using HSAmy mutated at Trp58, Trp59, and Tyr151.
Subsite mapping were carried out using both crystallographic analysis as well as hydrolytic
pattern analysis using end-labeled substrates clearly revealed that oligosaccharide binding was
affected in these mutants. Glucose production was significant in these mutants while negligible
in HSAmy. At lower concentrations of substrate, glucose production by W58 mutants was even
higher suggesting that preponderance of binding mode in which subsites –3 and –2 are
unoccupied. 2) The aromatic residues, Phe256 and Phe295 are prominent at the chloride-binding
site. Kinetic and crystal structure analysis of Phe256 and Phe295 mutants were used to
determine their role in the hydrolytic activity of HSAmy which show that the strategic location
of the Phe256 helps orient a water chain that may be involved in starch hydrolysis. From these
studies, we conclude that aromatic residues in HSAmy play a crucial role in the substrate
binding, enzyme activity and catalysis.
This work was supported by USPHS Grant DE12585 (NR) and T032005 and M041829 from the Hungarian Scientific
Research Fund (LK).
22
INVITED LECTURE - L8
Structure/function relationships determining
product and reaction specificities in cyclodextrin
glycosyltransferase
Hans LEEMHUIS & Lubbert DIJKHUIZEN
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB),
Kerklaan 30, 9751 NN Haren, The Netherlands; e-mail: [email protected]
Cyclodextrin glycosyltransferase (CGTase) converts starch into cyclodextrins and displays
relatively low hydrolytic activity. CGTase structural factors determining substrate/product
specificity and hydrolysis/transglycosylation reaction specificity have been identified
(LEEMHUIS & DIJKHUIZEN, 2003a,b,c).
Several examples of natural CGTase enzymes with rather unusual reaction specificities
or metabolic roles have been characterized. The Bacillus stearothermophilus maltogenic αamylase for instance is structurally very similar to CGTase, but converts starch into maltose and
has a very high hydrolytic activity (DAUTER et al., 1999). Introduction of the main structural
differences in either enzyme changed reaction specificities as predicted (BEIER et al., 2000;
LEEMHUIS et al., 2003).
Acarviosyl transferase (ATase) from the actinomycete Actinoplanes sp. SE50/110
transfers the acarviosyl moiety of the diabetic drug acarbose to sugar acceptors (WEHMEIER,
2003). ATase exhibits 42% sequence identity with CGTase but is virtually inactive on starch. In
contrast, ATase is the only known enzyme to efficiently use acarbose as substrate, a strong
inhibitor of CGTase and of most other α-amylase family enzymes. This distinct reaction
specificity makes ATase an interesting enzyme to further investigate the variation in reaction
specificity of α-amylase family enzymes.
Here we show that mutant G146H, introducing the typical His of the conserved
sequence region I of the α-amylase family, changed ATase into an enzyme with 4-αglucanotransferase and cyclodextrin forming activities, similar to CGTase. Moreover, a single
mutation, that removed this typical His side-chain in CGTase (H141A), introduced acarviosyl
transferase activity in CGTase (LEEMHUIS et al., 2004).
BEIER, L., SVENDSEN, A., ANDERSEN, C., FRANDSEN, T.P., BORCHERT , T.V. & CHERRY, J.R. 2000. Protein Eng. 13: 509513.
DAUTER, Z., D AUTER, M., BRZOZOWSKI, A.M., CHRISTENSEN, S., BORCHERT, T.V., BEIER, L., WILSON, K.S. & DAVIES, G.J.
1999. Biochemistry 38: 8385-8392.
LEEMHUIS, H. & DIJKHUIZEN, L. 2003a. J. Appl. Glycosci. 50: 263-271.
LEEMHUIS, H. & DIJKHUIZEN, L. 2003b. Biocatal. Biotransform. 21: 261-270.
LEEMHUIS, H. & DIJKHUIZEN, L. 2003c. in: Proc. 3rd Eur. Symp. Recent Advances in Enzymes in Grain Processing, pp. 3-8.
LEEMHUIS, H., KRAGH, K.M., DIJKSTRA, B.W. & DIJKHUIZEN, L. 2003. J. Biotechnol. 103: 203-212.
LEEMHUIS, H., WEHMEIER, U.F. & DIJKHUIZEN, L. 2004. Biochemistry (in press).
WEHMEIER, U.F. 2003. Biocatal. Biotransform. 21: 279-284.
23
ORAL T ALK - L9
Structural investigation and homology modeling
studies of a Ca-independent α-amylase purified
from Bacillus sp. KR-8104: active and stable at low
pH
Reza H. SAJEDI, Hossein NADERI-MANESH, Khosro KHAJEH, Madjid TAGHDIR &
Bijan RANJBAR
Department of Biochemistry and Biophysics, Faculty of Science, Tarbiat Modarres University, P. O. Box
14115-175, Tehran, Iran; e-mail: [email protected]
In modern industrial biotechnology, amylases have very important role in the starch, detergent,
beverage, and textile technology. In the present study, the α-amylase from Bacillus sp. KR-8104
isolated from soil samples of potato farms in Iran was purified by ion exchange and hydrophobic
interaction chromatography. The amino acid sequence of the enzyme was obtained from
nucleotide sequencing of PCR products of encoding gene. Enzyme kinetic studies, irreversible
thermoinactivation, Tm measurements based on CD results of thermal unfolding, intrinsic
fluorescence, sequence alignment, and homology modeling (the crystal structure of BLA as a
template) had been carried out to elucidate the effect of Ca2+, EDTA, and different values of pH
on enzyme activity and stability. Catalytic activity, kinetic and thermodynamic stability of
enzyme shows none or very little changes at the presence or absence of Ca2+ and EDTA. Its pH
profile also shows a very broad pH range of activity (3.5-7.0) and its optimum pH is 4.0.
Independence of Ca2+ ion and low pH performance are two very important properties in αamylases for both basic research and industrial processes. We discuss the reasons for these
properties, based on the sequence alignment and molecular modeling.
The authors express their gratitude to the research council of Tarbiat Modarres University and Ministry of Sciences,
Researches, and Technology for financial support during the course of this project.
SAJEDI, R.H., N ADERI-MANESH, H., MORADIAN, F., KHAJEH, K., AHMADVAND, R., RANJBAR, B. & ASOODEH, A. 2004.
Enzyme Microb. Technol. submitted.
MACHIUS , M., D ECLERCK, N., HUBER, R. & WIEGAND, G. 1998. Structure 6: 281-292.
NIELSEN, J.E., BORCHERT , T.V. & VRIEND, G. 2001. Protein. Eng. 14: 505-512.
24
ORAL T ALK - L10
The α-amylase amino acid feature in different
kingdoms
1
2
2
Ge-xin ZHANG , Yu CAO , Wei-jiang LI & Wen-bo XU
3
1
School of Chemical and Material Engineering, Southern Yangtze University, Wuxi, Jiangsu,
China, 214036; e-mail: [email protected]
Key Laboratory of Industrial Biotechnology of Ministry of Education, Southern Yangtze
University, Wuxi, Jiangsu, China, 214036
3
School of Information Technology, Southern Yangtze University, Wuxi, Jiangsu, China, 214036
2
Carl Woese’s articles have changed the way in which life on earth is viewed; there are three
kingdoms in life world. α-Amylase (EC 3.2.1.1) is a key enzyme, which hydrolyzes α-D-(1,4)
glucosidic bonds in starch and amylose at random. α-Amylases belong to the super family
(family) of α-amylase, members of which are found in all three kingdoms of life and function in
diverse cellular processes, often via chaperone-like activities. More and more applications of αamylases make it an increasing interest in biotechnology, such as improving agricultural silage
production, clarifying juices and wines, extracting coffee, using in pharmaceutical industry and
as artificial low sweetener, in paper and pulp industry, in industrial ethanol production.
α-Amylases belong to the GH_57 (Glycosyl hydrolase family 57), members of which are found
in all three kingdoms of life, archaea, bacteria, and eukaryota. Is there respective sequence
feature α-amylase in different kingdom? Have they common feature?
We downed all sequences, which are relational α-amylase from SWISS-PROT (January
2004). After clearing up, we found 22 archaea, 119 bacteria, and 199 eukaryota α-amylase
sequences. According their feature table, we deleted signal peptide in sequence. Those
sequences consisted of our α-amylase’s bank. After analysis, we found that there are 2 kinds
cases about relation between percent amino acid and every kingdoms’ α-amylase:
(1) The α-amylases average length is 525.35484; and archaea, bacteria, and eukaryota
average length is 508.68182, 609.79832, 477.17085.
(2) Comparing with common protein, the α-amylases average amino acid percent H, I, M,
P, Q, R, S, T, V, is the same. A, C, E, K, L is bigger. C is 2. D, F, G, N, W, Y is
smaller. W is half.
(3) In 3 kingdoms, F, H, M, P, R, V, W is the same. E, L, Y, is descending from archaea
to eukaryota. G, N, S, is increasing. In C, archaea is the same bacteria, is half of
eukaryota. In A, bacteria is the same eukaryota, archaea is 2/3 of them.
(4) With E, L, Q, and T pair percent, we can distinguish the α-amylases kingdom.
This study was supported by the Science Foundation of Southern Yangtze University.
25
ORAL T ALK - L11
Domain architecture of Lipomyces kononenkoae
α-amylases: possible role of N-terminal domain
1
2
Nivetha RAMACHANDRAN , Isak S. PRETORIUS & Ricardo CORDERO OTERO
1
1
Institute for Wine Biotechnology, Victoria street, J.H. Neethling building, Stellenbosch University,
Stellenbosch 7600 South Africa; e-mail: [email protected]
The Australian Wine Research Institute, Adelaide, SA 5064, Australia
2
The α-amylases from Lipomyces kononenkoae LKA1 and LKA2 have unique α-1, 4 and α-1, 6
activity on starch. LKA1 has a comparable activity on pullulan while LKA2 has a significant
dextrinase activity. Here, we report the possible role of N-terminal regions of LKA1 and LKA2
in raw starch adsorption, specificity and thermal stability. In silico homology modeling of LKA1
and LKA2 was used to develop a putative domain organization and the presumed tertiary
structure was modeled using TAKA amylase as reference. Taking into account, computer
simulations and structure data, functional N-terminal deletion mutants of LKA1 and LKA2
(LKA1∆N and LKA2∆N respectively) were created. Native and mutant amylolytic peptides
were analyzed for their raw starch binding function, thermal stability, effect of calcium ion on
activity, and its contribution towards substrate specificity. LKA1∆N shows a 95% reduction of
raw starch substrate adsorption, 20% less hydrolytic activity in complex starches, and 30% less
thermal stability than the wild-type enzyme. In contrast, LKA2∆N did not show significant
variations in raw starch adsorption or thermal stability. Furthermore, the mutated LKA2 lost
45% of original hydrolytic activity on dextrin. The results are discussed in terms of possible role
of N-terminal regions of LKA1 and LKA2 in raw starch adsorption, substrate specificity and
thermal stability. The data presented could be of assistance in the understanding of multidomain architecture of amylolytic enzymes.
26
ORAL T ALK - L12
Functional characteristics of the Lactobacillus
amylovorus α-amylase starch binding domain
Monserrat SANTIAGO, Larissa LINARES, Sergio SÁNCHEZ &
Romina RODRÍGUEZ-SANOJA
Depto. de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, UNAM, Ciudad
Universitaria, C.P. 04510, A.P. 70228, México, D.F.; e-mail: [email protected]
Lactobacillus amylovorus α-amylase (AmyA) presents an uncommon primary structure,
constituted by two parts: the catalytic domain (amino acids 1 to 474) and the C-terminal domain
(475-953) (GIRAUD & CUNNY, 1997). The catalytic domain is similar to other Gram positive
bacterial α-amylases, it contains the conserved regions described for these enzymes sharing 65.5
and 61.5% identity with Bacillus subtilis and Streptococcus bovis α-amylases, respectively
(MORLON-GUYOT et al., 2001). The C-terminal domain is responsible for starch binding
capability (RODRÍGUEZ SANOJA et al., 2000). Structurally this domain is different from other
reported α-amylases starch binding domains (SBD) since it is composed of five identical
repeated units (RU) of 91 amino acids each one.
In a previous work a truncated AmyA, encoding the N-terminal region (first 474 aa)
comprising the catalytic domain and excluding the putative SBD was expressed in a nonamylolytic Lactobacillus plantarum strain. This protein was unable to fix or hydrolyze raw
starch (RODRÍGUEZ-SANOJA et al., 2000). In regard to the SBD domain, this work examines the
importance of the repeats units on the starch binding capability. For this purpose, we expressed
either one or five RU of the C-terminal region in Escherichia coli, as independent domains.
Protein binding assays showed that the five repeated units alone bind to insoluble starch,
suggesting that the catalytic domain is not required for adsorption. Moreover, one repeated unit
was also bound to the starch grain. The whole L. amylovorus α-amylase presented a three times
greater Kad than 1RU, suggesting that the increase in the number of RU also increases the
capacity of adsorption. In addition, if we compared against the adsorption of the 5RU we
observed that all the added protein is adsorbed obtaining a Kad of 1, which was greater than the
one obtained for 1RU and even greater than the one of the whole amylase, pointing to an
accumulative effect between the units and a steric effect of the catalytic domain on binding.
These results suggest that each UR is acting as an independent fixation module, condition
observed in cellulases and chitinases but not in amylases (SIMPSON et al., 1999; WU et al., 2001).
This work was supported by CONACyT, México Grant 41222-Z. We thank Beatriz Ruiz and Rafael Cervantes for technical
assistance.
GIRAUD, E. & CUNY, G. 1997. Gene 198: 149-157.
MORLON-GUYOT, J., M UCCIOLO, R.F., RODRÍGUEZ-SANOJA, R. & GUYOT, J.P. 2001. DNA Seq. 12: 27-37.
RODRÍGUEZ-S ANOJA. R., MORLON-GUYOT, J., JORE, J., … & GUYOT, J.P. 2000. Appl. Environ. Microbiol. 66: 3350-3356.
SIMPSON, P.J., BOLAM, D.N., COOPER, A., … & WILLIAMSON, M.P. 1999. Structure 7: 853-864.
WU, M.L., CHUANG, Y.C., CHEN, J.P., CHEN, C.S. & CHANG, M.C. 2001. Appl. Environ. Microbiol. 69: 5100-5106.
27
INVITED LECTURE - L13
Binding of carbohydrates and protein inhibitors at
the surface of α-amylases
1,2
1,2
1,2
Sophie BOZONNET , Birgit C. BØNSAGER , Birte KRAMHØFT , Haruhide
1
2
1
1
MORI , Maher ABOU HACHEM , Martin W ILLEMOËS , Morten T. JENSEN ,
1
3
4
4
Peter K. NIELSEN , Nathalie JUGE , Xavier ROBERT , Samuel TRANIER ,
4
4
1
Nushin AGHAJARI , Richard HASER & Birte SVENSSON
1
Biochemistry and Nutrition Group, BioCentrum-DTU, Technical University of Denmark, Søltofts Plads,
Building 224, DK-2800, Kgs. Lyngby, Denmark; e-mail: [email protected]
2
Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500, Valby, Denmark
3
Institute of Food Research, Norwich, England
4
Laboratoire de BioCrystallographie, IBCP, CNRS, Lyon, France
The present work focuses on i) structure-guided mutation involving substrate binding at a
certain distance from the catalytic site, ii) polysaccharide hydrolysis by a multiple attack
mechanism, and iii) interaction of α-amylases with proteinaceous inhibitors. Subsite mapping
showed that barley α-amylases have a 10 subsites long binding cleft with 6 glycone (–6 to –1)
and 4 aglycone (+1 to +4) binding subsites (AJANDOUZ et al.). Substrates beyond a certain size
are envisaged to utilize additional binding areas and/or binding modules separate from the
catalytic domain. Such secondary binding sites are seen in crystal structures of barley αamylases 1 and 2 (KADZIOLA et al., 1998; ROBERT et al., 2003). Mutation of outer subsites –6
and +4 highly modified substrate specificity. Tyr105→Ala (subsite –6) thus has 140%, 15%,
and <1% activity toward insoluble starch, maltodextrin of DP17, and 2-chloro-4-nitrophenyl
maltoheptaoside, and Thr212→Tyr (subsite +4) has 32%, 370%, and 90% activity of AMY1
wild-type on the same substrates (BAK-JENSEN et al., 2004). This engineering of aromatic
residues is rationalized using modeled AMY1 maltodextrin complexes and guides future design
of AMY1/2 mutants. Tyr380 is situated at a newly discovered “sugar tongs” binding site in
domain C (ROBERT et al., 2003) and site-directed mutagenesis shows that this residue plays a
role in binding as measured by surface plasmon resonance. Surprisingly, although Tyr380 is
conserved, AMY2 lacks binding at the “sugar tongs”. Barley α-amylase applies a multiple
attack mechanism and hydrolyses amylose by 3 successive cleavages once the productive
enzyme-substrate complex is formed (KRAMHØFT et al., in prep.). This property appeared
unchanged in Tyr380 mutants. In contrast, 4 catalytic events per enzyme-substrate encounter
were obtained by Tyr105→Ala at the high-affinity subsite –6 or by the fusion AMY1-SBD with
a starch-binding domain of carbohydrate binding module family 20. AMY1-SBD shows 15-fold
increased activity on granular starch (JUGE et al., in prep.). Remarkably, AMY1 has even higher
multiple attack of 5 towards amylopectin. It is suggested that AMY1 hydrolyses several
substrate chains in the same enzyme-substrate encounter by “jumping” between amylopectin A
chains in a reaction that seems to involve the “sugar tongs”. In interactions with proteinaceous
inhibitors amylolytic enzymes use a variety of mechanisms. These will be discussed for
different enzymes and inhibitors on the basis of structure-guided mutational analysis and
engineering and measurements of binding affinities.
28
The work has been supported by the EU-projects AGADE (BIO4-CT98-0022), GEMINI (QLK-2000-00081), and CEGLYC
(QLK3-CT-2001-00149) and the Danish Natural Science Research Council.
AJANDOUZ, E.H., ABE, J., SVENSSON, B. & M ARCHIS-MOUREN, G. 1992. Biochim. Biophys. Acta 1159: 193-202.
BAK-JENSEN, K.S., ANDRÉ, G., GOTTSCHALK, T.E., … & SVENSSON, B. 2004. J. Biol. Chem. 279: 10093-10102.
JUGE, N., NØHR, J., LE GAL-COËFFET, M.-F., … & SVENSSON, B. In preparation.
KADZIOLA, A., SØGAARD, M., S VENSSON, B. & HASER, R. 1998. J. Mol. Biol. 278: 205-217.
KRAMHØFT, B., BAK-JENSEN, K.S., MORI, H., JUGE, N., NØHR, J. & SVENSSON, B. In preparation.
ROBERT, X., HASER, R., GOTTSCHALK, T.E., … & AGHAJARI, N. 2003. Structure 11: 973-984.
29
INVITED LECTURE - L14
Molecular analysis of α-glucosidase (GH-family 31)
Atsuo KIMURA, Masayuki OKUYAMA, Hiroyuki NAKAI, Haruhide MORI &
Seiya CHIBA
Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan;
e-mail: [email protected]
α-Glucosidase (EC 3.2.1.20) catalyses the hydrolytic reaction to liberate α-glucose from the
non-reducing end of the substrate. The enzyme recognizes many substrates, such as maltooligosaccharides (homogeneous substrate), p-nitrophenyl α-glucoside and sucrose
(heterogeneous substrate), starch and glycogen (polymer substrate). The substrate specificity
divided α-glucosidases into three groups (CHIBA & SHIMOMURA, 1978; CHIBA, 1988): i) The
first enzyme group (family-I) showed the high activity to heterogeneous substrate and the low
activities to homogeneous and polymer substrates; ii) The activity of the second enzyme group
(family-II) was high in the homogeneous substrate and low in the heterogeneous and polymer
substrates; iii) In the third enzyme group (family-III), the activity to homogeneous substrate as
well as polymer substrate was high and that to heterogeneous substrate was low. Interestingly,
the first enzyme group is classified into α-amylase family (GH-family 13), and both of the
second and the third groups are classified into GH-family 31.
α-Glucosidase family-II and family-III, being sorted into GH-family 31, distribute
widely in animals, plants, molds, yeasts, bacterium, and archaea. The catalytic amino acids of
Schizosaccharomyces pombe α-glucosidase were identified to be Asp481 and Asp647 by the
site-directed mutation approach (OKUYAMA et al., 2001). Each of Asp481 and Asp647 were in
the highly conserved sequences of “region A” and “region B” seen in the α-glucosidase familyII and family-III, respectively (OKUYAMA et al., 2001). It was found that Asp481Gly catalysed
the α-glycosynthase-reaction to form p-nitrophenyl α-maltoside and p-nitrophenyl αisomaltoside from β-glucosyl fluoride and p-nitrophenyl α-glucoside (OKUYAMA et al., 2002).
Yeast and mold α-glucosidases are classified into the family-II to have less or no
activity to polymer substrate. Plant α-glucosidase is the typical family-III enzyme to hydrolyse
starch. Recently we found the starch granule-binding region (domain, probably) in the Cterminal side of the plant α-glucosidases. A chimeric enzyme of yeast α-glucosidase, of which
the C-terminal region was replaced by that of plant α-glucosidase, acquired the starch granulebinding ability as well as starch granule-hydrolysing activity.
CHIBA, S. & S HIMOMURA, T. 1978. J. Jpn. Soc. Starch Sci. 25: 105-112.
CHIBA, S. 1988. in: Handbook of Amylases and Related Enzymes (ed. by The Amylase Research Society of Japan), pp. 104105.
OKUYAMA, M., OKUNO, A., S HIMIZU, N., MORI, H., KIMURA, A. & CHIBA, S. 2001. Eur. J. Biochem. 268: 2270-2280.
OKUYAMA, M., MORI, H., WATANABE, K., KIMURA, A. & CHIBA, S. 2002. Biosci. Biotechnol. Biochem. 66: 928-933.
30
ORAL T ALK - L15
Three-dimensional structure of glucodextranase,
glycoside hydrolase family 15 enzyme
1
1
1,2
Masahiro MIZUNO , Kazuhiro ICHIKAWA , Takashi TONOZUKA , Atsushi
1,2
1,2
NISHIKAWA & Yoshiyuki SAKANO
1
United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, 3-5-8,
Saiwai-cho, Fuchu, Tokyo 183-8509, Japan; e-mail: [email protected]
2
Department of Applied Science, Tokyo University of Agriculture and Technology, 3-5-8, Saiwai-cho,
Fuchu, Tokyo 183-8509, Japan
A glucodextranase (GDase) from Arthrobacter globiformis I42 hydrolyzes α-1,6-glucosidic
linkages of dextran from the non-reducing end to produce β-D-glucose (SAWAI et al., 1976).
Here we describe the crystal structures of GDase of the unliganded form and the complex with
acarbose. The structure of GDase is composed of four domains N, A, B and C. Domain A forms
an (α/α)6-barrel structure and domain N consists of 17 antiparallel β-strands, and both domains
are conserved in bacterial glucoamylases (GAs) and appear to be mainly concerned with
catalytic activity (MIZUNO et al., 2004). The structure of GDase complexed with acarbose
revealed that the positions and orientations of the residues at subsites -1 and +1 are nearly
identical between GDase and GA; however, the residues corresponding to subsite +2, which
form the entrance of the substrate-binding pocket, and the position of the open space and
constriction of GDase are different from those of GAs. On the other hand, domains B and C are
not found in the bacterial GAs. The primary structure of domain C is homologous with a surface
layer homology domain of pullulanases, and the three-dimensional structure of domain C
resembles the carbohydrate-binding domain of some glycohydrolases. The hydrophobicity of
domain B is higher than that of the other three domains. These findings suggest that domains B
and C serve the function of cell wall anchors and contribute to the effective degradation of
dextran at the cell surface.
SAWAI, T., YAMAKI, T. & OHYA, T. 1976. Agric. Biol. Chem. 40: 1293-1299.
MIZUNO, M., TONOZUKA, T., SUZUKI , S., UOTSU-TOMITA, R., KAMITORI, S., NISHIKAWA, A. & S AKANO, Y. 2004. J. Biol.
Chem. 279: 10575-10583.
31
ORAL T ALK - L16
Structure of the complex of S. fibuligera
glucoamylase with acarbose indicates the presence
of the starch-binding site in the catalytic domain
Jozef ŠEVČÍK, Eva HOSTINOVÁ, Adriana SOLOVICOVÁ & Juraj GAŠPERÍK
Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84551 Bratislava,
Slovakia; e-mail: [email protected]
Various strains of food-born yeast Saccharomycopsis fibuligera produce extracellular
glucoamylases with a high degree of homology in primary and tertiary structures that differ in
physico-chemical properties. Glucoamylase Glm produced by S. fibuligera IFO 0111 strain is
the only yeast amylolytic enzyme capable of raw starch digestion. Glucoamylase Glm, highly
homologous to Glu from S. fibuligera HUT7212, has a unique structure because it does not
contain a separate starch-binding domain typical for all known raw starch degrading amylases.
Our preliminary results show that the raw starch-binding site of Glm (HOSTINOVÁ et al., 2003) is
an integral part of the intact, “single domain” enzyme corresponding to the catalytic domains of
fungal glucoamylases.
Glucoamylase Glu produced by S. fibuligera HUT7212 strain is one of the bestdescribed glucoamylases. The enzyme although unable of raw starch digestion has a good
affinity towards this substrate. Structure of the enzyme in a complex with TRIS molecule bound
in the active site has been determined at 1.7 (ŠEVČÍK et al., 1998) and 1.1 Å resolution. The
enzyme consists of 492 residues and has 14 α-helices, 12 of which form an (α/α)6 barrel.
Here we present the structure of Glu in a complex with the specific amylase inhibitor
acarbose. The enzyme was crystallized from a protein solution of 10 mg/ml in 50 mM acetate
buffer at pH 5.4 and 15% PEG 8K. Crystals of the glucoamylase-acarbose complex were
prepared by diffusion. For the soaking, 1 µl of the mother liquor enriched by acarbose in
concentration 10 mM was added into drops (5 µl) containing native crystals. Diffraction data
were collected at room temperature on the EMBL synchrotron beam line X11 to 1.6 Å
resolution with a MAR Research (Hamburg) imaging plate scanner and processed with DENZO.
For solving the structure molecular replacement method was used with the structure of Glu
(1AYX) as a model. The structure was refined with the program REFMAC. Refinement
converged with R factor of 13.5%.
In the enzyme structure two acarbose molecules have been localized: one at the active
site and the other on the surface of the enzyme molecule interacting with Arg15, His447,
Asp450 and Thr462. From comparison of Glu with the modeled structure of Glm we deduce that
acarbose molecule which does not interact with the active site of Glu might be bound in the
region of the putative starch binding site. To prove this hypothesis, the double mutant of Glu
residues interacting with acarbose (H447A, D450A), was prepared. The recombinant protein,
expressed in Saccharomyces cerevisiae was isolated, characterized and compared with the wild
type enzyme. The mutant enzyme retained, on soluble starch, the full catalytic activity of the
wild type enzyme but its ability to adsorb on raw starch decreased.
HOSTINOVÁ, E., SOLOVICOVÁ, A., D VORSKÝ, R. & GAŠPERÍK, J. 2003. Arch. Biochem. Biophys. 411: 189-195.
ŠEVČÍK, J., SOLOVICOVÁ, A., HOSTINOVÁ, E., GAŠPERÍK, J., WILSON, K.S. & D AUTER, Z. 1998. Acta Cryst. D54: 854-866.
32
ORAL T ALK - L17
Probing the structure of glucan lyases - the lytic
members of GH-31 - by sequence analysis, circular
dichroism and proteolysis
1
1
2
2
Heidi A. ERNST , Leila LO LEGGIO , Shukun YU , Clive P. W ALTER , Christine
3
3
1,4
FINNIE , Birte SVENSSON & Sine LARSEN
1
Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø,
Denmark; e-mails: [email protected]; [email protected]
2
Danisco Innovations, Danisco A/S, Langebrogade 1, PO Box 17, DK-1001 Copenhagen K, Denmark
3
Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Valby, Denmark
4
European Synchrotron Radiation Facility (ESRF), 6 Rue Jules Horowitz, 38000 Grenoble, France
The glucan lyase (GL) is a unique polysaccharide lyase, which produces 1,5-anhydro-fructose
by lytic degradation of α-1,4-glucans such as maltose, maltooligosaccharides and the larger
storage polymers starch and glycogen (YU et al., 1999). The enzyme works by a catalytic
mechanism completely different from other known polysaccharide lyases (YU et al., 1999; LEE
et al., 2003). Notably, GL belongs to glycoside hydrolase family 31 in the sequence-based
classification system for carbohydrate-active enzymes (COUTINHO & HENRISSAT, 1999), rather
than one of the lyase families. No structure is yet known for a representative of GH family 31
and crystallization trials for GL are in progress. Here, we report parallel studies to probe the
structure of glucan lyases.
GLs are large proteins consisting of 1030-1080 residues depending on origin. A study of
the domain organization of the protein using bioinformatic approaches revealed remote
homology between the central, catalytic domain and the (β/α)8-barrel enzymes in GH family 13,
as also shown by others (RIGDEN, 2002). This indicates that the protein must be composed of at
least three structural domains. No structural homologues could be identified for the N- and Cterminal regions, which are predicted to consist mostly of β-strands. The overall secondary
structure content in this model is in good agreement with circular dichroism data obtained from
two GL isozymes from the red alga Gracilariopsis lemaneiformis. The two algal enzymes have
been studied by limited proteolysis using a wide range of proteases in order to further elucidate
the domain structure. The GLs were found to be surprisingly resistant to proteolytic degradation,
requiring relatively high protease concentrations and long incubation times for cleavage to
occur. Two cleavage sites have been identified in the N-terminal part of the protein, while the
central domain and the C-terminal region do not seem to be susceptible to proteolytic attack.
These results suggest that GLs are compact in structure, unlike many carbohydrate-modifying
enzymes consisting of modules connected by long flexible linkers.
We would like to acknowledge the EU for financial support to the NEPSA project.
Y U, S., BOJSEN, K., S VENSSON, B. & M ARCUSSEN, J. 1999. Biochim. Biophys. Acta 1433: 1-15.
LEE, S.S., Y U, S. & WITHERS, S. 2003. Biochemistry 42: 13081-13090.
COUTINHO, P.M. & HENRISSAT, B. 1999. http://afmb.cnrs-mrs.fr/cazy/CAZY/.
RIGDEN, D.J. 2002. FEBS Letters 523: 17-22.
33
INVITED LECTURE - L18
Characterization of a GH57 amylopullulanase: a
survey of functionality and active site organisation
1
Michael J. O’DONOHUE & Florent CHANG-PI-HIN
1,2
1
UMR FARE, Institut National de la Recherche Agronomique, 8, rue Gabriel Voisin, BP 316, 51688 cedex
02, France; e-mail: [email protected]
2
Present address: Unité de Biochimie, Place L. Pasteur, 1, B-1348 Louvain-la-Neuve, Belgique
Family 57 is composed of an ensemble of mainly amylolytic enzymes produced, for the most
part, by extremophilic microorganisms, notably arachaebacteria. From the outset, the status of
this family has been the subject of discussion because the relationship between GH57 and the
families that comprise the α-amylase family is unclear. However, two recent major
contributions from IMAMURA et al. (2001; 2003) as well as other work from several groups
including our own (ZONA et al., 2004; VAN LIESHOUT et al., 2003) and KANG et al. (2004) has
started to clarify this question and lead to a better understanding of structure/function
relationships in GH57 enzymes.
At the time of writing, the CAZy database contains 44 entries for GH-57. Of these, only
11 correspond to known enzyme activities. Four activities are represented: α-amylase 3.2.1.1,
amylopullulanase, 3.2.1.41, α-galactosidase 3.2.1.22 and 4- α-glucanotransferase, 2.4.1.25. The
last of these activities is represented by one enzyme, produced by the extremophilic
archaebacterium Thermococcus litoralis, that to date constitutes the most well studied member
of the family. The 4- α-glucanotransferase (TLGT) is a transglycosylating enzyme that acts on
the α-1,4 in α-D-glucans. Importantly, previous studies have led to the isolation and cloning of
the gene encoding TLGT, the identification (by the use of in vitro mutagenesis and a suicide
inhibitor) of its catalytic machinery and more recently the determination of its 3D structure.
In our group, we have being studying another GH57 enzyme that is a representative
member of a recently defined GH57 amylopullulanase subgroup (ZONA et al., 2004). This
enzyme, produced by Thermococcus hydrothermalis and designated ThApu, displays dual bond
cleaving capacity, acting both on α-1,4 and α-1,6 glucosidic bonds. As one might expect,
ThApu also displays extreme thermoactivity and thermostability. During our work, using a
variety of techniques, we have characterized different aspects of ThApu activity and provided a
partial identification of its catalytic residues. In this presentation, we will describe our data and
propose a working model for active site organization in ThApu. Moreover, we will discuss our
results in the light of the structural data for TLGT and that available for other α-(1,6)-acting
enzymes from GH13.
IMAMURA, H., F USHINOBU, S., JEON, B.S., WAKAGI, T. & M ATSUZAWA, H. 2001. Biochemistry 40: 12400-12406.
IMAMURA, H., F USHINOBU, S., Y AMAMOTO, M., … & MATSUZAWA, H. 2003. J. Biol. Chem. 278: 19378-19386.
KANG, S., VIEILLE, C. & ZEIKUS, J.G. 2004. Appl. Microbiol. Biotechnol. in press.
VAN LIESHOUT , J.F.T., V ERHHS, C.H., ETTEMA, T.J.G., … & D E VOS, W.M. 2003. Biocatal. Biotransform. 21: 243-252.
ZONA, R., CHANG-PI-HIN, F., O’DONOHUE, M.J. & JANECEK, S. 2004. Eur. J. Biochem. 271: 2863-2872.
34
INVITED LECTURE - L19
CEASE: Combinatorial Engineering to enhance
Amylosucrase performance
Bart A. VAN DER VEEN, Gabrielle POTOCKI–VÉRONÈSE, Cécile ALBENNE, Gilles
JOUCLA, Pierre MONSAN & Magali REMAUD-SIMEON
Centre de Bioingénierie Gilbert Durand, UMR CNRS 5504, UMR INRA 792, INSA, 135 avenue de
Rangueil, 31077 Toulouse Cedex 4, France; e-mail: [email protected]
Amylosucrase is a glucosyltransferase belonging to family 13 of glycoside hydrolases and
catalyses the formation of an amylose-type polymer from sucrose. Its potential use as an
industrial tool for the synthesis or the modification of polysaccharides, however, is limited by its
low catalytic efficiency on sucrose alone, its low stability, and its side reactions resulting in
sucrose isomer formation. Although some improvement can be obtained by site directed
mutagenesis (ALBENNE et al., 2004), not all determinants for these features are apparent from
structural and mutational analyses. Therefore, combinatorial engineering of amylosucrase
(CEASE) was started in the EU project CEGLYC (Combinatorial Engineering of GLYCoside
hydrolases of the α-amylase family), in order to generate more efficient variants of the enzyme
(VAN DER VEEN et al., 2004). In this project several strategies are being used to isolate novel
enzymes: directed evolution, through random mutagenesis, gene shuffling, and selective
screening; site-directed random mutagenesis, randomly changing specific residues involved in
the substrate binding pocket; genome mining, making use of the information made available in
the sequenced genomes. A convenient zero background expression cloning strategy was
developed for the generation of mutant gene libraries by error prone PCR, using Taq polymerase
with unbalanced dNTP’s or MutazymeTM, followed by recombination of the PCR products by
DNA shuffling. A selection method was developed to allow only the growth of amylosucrase
active clones on solid mineral medium containing sucrose as unique carbon source. Automated
protocols were designed to screen amylosucrase activity from mini-cultures using DNS staining
of reducing sugars and iodine staining of amylose-like polymer. Several variants with increased
activity, improved specificity, and higher thermostability have been isolated. Saturation
mutagenesis of selected residues in the amylosucrase binding pocket was carried out using
specific primers for several stretches of such residues. Again shuffling techniques were applied
to combine the mutations in the various residues, yielding large variant libraries, which were
analysed using the developed selection and screening procedures. Genome mining for
amylosucrases yielded several gene sequences of putative amylosucrases, however, with various
annotations. The encoded proteins share 35-45% identity with N. polysaccharea amylosucrase
(AS) and contain several amino acid residues proposed to be involved in AS specificity. The
putative amylosucrase identified in the Deinococcus radiodurans genome shares 42% identity
with N. polysaccharea amylosucrase, but was initially annotated as an α-amylase encoding
gene. The gene (NP_294657.1) was cloned and expressed in E. coli, and the protein was shown
to be a novel amylosucrase enzyme.
This work was supported by the EU project N° QLK3-CT-2001-00149; Combinatorial Engineering of GLYCoside
hydrolases from the α-amylase superfamily (CEGLYC).
ALBENNE, C., SKOV, L.K., MIRZA, O., … & REMAUD-SIMEON M. 2004 J. Biol. Chem. 279: 726-734.
VAN DER V EEN, B.A., POTOCKI–VÉRONÈSE, G., ALBENNE, C., … & REMAUD-SIMEON, M. 2004. FEBS Lett. 560: 91-97.
35
ORAL T ALK - L20
Dissociation/association properties of cyclodextrinhydrolyzing enzyme: an oligomeric state analysis
by pH and salt
1
1
2
1
Hee-Seob LEE , Jin-Soo KIM , Cheon-Seok PARK & Kwan-Hwa PARK
1
National Laboratory for Functional Food Carbohydrates, Center for Agricultural Biomaterials, and
Department of Food Science and Technology, Seoul National University, Seoul 151-742, Korea;
e-mail: [email protected]
2
Department of Food Science and Technology, Kyunghee University, Yongin 449-701, Korea
To understand the effect of pH and salt on the quaternary structure of cyclomaltodextrinase
(CDase) I-5, dissociation/association process of the oligomeric state of enzyme was studied at
various pHs, and in the presence of KCl. CDase I-5 exists exclusively in the state of dodecamer
by unification of six dimeric subunits at pH 7.0 whereas it prefers to be in a dimeric state at pH
6.0 (LEE et al., 2002). Thus, a dissociation-association phenomenon between dimer and
dodecamer is a pH-dependent process.
A series of mutant CDases having the combination of three histidine residues
substitution in N- and C-terminal regions (H49, H89, and H539) were constructed. The
dissociation processes of wild type and mutant CDase I-5 were compared at pH 6.0 by gel
permeation chromatography. The dissociations of the mutant enzymes occurred significantly
faster at pH 6.0 than that observed in the wild type enzyme. The results indicated that histidine
residues in N- and C-terminal regions played a significant role in dodecamerization of CDase I5.
The dissociation of wild type CDase from dodecamer to dimer in the presence of KCl at
pH 7.0 was extremely faster than that in the absence of salt. The rate constant of dissociation
from dodecamer to dimer was determined at various concentration of KCl by stopped-flow
spectrophotometer. These results suggest a possibility that the oligomeric state of CDase is
controlled by both pH and salt in natural environment (PARK et al., 2000).
PARK, K.H., KIM, T.J., CHEONG, T.K, KIM, J.W., OH, B.H. & SVENSSON, B. 2000. Biochim. Biophys. Acta 1478: 165-185.
LEE, H.S, KIM, M.S., CHO , H.S., KIM, J.I., KIM, T.J., CHOI, J.H., P ARK, C., LEE, H.S., OH, B.H. & PARK, K.H. 2002. J. Biol.
Chem. 277: 21891-21897.
36
ORAL T ALK - L21
Molecular evolution techniques as tools to improve
barley α-amylase AMY2 expression
Sophie BOZONNET1,2, Tae-Jip KIM1 & Birte SVENSSON1,2
1
Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby,
Denmark
2
Biochemistry and Nutrition Group, BioCentrum-DTU, Technical University of Denmark, Søltofts Plads Building 224, DK-2800 Kgs. Lyngby, Denmark; e-mail: [email protected]
Germinating barley seeds produce two α-amylase isozyme families distinguished by low
(AMY1) and high (AMY2) pI. Recombinant AMY1 and AMY2 are produced in yeasts, but
whereas reAMY1 can be obtained up to 50 mg/L using Pichia pastoris as host, the level of
production for reAMY2 only reaches 1 mg/L (JUGE et al., 1996).
We took advantage of the high level of genetic and structural similarity between AMY1
and AMY2 (80% sequence identity) to apply in vitro molecular evolution techniques as a
strategy to create α-amylase variants with better expression efficiency. Stemmer’s gene
shuffling method (STEMMER, 1994) was applied on templates selected amongst chimeric αamylases (KIM et al., 2003), chosen for their observed low activity on starch plates. In vivo
recombination was used for direct cloning into the expression host Saccharomyces cerevisiae.
Screening methods for the isolation of potential candidates were optimised and led us to obtain
α-amylase variants with improved activity on starch plates. Then, the amy genes from selected
clones were subcloned from their S. cerevisiae vector to a new vector allowing expression in P.
pastoris. The 4 new variants could all be produced in higher amount than parental reAMY2,
reaching concentrations up to 22 mg/L. Calcium dependency profiles proved high similarities
with reAMY2, showing optimal calcium concentration at 10 mM CaCl2, compared to 0.1 mM
for reAMY1. Concerning inhibition by barley α-amylase/subtilisin inhibitor (BASI), a key
distinctive between AMY1 and AMY2, two variants were inhibited and two were insensitive to
BASI. This pinpointed the influence of H295AMY2 in the interaction between inhibitor and
enzyme. DNA sequencing revealed that, even though a good level of shuffling of parental DNA
is obtained, the most highly active mutants are AMY2-like proteins, with protein identity that
could reach 98%. In addition, most of the observed mutations were silent, improving locally the
codon usage of the amy gene. This result points out the interest to focus on the local
improvement of codon usage in AMY2 by rational mutagenesis as well as combinatorial
engineering. Therefore, a new molecular evolution technique known as Degenerate
Oligonucleotide Gene Shuffling (DOGS) method (GIBBS et al., 2001) was used. It is based on
the design of primers in which the local coding sequence was improved according to the Pichia
codon usage table, aiming at producing AMY2 proteins with a much higher expression level.
Enzymatic and chemical properties of the different shuffling products will be reported.
This work was supported by the EU 5th Framework program to the project CEGLYC (QLK3-CT-2001-00149).
GIBBS, M.D., N EVALAINEN, K.M. & BERGQUIST, P.L. 2001. Gene 271: 13-20.
JUGE, N., ANDERSEN, J.S., T ULL, D., ROEPSTORFF, P. & S VENSSON, B. 1996. Protein Expr. Purif. 8: 204-214.
KIM, T.-J., BOZONNET, S., NIELSEN, P.K. & SVENSSON, B. 2003. In: Carbohydrate: Enzymes and Food Functionality,
Proceedings 2003 Agricultural Biotechnology Symposium, pp. 9-17.
STEMMER, W.P. 1994. Proc. Natl. Acad. Sci. US A 91: 10747-10751.
37
ORAL T ALK - L22
Engineering the reaction specificity of GH77
amylomaltase from Thermus thermophilus HB8 by
laboratory evolution
1,2
1,2
1,2
Thijs KAPER , Wieger EEUWEMA , Gert-Jan EUVERINK , Marc van der
1,3
1,2
MAAREL & Lubbert DIJKHUIZEN
1
Centre for Carbohydrate Bioengineering, TNO-University of Groningen, The Netherlands;
e-mail: [email protected]
2
Microbial Physiology, University of Groningen, Haren, Kerklaan 30, 9751 NN Haren, The Netherlands
3
Innovative Ingredients and Products Department, TNO-Nutrition and Food Research, Rouaanstraat 27,
9723 CC Groningen, The Netherlands
Glycoside hydrolases of family 77 are 4-α-glucanotransferases that belong to the α-amylase
superfamily. Their in vivo function is to disproportionate malto-oligosaccharides. In microorganisms they are known as amylomaltases, while their homologues in plants are named
D(isproportionating)-enzymes. Amylomaltases have applications in the starch industry (KAPER
et al., 2004). However, the high temperatures needed for the solubilization of starch require high
intrinsic enzyme stability. We therefore focus on amylomaltases from thermophilic organisms,
such as Thermus thermophilus. At 70 oC and pH 5.5, the T. thermophilus amylomaltase (Tt
AMase) is stable, active, and displays the highest transferase versus hydrolysis ratio (5000 : 1)
in the α-amylase superfamily. Acting on native potato starch, Tt AMase converts it to a product
that forms a strong thermo-reversible gel with gelatin-like properties (BINNEMA & EUVERINK,
1998).
The unique reaction specificity of Tt AMase has been addressed by laboratory evolution.
The Tt AMase encoding gene was subjected to two rounds of error-prone PCR and the resulting
variant enzymes were screened for increased hydrolysis of starch. Several variants with
increased hydrolytic activity (up to 15-fold) and altered ratios of transferase versus hydrolytic
activity (down to 34 : 1) were isolated. The mutated residues that were responsible for the
changes in activity were concentrated in the active site and the ‘second shell’.
BINNEMA, D.J. & E UVERINK, G.J.W. 1998. Patent application WO9815347.
KAPER, T., VAN DER MAAREL, M.J., E UVERINK, G.J. & DIJKHUIZEN, L. 2004. Biochem. Soc. Trans. 32: 279-282.
38
ORAL T ALK - L23
Specificity engineering of a maltogenic α-amylase
Lars BEIER, Carsten ANDERSEN, Anders VIKSØ-NIELSEN, Barrie E. NORMAN,
Sven PEDERSEN & Torben V. BORCHERT
Novozymes A/S, Krogshoejvej, DK-2880 Bagsvaerd, Denmark; email: [email protected]
α-Amylases (EC 3.2.1.1) are a family of endo-amylases that catalyze the hydrolysis of α-1,4glycosidic linkages in polymers of α-D-glucose and are thus needed for most organisms, since
starch and similar polymers are very often preferred as energy source. Despite great differences
in primary structure α-amylases from different organisms exhibit similar three-dimensional
structures. The α-amylase family has a (β/α)8- or TIM-barrel as the catalytic domain. Hence, the
α-amylases belong to family 13 in the glycosyl hydrolase classification (COUTINHO &
HENRISSAT, 1999) as all other starch-degrading enzymes containing a TIM-barrel.
The family 13 enzymes show a great variety regarding the product pattern resulting from
starch hydrolysis; e.g. α-amylases produce oligosaccharides with different degree of
polymerization while cyclodextrin glycosyltransferases (CGTases) produce both cyclodextrins
(CDs) and oligosaccharides. However, it is not always possible to identify a family 13 enzyme
both having the optimal product pattern and being able to perform at the relevant industrial
conditions. The answer to this issue is to optimize the current best enzyme towards the optimal
properties. Both protein engineering and directed evolution can be utilized for such tasks.
Stability issues are often addressed successfully whereas specificity typically represents
a more complicated property to optimize for due to the intrinsic coupling of the active site and
substrate-binding pocket determining the specificity of the enzyme.
We describe two efforts to engineer the specificity of a maltogenic α-amylase, a rational
approach and a random approach. In the rational approach the maltogenic α-amylase was
converted into a CGTase capable of producing CDs (BEIER et al., 2000), while in the random
approach the specificity of the maltogenic α-amylase was changed by directed evolution to
optimize the ratio between the various products.
We thank the European Commission for the grant QLK3-CT-2001-00149 generously supporting this work.
BEIER, L., SVENDSEN, A., ANDERSEN, C., FRANDSEN, T.P., BORCHERT , T.V. & CHERRY, J.R. 2000. Protein Eng. 13: 509513.
COUTINHO, P.M. & HENRISSAT, B. 1999. http://afmb.cnrs-mrs.fr/~cazy/CAZY/.
39
INVITED LECTURE - L24
Structural studies of sucrose-utilizing enzymes
from glycoside hydrolase family 13
1
1
2
2
Lars K. SKOV , Desireé SPROGØE , Cécille ALBENNE , Magali REMAUD-SIMEON ,
2
2
3
Bart A. VAN DER VEEN , Pierre MONSAN , Lambertus A. M. VAN DEN BROEK ,
3
1
Alphons G. J. VORAGEN & Michael GAJHEDE
1
Biostructural Research, Department of Medicinal Chemistry, The Danish University of Pharmaceutical
Sciences, Universitetsparken 2, DK 2100 Copenhagen, Denmark; e-mail: [email protected]
2
Centre de Bioingénierie Gilbert Durand, UMR CNRS 5504, UMR INRA 792, INSA, 135, avenue de
Rangueil, F-31077 Toulouse, France
3
Laboratory of Food Chemistry, Wageningen University, P.O. Box 8129, 6700 EV Wageningen, The
Netherlands
The size variation of carbohydrates that are substrates for enzymes from glycoside hydrolase
(GH) family 13 is quite remarkable. These include huge polymers like α-amylose down to small
disaccharides like sucrose and trehalose. However, the individual enzyme can be very specific in
both substrate selection and product formation. How this specificity is tailored by the protein
structure is a fundamental question that needs to be addressed before enzymes with improved
properties can be made by rational design. We will here focus on enzymes that utilize sucrose as
glycosyl donor, but give very different products. In GH 13 these include amylosucrase,
isomaltulose synthase, sucrose hydrolase and sucrose phosphorylase. For several of these highresolution structures are available (SKOV et al., 2001, SPROGOE et al., 2004; ZHANG et al., 2003)
and for amylosucrase structures of enzyme:carbohydrate complexes have also been determined
(MIRZA et al., 2001; SKOV et al., 2002). This will be the experimental background for an analysis
of similarities and differences.
Part of this work was supported by the EU project Combinatorial Engineering of Glycoside hydrolases (CEGLYC, QLK3CT-2001-00149). DS wishes to thank Novozymes A/S for a Novozymes Scholarships.
MIRZA, O., S KOV, L.K., REMAUD-SIMEON, M., POTOCKI DE MONTALK, G., ALBENNE, C., MONSAN, P. & GAJHEDE, M. 2001.
Biochemistry 40: 9032-9039.
SKOV, L.K., MIRZA, O., HENRIKSEN, A., POTOCKI DE MONTALK, G., REMAUD-SIMEON, M., S ARCABAL, P., WILLEMOT,
R.M., MONSAN, P. & GAJHEDE, M. 2001. J. Biol. Chem. 276: 25273-25278.
SKOV, L.K., MIRZA, O., SPROGOE, D., DAR, I., REMAUD-SIMEON, M., ALBENNE, C., MONSAN, P. & GAJHEDE, M. 2002. J.
Biol. Chem. 277: 47741-47747.
SPROGOE, D., VAN DEN BROEK, L.A.M., MIRZA, O., KASTRUP, J.S., VORAGEN, A.G.J., GAJHEDE, M. & SKOV, L.K. 2004.
Biochemistry 43: 1156-1162.
ZHANG, D.H., LI, N., LOK, S.M., ZHANG, L.H. & SWAMINATHAN, K. 2003. J. Biol. Chem. 278: 35428-35434.
40
INVITED LECTURE - L25
Laforin preferentially binds the neurotoxic starchlike polyglucosans, which form in its absence in
progressive myoclonus epilepsy
Elayne M. CHAN, Cameron A. ACKERLEY, Hannes LOHI, Leonarda IANZANO,
Miguel A. CORTEZ, Patrick SHANNON, Stephen W. SCHERER & Berge A.
MINASSIAN
The Hospital for Sick Children and Department of Molecular and Medical Genetics, the University of
Toronto, Toronto, Canada; e-mail: [email protected]
Lafora disease (LD) is a fatal and the most common form of adolescent-onset progressive
epilepsy. Fulminant endoplasmic reticulum (ER)-associated depositions of starch-like longstranded, poorly branched glycogen molecules [known as polyglucosans, which accumulate to
form Lafora bodies (LBs)] are seen in neuronal perikarya and dendrites, liver, skeletal muscle
and heart. The disease is caused by loss of function of the laforin dual-specificity phosphatase or
the malin E3 ubiquitin ligase. Towards understanding the pathogenesis of polyglucosans in LD,
we generated a transgenic mouse overexpressing inactivated laforin to trap normal laforin's
unknown substrate. The trap was successful and LBs formed in liver, muscle, neuronal
perikarya and dendrites. Using immunogold electron microscopy, we show that laforin is found
in close proximity to the ER surrounding the polyglucosan accumulations. In neurons, it
compartmentalizes to perikaryon and dendrites and not to axons. Importantly, it binds
polyglucosans, establishing for the first time a direct association between the disease-defining
storage product and disease protein. It preferentially binds polyglucosans over glycogen in vivo
and starch over glycogen in vitro, suggesting that laforin's role begins after the appearance of
polyglucosans and that the laforin pathway is involved in monitoring for and then preventing the
formation of polyglucosans. In addition, we show that the laforin interacting protein,
EPM2AIP1, also localizes on the polyglucosan masses, and we confirm laforin's intense binding
to LBs in human LD biopsy material.
41
INVITED LECTURE - L26
An overview of clan GH-H and distantly-related
families
E. Ann MACGREGOR
2 Nicklaus Green, Livingston, West Lothian, EH54 8RX, U.K.;
e-mail: [email protected]
In 1991 Bernard Henrissat (HENRISSAT, 1991) proposed a scheme for classifying glycoside
hydrolases, based on sequence rather than specificity of action. Up to now, 97 families of
glycosidases or transglycosidases have been identified, and some of these have been grouped
into clans, where a clan contains families closely related in three-dimensional structure and
catalytic mechanism.
A short history of clan GH-H, consisting of families 13, 70 and 77 - the so-called
α-amylase families - will be given. Relationships between the enzymes of this clan and families
such as 14, 27 and 31 will be explored, with emphasis on tertiary structure and active site
amino-acid residues.
HENRISSAT, B. 1991. Biochem. J. 280: 309-316.
42
INVITED LECTURE - L27
The thermohalophilic amylases of Halothermothrix
orenii
Bharat K. C. PATEL
Microbial Diversity and Discovery Group, School of Biomolecular and Biomedical Sciences, Faculty of
Scienc, Griffith University, Brisbane 4111, Queensland, Australia; e-mail: [email protected]
The anaerobe, Halothermothrix orenii (CAYOL et al., 1994), expresses environmental adapted
enzymes for growth at both high temperatures (growth maxima of 68 oC) and high salt
concentrations (NaCl concentrations up to 20%). Molecular analysis indicates that the proteins
from H. orenii lack the highly charged acidic amino acids characteristic of the “true” halophilic
members of the family Haloanaerobiaceae, with which it shares a close phylogenetic
relationship (MIJTS & PATEL, 2001a). Two amylase genes (amyA and amyB) were cloned and
the genes, including the upstream and downstream regions of up to 7 kb, were sequenced to
determine gene organisation (MIJTS & PATEL, 2001b). AmyA and AmyB were distantly related
to each other within the radiation of phylum “firmicutes” The characterisation of over-expressed
AmyA and AmyB showed marked differences in their thermostability, halophilicity and
substrate spectrum (MIJTS & PATEL, 2001, 2002). Analysis of data of crystals of AmyA and
AmyB which diffract to 1.8 and 2.5 Å respectively (LI et al., 2002; TAN et al., 2003), and other
proteins, suggests that the bacterium has specific structural properties and survival strategies
which assists it to adapt to fluctuating extreme environmental conditions.
CAYOL, J-L, OLLIVIER, B., PATEL, B.K.C., PRENSIER, G., GUEZENNEC, J. & GARCIA, J-L. 1994. Int. J. Bacteriol. 44: 534540.
LI, N., P ATEL, B.K.C., MIJTS, B. & SWAMINANTHAN, K. 2002. Acta Cryst. D58: 2125–2126.
MIJTS, B. & PATEL, B.K.C. 2002. Microbiology 148: 2343-2349.
MIJTS, B. & PATEL, B.K.C. 2001a. Extremophiles 5: 61-69.
MIJTS, B. & PATEL, B.K.C. 2001b. PhD thesis, Griffith University, Brisbane, Australia.
TAN, T.C., YIEN, Y.Y., P ATEL, B.K.C., MIJTS, B.N. & SWAMINATHAN, K. 2003. Acta Cryst. D59: 2257-2258.
43
ORAL T ALK - L28
Transglycosylations catalysed by α-amylases
Lili KANDRA
Institute of Biochemistry, Faculty of Sciences, University of Debrecen, H-4010 Debrecen, PO Box 55,
Hungary; e-mail: [email protected]
Enzyme-catalyzed synthesis of oligosaccharides is a very attractive method because it allows the
formation of well-defined oligosaccharides selectively without using any protection of hydroxyl
groups. Many different oligosaccharides have already been synthesized by enzymatic transfer
reactions. In addition, on the basis of the advances in genetic engineering, it is becoming
possible to produce a wider range of enzymes on a large scale, expending the number of
enzymes available for synthetic reactions. Despite the increasing work carried out with
glycosidases, little is known about the structural requirement for the binding of sugar acceptors
to the enzyme, which is essential to improve the synthetic utility of this methodology.
Human amylases of both salivary (HSA) and pancreatic origins (HPA) have been
extensively studied from the viewpoint of clinical chemistry because they are important as
indicators in evaluating diseases of pancreas and salivary glands. Furthermore, they are used as
targets for drug design in attempts to treat diabetes, obesity and other sugar metabolic disorders.
In addition, they could be useful for the synthesis of oligosaccharides by transglycosylation.
Synthesis of 4-nitrophenyl 1-thio-β-D-maltoside, maltotrioside and maltotetraoside in
yields up to 60%, has been achieved by Tyr151Met (Y151M) mutant of human salivary αamylase. Y151M is capable of transferring maltose and maltotriose residues from maltotetraose
donor onto different p-nitrophenyl glycosides. 1H and 13C-NMR studies revealed that the
mutated enzyme reserved the stereo- and regioselectivity. The glycosylation took place at
position 4 of the glycosyl acceptor forming the α(1-4)glycosidic bond, exclusively.
Many α-amylase inhibitors have been obtained from microbial and plant origins and
have also been found in synthetic substances. They have been proved of great value because of
their various uses as tools for the investigation of the active site of α-amylases, as reagents for
the measurement of α-amylase isoenzyme activities by selective inhibition, or as oral agents for
the treatment of diabetes, obesity, hyperlipidemia and caries.
The glucopyranosylidene-spiro-thiohydantoin (G-TH) is a very efficient competitive
inhibitor of phosphorylase with a µM range of Ki. We found that it was a mixed noncompetitive inhibitor of HSA with the inhibition constants in a mM range. Since this is a small
molecule the long enough active site facilitates accommodating in different binding modes
decreasing its inhibitor activity. It was envisaged that the longer spiro-thiohydantoin analogues
should be more effective inhibitors than G-TH. G-TH was transglycosylated with acarbose using
a new thermostable maltogenic amylase from Bacillus stearothermophilus (BSMA). Synthesis
and structural analysis of the acceptor products will be discussed in the presentation. NMR
studies revealed that the main product is a glucopyranosylidene-spiro-thiohydantoinyl-α-(1-6)acarviosin-glucoside (PTS-G-TH).
The new PTS-G-TH is an extremely effective inhibitor, much more effective than
acarbose, with Ki value three orders of magnitude lower than isoacarbose and four orders of
magnitude lower than acarbose.
44
ORAL T ALK - L29
Two novel cyclodextrin-degrading enzymes isolated
from thermophilic bacteria, have similar domain
structures but differ in oligomeric state and activity
profiles
Pernilla TURNER1, Antje LABES2, Olafur H. FRIDJONSSON3, Gudmundur O.
3
2
3
1
HREGGVIDSON , Peter SCHÖNHEIT , Jakob K. KRISTJANSSON , Olle HOLST &
1
Eva NORDBERG KARLSSON
1
Department of Biotechnology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden;
e-mails: [email protected], [email protected]
2
Institute for General Microbiology, Christian-Albrechts-University Kiel, Am Botanischen Garten 1-9, DE24118 Kiel, Germany
3
Prokaria Ltd, Gylfaflöt 5, IS-112 Reykjavik, Iceland
Two novel enzymes belonging to the α-amylase family and exhibiting cyclomaltodextrinase
specificity have been expressed and characterized. The sequences encoding the enzymes were
isolated from the genomic DNA of two thermophilic bacterial strains originating from Icelandic
hot springs and belonging to the genera Anoxybacillus (AfCda13) and Thermoactinomyces
(TsCda13) respectively. The genes were amplified using a consensus primer strategy utilizing
two of the four conserved regions present in glycoside hydrolase family 13. No identifiable
signal peptide was present in the ORFs encoding the respective enzyme, indicating an
intracellular location in both cases, and their physiological function to be intracellular
cyclodextrin degradation. The domain structure of both enzymes was also similar, including an
N-terminal domain, the catalytic module composed of the A- and B-domain, and a C-terminal
domain. Despite the similar domain composition, the two enzymes displayed differences in
oligomeric state with AfCda13 being a dimeric protein, while TsCda13 was a monomer. The two
enzymes also displayed significantly different activity profiles, despite being active on the same
range of substrates. It was shown that the enzyme displaying the highest cyclodextrin activity
was dimeric (AfCda13), indicating that the difference in oligomeric state is a key factor for the
relatively higher activity on cyclodextrin substrates.
This work was supported by the European Union (Contract no QLK3-CT-2000-01068). Additional funding was also
received from Stiftelsen Bengt Lundqvist Minne, Ernhold Lundströms Stiftelse, Bertil Anderssons fond and Hakon Hanssons
Stipendiefond.
45
ORAL T ALK - L30
Characterisation of putative α-amylases from apple
(Malus domestica) and Arabidopsis thaliana
1,2
1
2
Duncan STANLEY , Kevin J. F. FARNDEN & Elspeth A. MACRAE
1
Department of Biochemistry, University of Otago, P.O. Box 56, Dunedin, New Zealand;
HortResearch, Mt Albert Research Centre, Private Bag 92169, Auckland, New Zealand;
e-mail: [email protected]
2
We have previously identified three distinct families of α-amylases in plants (STANLEY et al.,
2002). The family one α-amylases are secreted enzymes that have been well characterised in
monocots, but there are no published reports describing the activity of the family two or family
three proteins. Family three α-amylases have a large (~480 amino acid) N-terminal extension,
relative to other α-amylases; the extension is found in some other carbohydrate active enzymes
and shares features with known starch-binding motifs. The family two and family three
α-amylases were predicted to localise to the cytosol and the chloroplast, respectively. The
N-terminal encoding region of each gene was fused to the gene for green fluorescent protein
(GFP), and transformed into plant cells. Fluorescence microscopy revealed that GFP fused to a
family two α-amylase was localised in the nucleus of the cell and in a small area adjacent to the
cell wall, thought to be the cytosol. GFP fused to a family three α-amylase was localised to
chloroplasts in leaves and non-photosynthetic plastids in roots. The three α-amylase genes of A.
thaliana display distinctive expression patterns, the plastid-targeted form in particular showed
strong diurnal regulation. The activities of family two and family three α-amylases were
explored by expressing them in Escherichia coli. A family two α-amylase was solubilised and
purified by fusion to maltose binding protein (MBP), and showed activity against amylopectin
in native PAGE gels, and against soluble and insoluble starch in agarose gels. The purified
protein was active at high pH, and was not inactivated by high concentrations of EGTA. The
full-length family three α-amylase was sparingly soluble and active against amylopectin in
native PAGE gels, but the N-terminal extension did not confer any significant starch-binding
properties when fused to GFP. It appears that the three gene families have distinct roles in
carbohydrate degradation in plant cells. Family one α-amylases are secreted from the cell, and
are important in germination of seeds and possibly in degrading excess starch following cell
pathogenesis. Family two α-amylases are localised to the cytosol and may degrade starch- and
glycogen-like molecules that are proposed to buffer cellular carbohydrate metabolism. Family
three α-amylases may initiate breakdown of native starch granules in the plastids of
photosynthetic and/or starch storage tissues.
STANLEY, D., FITZGERALD, A.M., F ARNDEN, K.J.F. & MACRAE, E.A. 2002. Biologia, Bratislava 57 (Suppl. 11): 137-148.
46
INVITED LECTURE - L31
Synthesis and study of potent α-amylase inhibitors
John F. ROBYT
Laboratory of Carbohydrate Chemistry and Enzymology, Department of Biochemistry, Biophysics, and
Molecular Biology, 4252 Molecular Biology Bldg., Iowa State University, Ames, Iowa 50011, USA;
e-mail: [email protected]
Acarbose is a natural product produced by various strains of Actinoplanes. It is a
pseudotetrasaccharide with an unsaturated cyclitol [2,3,4-trihydroxy-5-(hydroxymethyl)-5,6cyclohexene in a D–gluco-configuration] attached to the nitrogen of 4-amino-4,6-dideoxy-D–
glucopyranose, which is linked α-1→4 to maltose. Acarbose has been shown to be a good
inhibitor of several carbohydrases. Modification of the maltose unit of acarbose has led to new
inhibitors for a number of exo-acting glucosidases. For example, the substitution of cellobiose or
lactose at the reducing-end of the pseudotetrasaccharide gave specific inhibitors for βglucosidases and β-galactosidases, for which acarbose itself was not an inhibitor. We have now
modified the nonreducing-end of acarbose by reacting cyclomaltohexaose and Bacillus
macerans cyclomaltodextrin glucanyltransferase (CGTase). This reaction gave three major
products, which were separated and purified by Bio-Gel P2 gel-permeation chromatography.
Hydrolysis of the purified products by β-amylase and glucoamylase showed that they were
composed of maltohexaose (G6), maltododecaose (G12), and maltooctadecaose (G18),
respectively, attached to the nonreducing-end of acarbose. 13C-NMR of the glucoamylase
product (D–glucopyranosyl-acarbose) had the D–glucose moiety attached α- to the C4-OH
group of the nonreducing-end cyclohexene ring of acarbose, indicating that the maltodextrins
were attached α-1→4 to the nonreducing-end cyclohexene ring of acarbose. Acarbose and the
acarbose analogues, 4IV–maltohexaosyl acarbose (G6-Aca) and 4IV–maltododecaosyl acarbose
(G12-Aca) were studied as potential inhibitors for four different α-amylases: Aspergillus oryzae,
Bacillus amyloliquefaciens, human salivary, and porcine pancreatic α-amylases. The three
inhibitors showed mixed, noncompetitive inhibition for all four α-amylases. Acarbose had
inhibition constants, Ki values, of 270, 13, 1.27, and 0.80 µM, respectively; the Ki values for
G6-Aca were 33, 37, 14, and 7 nM, respectively; and Ki values for G12-Aca were 59, 81, 18,
and 11 nM, respectively. The G6-Aca and G12-Aca are the most potent α-amylase inhibitors
heretofore observed, with Ki values 1 to 3 orders of magnitude more potent than acarbose,
which itself is 1 to 3 orders of magnitude more potent than any other known α-amylase
inhibitors. Neither of the two maltodextrin chains, attached to acarbose, were hydrolyzed by the
α-amylases.
47
ORAL T ALK - L32
The conclusive proof that supports the concept of
the α-amylase family: structural similarity and
common catalytic mechanism
Takashi KURIKI1, Hironori HONDOH2 & Yoshiki MATSUURA3
1
Biochemical Research Laboratory, Ezaki Glico Co., Ltd., 4-6-5 Utajima, Nishiyodogawa-ku, Osaka 5558502, Japan; email: [email protected]
Faculty of Science and Engineering, Ritsumeikan University, 1-1-1 Noji-higashi, Kusatu, Shiga 525-8577,
Japan
3
Institute for Protein Research, Osaka University, 3-2 Yamada-oka, Suita, Osaka 565-0871, Japan
2
We previously found a new enzyme, neopullulanase (EC 3.2.1.135) from Bacillus
stearothermophilus (KURIKI et al., 1988), and showed that it catalyzes the hydrolysis of α-1,4and α-1,6-glucosidic linkages (IMANAKA & KURIKI, 1989), as well as transglycosylation to form
α-1,4- and α-1,6-glucosidic linkages (TAKATA et al., 1992). The replacement of several amino
acid residues that constitute the active center of the neopullulanase showed that one active
center of the enzyme participated in all four reactions described above (KURIKI et al., 1991).
Pointing out the structural similarity and the common catalytic mechanism of the enzymes that
catalyze these four reactions, we proposed and defined a general idea for the α-amylase family
(TAKATA et al., 1992). Based on the concept of the α-amylase family, we controlled the
substrate preference and transglycosylation activity of the neopullulanase (KURIKI et al., 1996)
and analyzed the regions that determined the specificity of maize branching enzyme isoforms
(KURIKI et al., 1997). We recently determined crystal structure of the neopullulanase and its
complexes with panose, maltotetraose, and isopanose (HONDOH et al., 2003). The active enzyme
forms a dimer in the crystalline state and in solution. The monomer enzyme is composed of four
domain, N, A, B, and C, and has a (β/α)8-barrel in domain A. The active site lies between
domain A and domain N from the other monomer. Possible acceptor binding for
transglycosylation was also observed. Here, we show the first evidence that one active center of
an enzyme participated in all four reactions; hydrolysis of α-1,4- and α-1,6-glucosidic linkages
and transglycosylation to form α-1,4- and α-1,6-glucosidic linkages on the structural basis.
These structural and mutational analyses show the conclusive proof that supports the concept of
α-amylase family: structural similarity and common catalytic mechanism.
HONDOH, H., KURIKI, T. & MATSUURA, Y. 2003. J. Mol. Biol. 326: 177-188.
IMANAKA, T. & KURIKI T. 1989. J. Bacteriol. 171: 369-374.
KURIKI, T., KANEKO, H., Y ANASE, M., TAKATA, H., S HIMADA, J., TAKADA, T., U MEYAMA H. & OKADA, S. 1996. J. Biol.
Chem. 271: 17321-17329.
KURIKI, T., OKADA, S. & I MANAKA, T. 1988. J. Bacteriol. 170: 1554-1559.
KURIKI, T., STEWART, D.C. & PREISS, J. 1997. J. Biol. Chem. 272: 28999-29004.
KURIKI, T., TAKATA, H., OKADA, S. & I MANAKA, T. 1991. J. Bacteriol. 173: 6147-6152.
TAKATA, H., KURIKI, T., OKADA, S., TAKESADA, Y., IIZUKA, M., MINAMIURA, N. & IMANAKA, T. 1992. J. Biol. Chem. 267:
18447-18452.
48
2nd Symposium on the Alpha-Amylase Family
LIST OF POSTERS
49
Maher ABOU HACHEM, Bent W. SIGURSKJOLD & Birte SVENSSON: Calcium binding to
barley α-amylase isozymes: insight into differences in calcium dependence and
implications on stability and function.
Cécile ALBENNE, Gabrielle POTOCKI-VÉRONÈSE, Bart VAN DER VEEN, Lars SKOV, Osman
MIRZA, Michael GAJHEDE, Gwénaelle ANDRÉ, Pierre MONSAN & Magali REMAUDSIMÉON: Structural analysis and molecular modelling to rationally design
amylosucrase from Neisseria polysaccharea.
Sikander ALI, Hamad ASHRAF, Irfana MARIAM & Ikram UL-HAQ: Stimulatory effect of
polyalcohols on α-amylase production by Bacillus licheniformis: a kinetic study.
Thomas R. M. BARENDS, Thijs KAPER, Lubbert DIJKHUIZEN & Bauke W. DIJKSTRA:
Structure of a covalent intermediate in Thermus thermophilus amylomaltase.
Lone BAUNSGAARD, Henrik LÜTKEN, René MIKKELSEN, Mikkel GLARING, Tam T. PHAM &
Andreas BLENNOW : A novel glucan water dikinase phosphorylates
phosphoglucans and is required for normal degradation of leaf starch.
Ezzedine BEN MESSAOUD, Mamdouh BEN ALI, Nizar ELLEUCH & Samir BEJAR:
Characterisation and molecular cloning of a maltoheptaose producing amylase
from a new isolated Bacillus subtilis US116 strain.
Kyung-Ah CHEONG, Shuangyan TANG, Sung-Jae YANG, Hye-Sun PARK, Hee-Seob LEE,
Jung-Wan KIM & Kwan-Hwa PARK: Molecular and enzymatic characterization of
a dimeric maltogenic amylase of Bacillus thermoalkalophilus ET2.
Camilla CHRISTIANSEN, Lone BAUNSGAARD, Birte SVENSSON & Andreas BLENNOW :
Function of starch binding domain.
Monika DOMAN-PYTKA & Jacek BARDOWSKI: An acidic pullulanase from a mesophilic
lactic acid bacterium Lactococcus lactis IBB500.
Atieh GHASEMI, Khosro KHAJEH, Hossein NADERI-MANESH, Mehdi NADERI-MANESH &
Bijan RANJBAR: Proteolysis of mesophilic and thermophilic α-amylases and
stabilization of these enzymes by their specific antibody.
Andrej GODÁNY, Viera HORVÁTHOVÁ & Štefan JANEČEK: Biochemical properties of
thermostable α-amylase from Thermococcus hydrothermalis expressed in
Escherichia coli.
Javad HAMEDI, Azam HASSANI-NASAB & Mohammad A. AMOOZEGAR: Relationship
between growth, erythromycin production and amylase activity in
Saccharopolyspora erythraea.
Suhaila. O. HASHIM, Osvaldo D. DELGADO, M. Alejandra MARTÌNEZ, Rajni-Hatti KAUL,
Maria ANDERSSON, Francis J. MULAA & Bo MATTIASSON: An alkaline active
maltohexaose forming α-amylase from Bacillus halodurans LBK 34: activity and
stability features of Amy 34.
50
Štefan JANEČEK, Birte SVENSSON & E. Ann MACGREGOR: Maltogenic amylase versus the
maltogenic α-amylase: why they should be clearly discriminated from each
other.
Khosro KHAJEH, Maryam MONSEF SHOKRI, Ahmad ASOODEH, Bijan RANJBAR, Saman
HOSSEINKHANI & Hossein NADERI-MANESH: Comparative studies on “molten
globule” states of a thermophilic α-amylase and its mesophilic counterpart.
Slavko KRALJ, Ewa STRIPLING, Peter SANDERS, Ineke G.H. VAN GEEL-SCHUTTEN &
Lubbert DIJKHUIZEN: A highly hydrolytic reuteransucrase from a probiotic
Lactobacillus reuteri strain.
Birte KRAMHØFT , Sophie BOZONNET , Morten T. JENSEN & Birte SVENSSON: Amylopectin
hydrolysis by barley α-amylase: role of the “sugar tongs” binding site.
Martin MACHOVIČ & Štefan JANEČEK: Hunting in the genomes: putative GH-77
amylomaltase from Borrelia burgdorferi contains just the catalytic triad
conserved.
Martin MACHOVIČ, Richard ZONA, Birte SVENSSON, E. Ann MACGREGOR & Štefan
JANEČEK: Bioinformatics of starch-binding domains from CBM-20 and CBM-21.
Irfana MARIAM, Hamad ASHRAF & Saeed Ahmad NAGRA: Kinetics of carbon sources
utilization by parental and mutant strains of Bacillus licheniformis for the
production of α-amylase using agricultural by-products.
Henrik NÆSTED, Kirsten BOJSEN & Birte SVENSSON: Purification and characterization of
recombinant high pI barley α-glucosidase.
Morten Munch NIELSEN & Vibeke BARKHOLT : The low in vitro starch digestibility of
cooked legumes is mainly determined by the organisation in plant tissue, not by
antinutritional factors.
Gabrielle POTOCKI-VERONESE, Sandra PIZZUT -SERIN, Cécile ALBENNE, Bart A. VAN DER
VEEN, Pierre MONSAN & Magali REMAUD-SIMEON: DR-AS: a novel amylosucrase
from Deinococcus radiodurans.
Sumitra RAMACHANDRAN, Ashok PANDEY, Viviana NAGY & George SZAKACS: Statistical
optimization of production medium for Aspergillus oryzae and properties of its αamylase produced in solid cultures.
Piotr SZWEDA, Beata GRZYBOWSKA & Józef SYNOWIECKI: New expression system of
Pyrococcus woesei α-amylase in Escherichia coli.
Takashi TONOZUKA, Kazuhiro ICHIKAWA, Rie UOTSU-TOMITA, Masahiro MIZUNO, Atsushi
NISHIKAWA & Yoshiyuki SAKANO: Bacterial and archaeal enzymes homologous to
glucoamylase.
Ikram-UL-HAQ, Hamad ASHRAF & Javed IQBAL: Thermostability of α-amylase produced
by Bacillus licheniformis and its evaluation as desizer in textile industry.
51
Rachel M. VAN DER KAAIJ, Yuan XIAOLIAN, Arthur RAM, Elena MARTENS, Harrie J. KOOLS,
Gert-Jan W. EUVERINK, Marc J.E.C. VAN DER MAAREL & Lubbert DIJKHUIZEN: New
glucan-acting enzymes in the fungus Aspergillus niger.
Shukun YU, Karsten, M. KRAGH, Katrine S. LARSEN & Susan MADRID: Heterologous
expression and characterization of α-1,4-glucan lyase of glycosidase family 31
Ge-xin ZHANG, Yin-shi PIAO, Jian-yao ZHU, Wei-jiang LI & Wen-bo XU: Relation
between amino acid percentage and optimal temperature of αamylase.
Ge-xin ZHANG, Yin-shi PIAO, Wei-jiang LI & Wen-bo XU: Relation between
amino acid percentage and optimal pH of α-amylase.
Richard ZONA & Štefan JANEČEK: Bioinformatics of the family GH-57: conserved
sequence regions, evolutionarily related subfamilies and SLH-like motifs.
Richard ZONA & Štefan JANEČEK: SLH motifs in the members of the α-amylase family:
relationships within the families of glycoside hydrolases.
52
2nd Symposium on the Alpha-Amylase Family
POSTER ABSTRACTS
53
POSTER - P1
Calcium binding to barley α-amylase isozymes:
insight into differences in calcium dependence and
implications on stability and function
Maher ABOU HACHEM1, Bent W. SIGURSKJOLD2 & Birte SVENSSON1
1
Biochemistry and Nutrition Group, BioCentrum-DTU, Technical University of Denmark, Søltofts Plads Building 224, DK-2800 Kgs. Lyngby, Denmark; e-mail: [email protected]
2
Department of Biochemistry, August Krogh Institute, University of Copenhagen, Universitetsparken 13,
DK-2100 Copenhagen, Denmark
Calcium ions, which play a key role in a diversity of biological processes, are frequently bound
to proteins with affinities (Ka) typically in the range 105-108 M-1. It is well known that calcium
binding is almost a universal property in the α-amylase family (glycoside hydrolase family 13).
In addition to a conserved calcium-binding site, some amylases contain additional Ca2+ or other
metal ions. In germinating barley seeds, two major α-amylase isozyme families referred to as
AMY1 and AMY2 are expressed for starch degradation and thus for the mobilisation of this
energy source. The two isozymes, whose high resolution structures have been determined, are
homologous sharing 80% sequence identity and a detailed analysis of their structures shows that
they are virtually identical with only minor local differences (ROBERT et al., 2002). Despite this
homology and high extent of structural conservation, the two isozymes display a number of
differences in their physico-chemical properties, their thermal and acid stabilities, their
sensitivity for barley α-amylase/subtilisin endogenous inhibitor (BASI), as well as their
requirements for calcium ions. The high-resolution structures of both isozymes have revealed
the presence of three Ca2+ superimposing perfectly along with their protein and solvent ligands.
Despite this spatial identity, earlier studies suggested that the calcium dependence profiles of
AMY1 and AMY2 are strikingly different (BOZONNET et al., 2003) and the factors eliciting
these differences in stability, enzymatic properties, and the role played by Ca2+ in modulating
these properties is yet to be explained. Hence a detailed study addressing calcium binding and
its effects has been undertaken and calorimetric and spectroscopic techniques have been
employed to shed light on this pivotal aspect regarding these two isozymes and similar systems.
Differential scanning calorimetry (DSC) has been employed to assess the implications of
calcium binding on the thermodynamic stability of the isozymes. The thermal unfolding of both
isozymes was irreversible under all conditions but the low pH and the high Ca2+ or Mg2+
resulted in severe aggregation. Calcium had a pronounced effect on the stability of both
isozymes as judged by their markedly increased Tm values. The latest results from this study will
be presented and discussed.
The Danish Natural Science Research Council (SNF) is thanked for financial support.
ROBERT, X., HASER, R., SVENSSON, B. & AGHAJARI, N. 2002. Biologia, Bratislava 57 (Suppl. 11): 59-70.
BOZONNET, S., KIM, T.-J., BØNSAGER, B., KRAMHØFT, B., NIELSEN, P.K., BAK-JENSEN, K.S. & SVENSSON, B. 2003.
Biocatal. Biotransform. 21: 209-214.
54
POSTER - P2
Structural analysis and molecular modelling to
rationally design amylosucrase from Neisseria
polysaccharea
1
1
1
Cécile ALBENNE , Gabrielle POTOCKI-VÉRONÈSE , Bart VAN DER VEEN , Lars
2
2
2
3
1
SKOV , Osman MIRZA , Michael GAJHEDE , Gwénaelle ANDRÉ , Pierre MONSAN
1
& Magali REMAUD-SIMÉON
1
Centre de Bioingénierie Gilbert Durand, UMR CNRS 5504, UMR INRA 792, INSA DGBA, 135, avenue de
Rangueil, 31 077 Toulouse Cedex 4, France; e-mail: [email protected]
2
Department of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences,
Universitetsparken 2, DK-2100 Copenhagen, Denmark
3
INRA – UPCM, BP 71627, 44316 Nantes Cedex 03, France
Amylosucrase from Neisseria polysaccharea (AS) is a transglucosidase from family 13 of
glycoside-hydrolases, which uses sucrose to synthesise an amylose-like polymer and to elongate
acceptor molecules such as glycogen that is a strong activator of the enzyme (POTOCKI DE
MONTALK et al., 1999). Since sucrose is a largely available and low cost agro-resource, AS is
very attractive for the synthesis of resistant amylose and for the modification of glycogen.
Three-dimensional structure analysis and molecular modelling were combined to investigate the
molecular basis of the catalysis and to rationally design AS.
Enzyme-substrate complex (MIRZA et al., 2001, SKOV et al., 2002) analyses enabled the
identification of active site residues responsible for AS specificity towards sucrose and
maltooligosaccharides that are used as acceptor molecules. Site-directed mutagenesis
experiments, combined with biochemical characterization, allowed the de novo amylose-like
polymer synthesis to be elucidated and improved (ALBENNE et al., 2004a).
To elucidate the glycogen activating effect on AS, glycogen-enzyme interactions were
modelled from the crystallographic AS: maltoheptaose complex (SKOV et al., 2002). To mimic a
glycogen branching, the strategy consisted in joining the two maltoheptaose molecules bound in
OB1 and OB2 by an α-1,6 ramification. Among the various docking positions built, four models
were retained from geometrical and energetic criteria. They all revealed that OB2 surface site
provides an anchoring platform at the enzyme surface to capture the polymer and direct the
branches towards OB1 acceptor site for elongation (ALBENNE et al., 2004b). Besides, robotic
calculations enabled to describe a back and force motion of an hairpin loop of the AS specific
B’-domain that may assist the elongation of glycogen branches (ALBENNE et al., 2004b). This
molecular modelling approach enabled to propose a dynamic mechanism of glycogen elongation
and to identify key residues distant from the active site, which can be used as new targets for AS
rational design.
ALBENNE, C., SKOV, L.K., MIRZA, O., … & REMAUD-SIMEON, M. 2004a. J. Biol. Chem. 279: 726-734.
ALBENNE, C., SKOV, L.K., MIRZA, O., … & ANDRÉ, G. 2004b. Proteins (submitted).
MIRZA, O., S KOV, L.K., REMAUD-SIMEON, M., … & GAJHEDE, M. 2001. Biochemistry 40: 9032-9039.
POTOCKI DE MONTALK, G., REMAUD-SIMÉON, M., WILLEMOT , R.M. & MONSAN, P. 1999. FEMS Microbiol. Lett. 186: 103108.
SKOV, L.K., MIRZA, O., SPROGØE, D., … & GAJHEDE, M. 2002. J. Biol. Chem. 277: 47741-47747.
55
POSTER - P3
Stimulatory effect of polyalcohols on α-amylase
production by Bacillus licheniformis: a kinetic study
1
1
2
Sikander ALI , Hamad ASHRAF , Irfana MARIAM & Ikram UL-HAQ
1
1
Biotechnology Research Centre, Department of Botany, Government College, University Lahore, Lahore
54000, Pakistan; e-mail: [email protected]
Institute of Chemistry, Quaid-e-Azam Campus, University of the Punjab, Lahore 54590, Pakistan
2
The present investigation deals with the selection of polyalcohols such as sorbitol, monitol,
glycerol, and propylene diol or poly ethylene glycol on the biosynthesis of α-amylase by
parental and mutant strains of Bacillus licheniformis. Of all the alcohols tested, significant
enhancement in enzyme formation was obtained by the addition of sorbitol (0.3 M) to the
fermentation medium. Kinetic study indicated that volumetric rate of enzyme and biomass
formation was found to be significant as the sorbitol was added in the fermentation medium.
The volumetric productivity and yield of the enzyme by cell mass formation was then studied in
stirred fermentor. The effect of sorbitol addition was carried out after every 4th h. The addition
of sorbitol, 28 h after inoculation gave significant enhancement in the stationary phase of the
bacteria that leads towards the maximum accumulation of α-amylase in the fermentation
medium. The addition of sorbitol at 0 h, after 28 h and without sorbitol was kinetically analysed
and it was found that although the volumetric rate of biomass formation was non-significantly
varied among the values, however, the volumetric rate of enzyme formation was significantly
higher as the sorbitol was added in the fermentation medium at 28 h. Thus, the yield of the
enzyme by cell mass formation was significantly improved.
56
POSTER - P4
Structure of a covalent intermediate in Thermus
thermophilus amylomaltase
1
2
2
Thomas R. M. BARENDS , Thijs KAPER , Lubbert DIJKHUIZEN & Bauke W.
1
DIJKSTRA
1
Laboratory of Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The
Netherlands; e-mail: [email protected]
2
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB),
Kerklaan 30, 9751 NN Haren, The Netherlands
The crystal structure of Thermus aquaticus amylomaltase has been described by PRZYLAS et al.
(2000a, 2000b) in both an uncomplexed form and complexed with acarbose. Now, the 2.2 Å
crystal structure of a covalent adduct between acarbose and amylomaltase on the nucleophilic
residue Asp293 has been solved (Fig. 1). The structure allows the investigation of the
conformations of the active site residues in the covalent intermediate state. Also, the water
structure around the active site can be investigated, to study why amylomaltases catalyse
disproportionation rather than hydrolysis.
Figure 1. A covalent intermediate in the active site of Thermus thermophilus amylomaltase.
The catalytic residues Asp293, Glu340 and Asp395 are shown, as well as the original difference electron
density calculated prior to inclusion of the sugar residues in the model.
PRZYLAS, I., T ERADA, Y., F UJII, K., T AKAHA, T., SAENGER, W. & STRÄTER, N. 2000a. Eur. J. Biochem. 267: 6903-6913.
PRZYLAS, I., TOMOO, K., T ERADA, Y., TAKAHA, T., OKADA, S., SAENGER, W. & STRÄTER, N. 2000b. J. Mol. Biol. 296: 873886.
57
POSTER - P5
A novel glucan water dikinase phosphorylates
phosphoglucans and is required for normal
degradation of leaf starch
Lone BAUNSGAARD, Henrik LÜTKEN, René MIKKELSEN, Mikkel GLARING, Tam T.
PHAM & Andreas BLENNOW
Center for Molecular Plant Physiology (PlaCe), Plant Biochemistry Laboratory, Department of Plant
Biology, The Royal Veterinary and Agricultural University, DK-1871 Frederiksberg C, Copenhagen,
Denmark; e-mail: [email protected]
Many key enzymes responsible for starch biosynthesis have been identified and the formation of
the final starch structures has turned out to be a complex process. The elucidation of the process
of degradation of transient starch in planta is still lacking behind, though many starch degrading
activities have been described in vitro. However, novel activities are still discovered, the most
recent one being the potato starch-phosphorylating enzyme α-glucan water dikinase GWD
which phosphorylates starch by a dikinase-type reaction mechanism in which the β-phosphate of
ATP is transferred to the glucan substrate (RITTE et al., 2002; MIKKELSEN et al., 2004). The
studies of plants with down regulated GWD activity have revealed a close link between starch
phosphorylating activity and starch breakdown in transient leaf starch, as exemplified by the
Arabidopsis sex1 (starch excess) mutant (Yu et al., 2001).
We have characterized a novel GWD, termed GWD3, which binds and phosphorylates
only phosphoglucans, which is a novel prerequisite for a glucan water dikinase reaction. The
molecular dissection of this starch binding protein shows a conserved CBM20 module in the Nterminus, which is unique for the GWD3-subgroup of GWDs. The analysis of the in vitro
activity of the enzyme and the regulatory role for this activity of this activity in planta will be
presented.
RITTE, G., LLOYD, J. R., ECKERMANN, N., ROTTMANN, A., KOSSMANN, J. & STEUP , M. 2002. Proc. Natl.Acad. Sci. USA 99:
7166-7171.
MIKKELSEN, R., BAUNSGAARD, L. & BLENNOW, A. 2004. Biochem. J. 377: 525-532.
Y U, T-S., KOFLER, H., HÄUSLER, R.E., HILLE, D., F LÜGGE, U-I., ZEEMAN, S.C., S MITH, A.M., KOSSMANN, J., LLOYD, J.,
RITTE, G., STEUP, M., LUE, W-L., CHEN J. & WEBER, A. 2001. Plant Cell 13: 1907-1918.
58
POSTER - P6
Characterisation and molecular cloning of a
maltoheptaose producing amylase from a new
isolated Bacillus subtilis US116 strain
Ezzedine BEN MESSAOUD, Mamdouh BEN ALI, Nizar ELLEUCH & Samir BEJAR
Laboratoire d’Enzymes et de Métabolites des Procaryotes, Centre de Biotechnologie de Sfax, BP "K", 3038
Sfax Tunisie; e-mail: [email protected]
Maltodextrins are used in nutritional, cosmetic and pharmaceutical industries. In fact, they serve
as coating agents, viscosity providers, flavor carriers, moisture controllers, crystallization
inhibitors, fat substitutes etc. They are normally composed by malto-saccharides with a wide
distribution of their degree of polymerization (DP). This may allow the product to be inadequate
for performance in diverse applications. Therefore, there is a growing interest to produce
maltodextrins consist of saccharides with a narrow DP distribution, appropriate for a specific
application and more adequate for a possible subsequent purification. For this aim, we need to
use atypical amylases able to generate maltodextrins with high content of a specific maltosaccharide.
Here we report the purification and characterization of an atypical amylase produced
from a newly identified Bacillus subtilis strain US116. The extra-cellular enzyme was purified
to homogeneity. Optimal conditions for the activity of the purified enzyme are pH 6 and 65 oC.
The action of the enzyme on starch forms predominantly maltohexaose (DP6) and
maltoheptaose (DP7). The study of its action showed that the minimum length of maltosaccharide cleaved by this enzyme was DP7 (BEN MESSAOUD et al., 2004). Hence this amylase
could be utilized for the production of maltodextrins with high content of maltoheptaose and/or
maltohexaose.
The cloning and sequencing of the corresponding gene will be also reported. The
comparison of the AmyUS116 amino acid sequence and its three-dimensional model structure,
with some reported typical amylases, will be also discussed.
BEN M ESSAOUD, E., BEN ALI, M., E LLEUCH, N., FOURATI M ASMOUDI, N. & BEJAR, S. 2004. Enzyme Microb. Technol. 34:
662-666.
59
POSTER - P7
Molecular and enzymatic characterization of a
dimeric maltogenic amylase of Bacillus
thermoalkalophilus ET2
1
1
1
1
Kyung-Ah CHEONG , Shuangyan TANG , Sung-Jae YANG , Hye-Sun PARK ,
1
2
1
Hee-Seob LEE , Jung-Wan KIM & Kwan-Hwa PARK
1
National Laboratory for Functional Food Carbohydrates, Center for Agricultural Biomaterials, and
Department of Food Science and Technology, Seoul National University, Seoul 151-742, Korea; e-mail:
[email protected]
2
Department of Biology, University of Incheon, Incheon 402-749, Korea
A gene encoding a thermostable maltogenic amylase was cloned from a thermophilic bacterium,
Bacillus thermoalkalophilus ET2 in Escherichia coli. The maltogenic amylase (BTMA) gene
encoded 588 amino acids for a protein with a molecular mass of 68,774 daltons. BTMA shared
78.2% identity with maltogenic amylase from B. stearothermophilus, 73.3% with
neopullulanase from B. stearothermophilus TRS40, 71.9% with maltogenic amylase from
Thermus 6501, 50.8% with maltogenic amylase from B. licheniformis ATCC27811 at the amino
acid sequence level (PARK et al., 2000). BTMA had the four amino acid sequences that were
known conserved among amylolytic enzymes and the extra amino-terminal domain that was
found in maltogenic amylases. BTMA had an optimal temperature of 70 oC, the highest among
maltogenic amylases reported so far. The enzyme had pH optimun at 8.0 and was stable between
pH 8.0 and 10.0. DSC analysis showed that Tm of BTMA at pH 8 was 76.7 oC with an enthalpy
value of 0.8 mJ/mg. BTMA exhibited both hydrolysis and transglycosylation activities for
various carbohydrates. It hydrolyzed β-cyclodextrin (β-CD) and soluble starch mainly to
maltose and pullulan to panose. Acarbose, a strong amylase inhibitor, was hydrolyzed by
BTMA to glucose and acarviosine-glucose. The KM and kcat values of BTMA for β-CD were
0.13mM and 165.8 s-1mM, respectively. The overall catalytic efficiency (kcat/KM) of the enzyme
was the highest toward β-CD. BTMA was present in a monomer-dimer equilibrium with a molar
ratio of 54: 46 in 50mM glycine-NaOH buffer (pH 8.0), which could be affected by the
concentration of KCl. High concentration of KCl could dissociate the dimer to monomer. In
analytical ultracentrifugation analysis, the equilibrium constant increased from 3.7 x 10-7 to 1.5
x 10-6 with increasing concentration of KCl. The activity toward β-CD decreased by
approximately 48% as the concentration of KCl increased from zero to 0.2 M, while the activity
toward soluble starch in contrast showed an increase of 139% (KIM et al., 2001). DSC analysis
showed that the peak temperature of denaturarion (Tp) of BTMA decreased to 74.2 oC in 1.0 M
KCl while it was 76.2 oC in the abscence of KCl. BTMA was less thermostable where it existed
mostly as monomer than as dimer. Therefore, the multisubstrate specificity and thermostability
of the enzyme was associated with structural differences between monomeric and dimeric form.
PARK, K.H., KIM, T.J., CHEONG, T.K, KIM, J.W., OH, B.H. & SVENSSON, B. 2000. Biochim. Biophys. Acta 1478: 165-185.
KIM, T.J., NGUYEN, V.D., LEE, H.S., KIM, M.J., CHO, H.Y., KIM, Y.W., MOON, T.W., PARK, C.S., KIM, J.W., OH, B.H.,
LEE, S.B., S VENSSON, B. & PARK, K.H. 2001. Biochemistry 40: 14182-14190.
60
POSTER - P8
Function of starch binding domain
Camilla CHRISTIANSEN1, Lone BAUNSGAARD1, Birte SVENSSON2 & Andreas
1
BLENNOW
1
Center for Molecular Plant Physiology (PlaCe), Plant Biochemistry Laboratory, Department of Plant
Biology, The Royal Veterinary and Agricultural University, DK-1871 Frederiksberg C, Copenhagen,
Denmark; e-mail: [email protected]
2
Biochemistry and Nutrition Group, BioCentrum-DTU, Technical University of Denmark, Søltofts Plads,
Building 224, DK-2800 Kgs. Lyngby, Denmark
Starch binding domains (SBDs) are carbohydrate binding modules present in certain starch
metabolising enzymes. SBDs are known from some of the GH13 α-amylase family, in certain
GH14 β-amylases, in the vast majority of the GH15 glucoamylase family and recently a putative
SBD have been identified in glucan water dikinase (GWD).
Several enzymes have SBD connected to the catalytic domain through a polypeptide
linker, others form a globular multidomain structure. Preliminary findings indicate that SBDs
promote degradation and phosphorylation of starch granules (GIARDINA et al., 2001; REIMANN et
al., 2004). SBD from glucoamylase from Aspergillus niger can bind to amylose chains forming
nano-size ring shaped-structures as seen by AFM. Such structures are also formed by
catalytically inactive glucoamylase (GIARDINA et al., 2001). Possibly the SBD has the capacity
to break native starch granular structures thereby exposing nano-sized starch motifs to act upon
by the enzyme catalytic domains. The general impact of SBD on starch degradation as well as
its mechanism of action, however, remains to be elucidated.
In the CAZy classification the SBDs grouped in family in CBM20 are positioned at the
C- or N-terminus of amylolytic enzymes (JANECEK et al., 2003). The current insight into
structure and function of CBM20 provides the basis for the proposed genetic and protein
engineering.
Sequence alignments and phylogenetic analysis of SBDs will guide the project into
strategies for the production of the genetic constructs. Starch binding modules of highly
diverged enzymes will be used as building blocks creating chimeric proteins.
GIARDINA, T., GUNNING, A.P., JUGE, N., FAULDS, C.B., FURNISS, C.S., SVENSSON, B., MORRIS, V.J. & WILLIAMSON, G.
2001. J. Mol. Biol. 313: 1149-1159.
JANECEK, S., S VENSSON, B. & M ACGREGOR, E.A. 2003. Eur. J. Biochem. 270: 635-645.
REIMANN, R., HIPPLER, M., MACHELETT, B. & APPENROTH, K.J. 2004. Plant Physiol. 135: 121-128.
61
POSTER - P9
An acidic pullulanase from a mesophilic lactic acid
bacterium Lactococcus lactis IBB500
1,2
Monika DOMAN-PYTKA
1
& Jacek BARDOWSKI
1
Institute of Biochemistry and Biophysics of Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw,
Poland; e-mail: [email protected]
2
Department of Food Technology, Agricultural University, Skromna 8, 20-950 Lublin, Poland
Mesophilic lactic acid bacteria belonging to the genus of Lactococcus are widely used in dairy
fermentations and therefore, these bacteria are well equipped in lactose and casein hydrolytic
enzymes. Despite the plants constitute a natural niche for lactococci, these bacteria are known
not to be able of hydrolysing starch, an abundant plant carbon source. However, in our recent
studies we identified lactococcal strain capable of producing a starch-degrading enzyme
(DOMAN-PYTKA et al., 2004). The enzyme is active in the range of 25-50 oC with an optimum at
40 oC and at pH 4.3.
This enzymatic protein was found to be encoded by a plasmid located gene that was
sequenced revealing that the enzyme is homologous to bacterial pullulanases with the highest
similarity to the enzymes from streptococci, like Streptococcus agalactiae or Streptococcus
pneumoniae. Typical features of enzymes of the amylase family have also been detected in the
785 amino acid sequence of this lactococcal pullulanase (DOMAN-PYTKA & BARDOWSKI, 2004).
Among them counterparts of 4 highly conserved regions I-IV, constituting an active centre and
common substrate-binding sites were identified.
Analysis of the nucleotide sequence of the pullulanase downstream region demonstrated
that the pul gene probably constitutes a polycistronic operon, encompassing three other putative
genes and followed by a rho-independent transcriptional terminator. A long, 636-bp non
translated nucleotide sequence containing several putative promoter sequences and a sequence
homologous to the catabolite responsive element (cre) preceding the translational start codon of
the pullulanase suggested a potential complex genetic control of the expression of this gene
(operon).
Several potential industrial applications of the pullulanase enzyme, its gene, as well as
bacterial cells capable of producing the enzyme are possible to be used.
This work was partly supported by the KBN grant No. 6 P06G 055 20.
DOMAN-P YTKA, M., RENAULT, P. & BARDOWSKI, J. 2004. Lait 84: 33-37.
DOMAN-P YTKA, M. & BARDOWSKI, J. 2004. Crit. Rev. Microbiol. 30: 107-121.
62
POSTER - P10
Proteolysis of mesophilic and thermophilic
α-amylases and stabilization of these enzymes by
their specific antibody
Atieh GHASEMI, Khosro KHAJEH, Hossein NADERI-MANESH, Mehdi NADERIMANESH & Bijan RANJBAR
Department of Biochemistry and Biophysics, Faculty of Science, Tarbiat Modarres University, P. O. Box
14115-175, Tehran, Iran; e-mail: [email protected]
An investigation was carried out to compare the proteolytic resistance of the mesophilic αamylase from Bacillus amyloliquifaciens (BAA) and its thermophilic counterpart from Bacillus
licheniformis (BLA). Correlation between sites of proteolytic cleavage and the threedimensional structure of the α-amylases, with the application of theoretical modeling of BAA,
allowed discussion of the flexibility and the stability of both enzymes. The thermophilic enzyme
shows higher resistance to trypsin, papain and thermolysin but it is sensitive to pronase.
Proteolytic digestion of the thermophilic enzyme leads to an increased activity of the enzyme at
room temperature even though there is proteolytic cleavage based on SDS-PAGE results.
Furthermore, the 18 kDa and 38 kDa fragments, resulting from tryptolytic digestion of
thermophilic α-amylase, were separated by HPLC and preparative gel electrophoresis. Then,
polyclonal antibody was raised against 18 kDa fragment. Thermal stability of BLA and its
mesophilic counterpart in the presence of antibody were studied after the antibody purification
by protein A-Sepharose. Results show that, in the presence of Ca2+ ion, antibody addition leads
to stabilization of BLA. But, despite significant homology between BLA and BAA, this
antibody decreases only thermal stability of BAA.
63
POSTER - P11
Biochemical properties of thermostable α-amylase
from Thermococcus hydrothermalis expressed in
Escherichia coli
Andrej GODÁNY1,2, Viera HORVÁTHOVÁ2 & Štefan JANEČEK1,2
1
Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84551 Bratislava,
Slovakia
Department of Biotechnologies, Faculty of Natural Sciences, University of SS Cyril and Methodius, SK91700 Trnava, Slovakia; e-mail: [email protected]
2
Thermostable α-amylases are very important enzymes especially for applications in starch
industry. These enzymes are used in the first step of starch hydrolysis. Due to lower optimal pH
for the activity of archaeal α-amylase from Thermococcus hydrothermalis pEAMY 101 (5.05.5) in comparison with the Bacillus α-amylases (6.0-6.5), this enzyme might have a potential in
the starch degradation process together with the fungal glucosidases currently used in the
saccharification step of degradation starch into glucose (LEVEQUE et al., 2000b). All archaeal αamylases are interesting also by the fact that their amino acid sequences exhibit most similarities
to those of plant α-amylases, the difference between their thermostability being ~ 50 oC
(JANEČEK et al., 1999).
The gene encoding the α-amylase from thermophilic archaeon Thermococcus
hydrotermalis pEAMY 101 has already been cloned and expressed in Escherichia coli, and the
protein partially characterized (LEVEQUE et al., 2000a). This work brings the results from the recloning of the gene into the Escherichia coli pET expression vector with the aim to achieve
higher, IPTG-controlled expression.
Six different variants of the structural α-amylase gene were prepared using PCR, cloned
and expressed in E. coli. The recombinant proteins containing His(6)-tag at the N-terminus were
purified using Ni-NTA column affinity chromatography procedure. The high specific activity
(~150-200 U/mg and ~600-1000 U/mg after thermal modification) of the recombinant αamylases was possible to determine directly from the supernantant. All constructs exhibited the
properties of the native α-amylase with the Topt of 85 oC and pHopt of 5.5.
The recombinant variants of the α-amylase were also tested for the conditions used in
the process of enzymatic hydrolysis of starch (using commercial maize starches). The results are
comparable with those achieved by commercial enzymes exploited in starch industry.
This work was supported in part by the VEGA grant No. 1/0101/03 from the Slovak Grant Agency. Prof. Abdel Belarbi
(UFR Sciences de Reims, Reims, France) is thanked for providing the α-amylase gene from T. hydrothermalis.
JANEČEK, Š., LEVEQUE, E., BELARBI, A. & HAYE, B. 1999. J. Mol. Evol. 48: 421-426.
LEVEQUE, E., HAYE, B. & BELARBI, A. 2000a. FEMS Microbiol. Lett. 186: 67-71.
LEVEQUE, E., JANEČEK, Š., HAYE, B. & BELARBI, A. 2000b. Enzyme Microb. Technol. 26: 3-14.
64
POSTER - P12
Relationship between growth, erythromycin
production and amylase activity in
Saccharopolyspora erythraea
Javad HAMEDI, Azam HASSANI-NASAB & Mohammad A. AMOOZEGAR
Microbiology Division, Department of Biology, Faculty of Science, University of Tehran, P.O. Box: 141556455, Tehran, Iran; e-mail: [email protected]
Erythromycin is a prototype of macrolide antibiotics. This antibiotic consists of a macrolactone
ring (erythonolide B), and two amino-sugars (L-cladinose and D-desosamine) (SCHFELD &
KIRST, 2002). Attachment of these sugars is required for the bioactivity of erythromycin. It was
shown that NDP-4-keto-6-deoxy-D-glucose, a nucleotide intermediate, is the precursor of these
amino-sugars (DOUMITH et al., 2000). Although dextrin and starch were routinely used for
erythromycin production by Saccharopolyspora erythraea (HAMEDI et al., 2002), however there
is no work on the amylase activity and its relation to erythromycin production. In this research,
S. erythraea was inoculated in seed medium and incubated at 30 oC for 48 h at 220 rpm. 5% of
the seed culture was inoculated into a basal fermentation medium (supplemented with various
concentrations of dextrin and starch) and incubated at 30 oC for 5 days at 220 rpm. Basal
fermentation medium consisted of sodium nitrate, KH2PO4, K2HPO4 and MnSO4. The biomass
and erythromycin concentrations, amylase activity and pH value were measured. The results
obtained showed that in a medium containing same concentration of dextrin/starch, amylase
activity in the dextrin containing media was higher than that in the starch containing media. In
the media containing 20 g/l dextrin/starch, amylase activities were 12.11 and 10.31 units,
respectively. Although in the media containing 1-20 g/l dextrin and 1-25 g/l starch, there were a
positive correlation between concentration of substrates and amylase activity, however in the
higher concentration of substrates the amylase activity was nearly constant. But increasing of
dextrin/starch concentrations caused the enhancing of the growth of S. erythraea and
erythromycin production. In the medium containing 105 g/l dextrin, relative amylase activity
and erythromycin concentration were 94% and 3.1 g/l, respectively. Erythromycin production in
the media containing starch was less than that in the dextrin containing media. In the starch
containing media, highest concentration of erythromycin (2.4 g/l) was found in 55 g/l starch
containing medium. In this medium, relative amylase activity was 92%.
DOUMITH, M., WEINGARTEN, P., WEHMEIER, U.F., SALEH-BAY, K., BENHAMOU, B., CAPDEVILA, C., MICHEL, J.M.
PIEPERSBERG, W. & RAYNAL, M.C. 2000. Mol. Gen. Genet. 264: 477-485.
HAMEDI, J., MALEKZADEH, F. & NIKNAM, V. 2002. Biotechnol Lett: 24: 697-700.
SCHFELD, W. & KIRST, H.A. 2002. Macrolide Antibiotics, Birkhauser, p. 3.
65
POSTER - P13
An alkaline active maltohexaose forming α-amylase
from Bacillus halodurans LBK 34: activity and
stability features of Amy 34
Suhaila. O. HASHIM1,2, Osvaldo D. DELGADO#, M. Alejandra MARTÌNEZ1,
1
2
1
Rajni-Hatti KAUL , Maria ANDERSSON, Francis J. MULAA & Bo MATTIASSON
1
Department of Biotechnology, Lund University, Box 124, SE-221 00 Lund, Sweden;
e-mail: [email protected]
Department of Biochemistry, University of Nairobi, Box 30197, 00100 Nairobi, Kenya
#
Present address: Planta Piloto de Procesos Industriales Microbiológicos, Av. Belgrano y Pasaj Caseros,
4000 Tucumán, Argentina
2
The gene encoding Amy 34, a maltohexaose forming α-amylase from Bacillus halodurans LBK
34 isolated from Lake Bogoria, Kenya (HASHIM et al., 2004a), has been cloned, sequenced and
expressed in E. coli (HASHIM et al., 2004b). Within the amino acid sequence of the mature
peptide of Amy 34, the four conserved regions and the three proposed catalytic residues within
the α-amylase family could be identified while a carbohydrate-binding module (family 25) is
also present at the C-terminal end of the sequence.
Occurrence of five isoforms was detected from both the wild type and recombinant
enzymes. The 119 kDa recombinant Amy 34 enzyme exhibits optimum activity at 60 oC and pH
10.5-11.5. Maltohexaose is the main initial product formed by the hydrolytic action of Amy 34
on starch, while it produces mainly maltotetraose from amylose, amylopectin and maltodextrin.
Pullulan, α- and β-cyclodextrin are not hydrolysed by the enzyme while γ-cyclodextrin is
hydrolysed to produce glucose, maltose and maltotetraose.
Differential Scanning Calorimetry (DSC) studies have revealed that the thermal unfolding
of Amy 34 is irreversible with four transitions, as determined by curve fitting using Gaussian
curves. A melting temperature, Tm of 70.8 oC is obtained at pH 9.0, which increases by 5 oC in
presence of 100-fold molar excess of CaCl2 and decreases by 10.4 oC upon heating in presence
of 100-fold molar excess of metal chelator, EDTA. Aggregation is observed when the enzyme is
heated in presence of 1000-fold molar excess CaCl2. The effect of calcium on the activity and
thermal unfolding of Amy 34 suggests that calcium plays a role in entropic stabilisation rather
than having a direct role in the catalytic activity of Amy 34.
Support from the Swedish International Development Cooperation Agency (Sida/SAREC) is gratefully acknowledged.
HASHIM, S.O., D ELGADO, O.D., HATTI-KAUL,R, M ULAA, F.J. & MATTIASSON, B. 2004a. Biotechnol. Lett. 26: 823-828.
HASHIM, S.O., HATTI-KAUL, R., ANDERSSON, M., M ULAA, F.J. & MATTIASSON, B. 2004b. Enzyme Microb. Technol. (in
press).
66
POSTER - P14
Maltogenic amylase versus the maltogenic
α-amylase: why they should be clearly
discriminated from each other
Štefan JANEČEK1, Birte SVENSSON2 & E. Ann MACGREGOR3
1
Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84551 Bratislava,
Slovakia; e-mail: [email protected]
Biochemistry and Nutrition Group, BioCentrum-DTU, Technical University of Denmark, Søltofts Plads,
Building 224, DK-2800, Kgs. Lyngby, Denmark
3
2 Nicklaus Green, Livingston, West Lothian, EH54 8RX, UK
2
The α-amylase family, consisting of the three glycoside hydrolase families GH-13, GH-70 and
GH-77 (clan GH-H), contains at present members with 30 specificities (COUTINHO &
HENRISSAT, 1999). All of them share several features such as stretches of sequence, structural
domains, catalytic machinery and reaction mechanism (MACGREGOR et al., 2001). Some
members are so closely related and similar that subfamilies have recently been established
(OSLANCOVÁ & J ANEČEK, 2002). On the other hand, there are two members, the maltogenic
amylase (a neopullulanase-like enzyme) and the maltogenic α-amylase (a CGTase-like
enzyme), that have both been called, often and confusingly, maltogenic amylase or maltogenic
α-amylase. Since these two enzymes differ from each other in their amino acid sequence, threedimensional structure, specificity and evolutionary relationships, their exact names should be
used carefully.
COUTINHO, P.M. & HENRISSAT, B. 1999. http://afmb.cnrs-mrs.fr/cazy/CAZY/.
MACGREGOR, E.A., JANEČEK, Š. & SVENSSON, B. 2001. Biochim. Biophys. Acta 1546: 1-20.
OSLANCOVÁ, A. & JANEČEK, Š. 2002. Cell. Mol. Life Sci. 59: 1945-1959.
OH, B.H. 2003. Biologia, Bratislava 58: 299-305.
67
POSTER - P15
Comparative studies on “molten globule” states of
a thermophilic α-amylase and its mesophilic
counterpart
Khosro KHAJEH, Maryam MONSEF SHOKRI, Ahmad ASOODEH, Bijan RANJBAR,
Saman HOSSEINKHANI & Hossein NADERI-MANESH
Department of Biochemistry and Biophysics, Faculty of Science, Tarbiat Modarres University, P. O. Box
14115-175, Tehran, Iran; e-mail: [email protected]
In recent years great interest has been generated in the process of protein folding. Recently,
formation of intermediates during the folding process has been proved with new experimental
strategies based on the characterization of different stages of the protein on the folding pathway.
α-Amylases are industrially important extra cellular enzymes with a number of
applications. The molten globule states of Bacillus amyloliquefaciens α-amylase (BAA) and its
thermophilic counterpart from B. licheniformis (BLA), which it was induced by acidic pH, were
studied by far- and near-UV circular dichroism, intrinsic fluorescent emission spectroscopy, 1anilino naphthalene-8-sulfonate (ANS) binding, light scattering and proteolytic digestion by
pepsin. At pH 3.0, both enzymes acquire partially folded state, which it shows characteristics of
molten globules, i.e., reduction of defined tertiary structure and almost no change in the
secondary structure. ANS binding and light scattering experiments show that at acidic pH, BAA
and BLA unfold in such a way that their hydrophobic surfaces are exposed to a greater extent
compared to the native forms. Refolding analysis and proteolytic digestion with pepsin show
that molten globule state of BLA is more stable than that of BAA. Gel filtration result indicates
that BAA has a more compact structure at pH 3 than at pH 7.5. However, molten globule state
of BLA, at acidic pH, is less compact than native state. The effect of polyols such as trehalose,
sorbitol, and glycerol on refolding from molten to native state was also studied. These polyols
are effective on refolding of mesophilic α-amylase but have no effect on BLA refolding. The
folding pathway and stability of intermediate state of thermophilic and mesophilic α-amylase
will be discussed.
68
POSTER - P16
A highly hydrolytic reuteransucrase from a
probiotic Lactobacillus reuteri strain
1,4
1,4
2
Slavko KRALJ , Ewa STRIPLING , Peter SANDERS , Ineke G.H.
1,2
VAN GEEL-SCHUTTEN
& Lubbert DIJKHUIZEN1,4
1
Centre for Carbohydrate Bioengineering TNO-University of Groningen
Innovative Ingredients and Products Department, TNO nutrition and food research, Rouaanstraat 27,
9723 CC Groningen, The Netherlands
3
Innovative Ingredients and Products Department, TNO nutrition and food research, Utrechtseweg 48,
3704 HE Zeist, The Netherlands
4
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB),
University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands; e-mail: [email protected]
2
Glucansucrases or glucosyltransferases (GTFs, EC 2.4.1.5) of lactic acid bacteria are large
extracellular enzymes responsible for the synthesis of α-glucans from sucrose. Much research is
performed on GTFs from oral streptococci and Leuconostoc species, whereas little is known
about the GTFs of lactobacilli. Reuteran has been characterized as a novel α-glucan with mainly
α-(1,4) glucosidic linkages (45%) produced by GTFA from Lactobacillus reuteri strain 121
(KRALJ et al., 2002). Recently the biochemical and molecular characteristics of this
reuteransucrase enzyme have been reported (KRALJ et al., 2004).
We have isolated a Lactobacillus reuteri strain from a ReuteriTM Tablet purchased from
the BioGaia Company. This probiotic strain produces a glucan in which the majority of the
linkages are of the α-(1,4) glucosidic type (70%). The L. reuteri BioGaia glucansucrase gene
was cloned, expressed in Escherichia coli and the GTFBIO enzyme was purified. The
recombinant GTFBIO enzyme and the L. reuteri strain BioGaia culture supernatants synthesized
identical glucan polymers with respect to binding type and size distribution. The preference of
GTFBIO for synthesizing α-(1,4) linkages is also evident from the oligosaccharides produced
from sucrose with different acceptor substrates, e.g. isopanose from isomaltose. GTFBIO has a
relatively high hydrolysis/transferase ratio. This is only the second example of the isolation and
characterization of a reuteransucrase and its reuteran product, both found in different L. reuteri
strains. GTFBIO synthesizes a reuteran with the highest amount of α-(1,4) linkages reported to
date.
KRALJ, S., V AN GEEL-SCHUTTEN, G.H., RAHAOUI, H., LEER, R.J., FABER, E.J., VAN DER MAAREL, M.J. & DIJKHUIZEN, L.
2002. Appl. Environ. Microbiol. 68: 4283-4291.
KRALJ, S., VAN GEEL-S CHUTTEN, G.H., V AN DER MAAREL, M.J. & DIJKHUIZEN, L. 2004. Microbiology 150: 2099-2112.
69
POSTER - P17
Amylopectin hydrolysis by barley α-amylase: role of
the “sugar tongs” binding site
1,2
1,2
1
Birte KRAMHØFT , Sophie BOZONNET , Morten T. JENSEN & Birte SVENSSON
1,2
1
Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark
Biochemistry and Nutrition Group, BioCentrum-DTU, Technical University of Denmark, Søltofts Plads,
Building 224, DK-2800 Kgs. Lyngby, Denmark; e-mail: [email protected]
2
Barley starch is composed of 75% amylopectin and 25% amylose (TANG et al., 2001). An
important starch-degrading enzyme is α-amylase (AMY, EC 3.2.1.1), a member of the
glycoside hydrolase family 13 acting specifically on α-1,4 linkages. Barley AMY exists in two
isoforms, AMY1 and AMY2 (SVENSSON et al., 1985). A surface-binding site on the C-terminal
domain denoted “sugar tongs”, which appears to be non-functional in AMY2, has recently been
identified in the AMY1 structure (ROBERT et al., 2003). Our results demonstrate a role of the
“sugar tongs” in amylopectin hydrolysis.
Amylolytic attacks on A-chains of amylopectin will yield oligosaccharides whereas
attacks on B-chains initially yield polysaccharide products (GÉRAD et al. 2000). The hydrolysis
of amylopectin by AMY1 was followed by measurement of rates of reducing value (RV)
formation after separation of oligosaccharides from polysaccharides by ethanol precipitation
(ROBYT & FRENCH, 1967, KRAMHØFT et al., in preparation). During the initial stage of substrate
degradation oligosaccharide products (RVsoluble) were released at a 4-fold higher rate than new
polysaccharides (RVpolysaccharide). This phase was followed by a second phase where RVsoluble
decreased, whereas RVpolysaccharide remained constant. Thus A-chains seem to be preferred
initially for enzymatic cleavage. Multiple attack is involved in hydrolysis of amylose by AMY1
(KRAMHØFT & SVENSSON, 1998) and the degree of multiple attack (DMA) can be calculated as
RVsoluble/RVpolysaccharide (KRAMHØFT et al., in preparation). Applying this principle to amylopectin
hydrolysis gave an apparent DMA of 4 in the initial stage of degradation. Mutations at Tyr380
and Ser378 from the “sugar tongs” binding site and addition of β-cyclodextrin (5 mM), which
binds to this surface binding site, both reduced the apparent DMA to 3. AMY2, without
functional “sugar tongs” also had a “DMA” of 3. It is concluded that the “sugar tongs” play a
particular role in hydrolysis of amylopectin. The major oligosaccharide product released in
amylose- as well as in amylopectin hydrolysis was maltoheptaose (KRAMHØFT et. al., in
preparation). Multiple attack implies that the substrate repositions in the substrate-binding site
for a number of successive catalytic events before dissociation of the enzyme substrate complex
(ROBYT & FRENCH, 1967). However, amylopectin A-chains are too short to allow 4 multiple
attacks on individual chains releasing maltoheptaose (HANASHIRO et al., 2002). We therefore
suggest that the enzyme “jumps” among A-chains, that the “sugar tongs” are required for this
“jumping”, and that they function in the initial preference of A-chains for B-chains.
TANG, H., ADO, H., WATANABE, K., TAKEDA, Y. & MITSUNAGA, T. 2001. Carbohydr. Res. 330: 241-248.
SVENSSON, B., M UNDY, J., GIBSON, R.M. & SVENDSEN, I. 1985. Carlsberg Res. Communic. 50: 15-22.
ROBERT, X., HASER, R., GOTTSCHALK, T., … & AGAHAJARI, N. 2003. Structure 11: 973-984.
GÉRAD, C., P LANCHOT, V., COLONNA, P. & BERTOFT, E. 2000. Carbohydr. Res. 326: 130-144.
ROBYT, J.F. & FRENCH, D. 1967. Arch. Biochem. Biophys. 122: 8-16.
KRAMHØFT, B. & SVENSSON, B. 1998. Progr. Biotechnol. 15: 343-347.
HANASHIRO, I., TAGAWA, M., S HIBABHARA, S., IWATA, K. & T AKEDA, Y. 2002. Carbohydr. Res. 337: 1211-1215.
70
POSTER - P18
Hunting in the genomes: two putative GH-77
amylomaltases from Borrelia genomes contain just
the catalytic triad conserved
Martin MACHOVIČ & Štefan JANEČEK
Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84551 Bratislava,
Slovakia; e-mail: [email protected]
The α-amylase family, the clan GH-H, consists of three glycoside hydrolase families GH-13,
GH-70, and GH-77 (MACGREGOR et al., 2001). As the number of sources and specificities
belonging to the family enormously increased, the number of invariant residues decreased and
has been established to be only 4 in the last few years. These were the three catalytic residues
(two Asp and one Glu; * in Fig. 1) plus the Arg (↑ in Fig. 1) in the position i-2 with respect to
the catalytic nucleophile Asp located near the strand β4 of the (β/α)8-barrel.
Amylomaltases (GH-77)
Clostridium butyricum
Chlamydomonas reinhardtii
Escherichi coli
Solanum tuberosum
Thermus aquaticus
Borrelia burgdorferi
Borrelia garinii
Conserved sequence regions
30_GQKYWQILP
119_GMQCWQLLP
178_GGSFIGLNP
61_GCSLWQVLP
40_GGRYWQVLP
49_SQSYWQMFA
49_SQSYWQMFA
204_DIPIYI
297_DMPIYV
374_DLAVGV
241_DMPIYV
213_DMPIFV
227_NVPFFI
227_NLPLFI
281_ILRIDHFRG
373_ECRIDHFRG
444_ALRIDHVMS
317_EFRIDHFRG
289_LVRIDHFRG
303_IIKIDHFRG
303_VIKIDHFRG
↑ *
328_EIIAEDLG
420_PILAEDLG
492_MVIGEDLG
364_NIIAEDLG
336_PVLAEDLG
350_KIWVEDFQ
350_KIWVEDFE
*
Acc. No.
380_YTGTHD
472_YPGTHD
542_VAATHD
416_YTGTHD
390_YTGTHD
402_YTGSGD
402_YTGIGD
*
AAB84229
AAG29840
M32793
X68664
AB016244
AAC66547
AAU07022
Figure 1. Selected conserved sequence regions of a few GH-77 amylomaltases.
Two Borrelia putative amylomaltases contain the ArgÆLys substitution (K305)
as well as the mutations in several other important positions (signified by bold).
A putative member of the GH-77, the amylomaltase, predicted in the complete genome
of the Lyme disease spirochete Borrelia burgdorferi (FRASER et al., 1997), was found to contain
invariantly conserved the catalytic triad only (MACHOVIČ & J ANEČEK, 2003). The sequence of
this amylomaltase possesses the otherwise invariant β4-strand Arg substituted by lysine (Fig. 1).
It could become the first relevant member of the α-amylase family with only 3 invariant
residues, i.e. the catalytic triad. The possibility of a sequencing error (ArgÆLys) is disregarded
since this protein exhibits mutations at several other important positions (Fig. 1). This is
supported by the other putative GH-77 amylomaltase from a related Borrelia garinii genome
(GLOECKNER et al., 2004; Fig. 1). Mutations of the β4-strand Arg in α-amylases and branching
enzyme resulted in substantial activity decrease (VIHINEN et al., 1990; LIBESSART & PREISS,
1998). Definitive answers concerning the eventual enzymatic function and exact specificity will
be possible to gain only after the cloning of the respective genes, their expression and the
biochemical characterisation of the protein encoded. These studies are now in progress.
FRASER, C.M., CASJENS, S., HUANG, W.M., … & V ENTER, J.C. 1997. Nature 390: 580-586.
GLOECKNER,G., LEHMANN,R., ROMUALDI,A., … & PLATZER,M. 2004. Unpublished.
LIBESSART, N. & P REISS, J. 1998. Arch. Biochem. Biophys. 360: 135-141.
MACGREGOR, E.A., JANEČEK, Š. & SVENSSON, B. 2001. Biochim. Biophys. Acta 1546: 1-20.
MACHOVIČ, M. & JANEČEK, Š. 2003. Biologia, Bratislava 58: 1127-1132.
VIHINEN, M., OLLIKKA, P., NISKANEN, J., … & MÄNTSÄLÄ, P. 1990. J. Biochem. 107: 267-272.
71
POSTER - P19
Bioinformatics of starch-binding domains from
CBM-20 and CBM-21
1
1
2
3
Martin MACHOVIČ , Richard ZONA , Birte SVENSSON , E. Ann MACGREGOR &
1
Štefan JANEČEK
1
Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84551 Bratislava,
Slovakia; e-mail: [email protected]
2
Biochemistry and Nutrition Group, BioCentrum-DTU, Technical University of Denmark, Søltofts Plads,
Building 224, DK-2800 Kgs. Lyngby, Denmark
3
2 Nicklaus Green, Livingston, West Lothian, EH54 8RX, UK
Amylolytic enzymes are multidomain proteins. Approximately 10% of them are able to bind and
degrade raw starch. Usually a distinct domain or module is responsible for this property. The
starch-binding domains (SBD) have been classified, according to sequence similarities, into
families of carbohydrate-binding modules (CBM) (COUTINHO & HENRISSAT, 1999). At present,
there are five SBD families: CBM-20, CBM-21, CBM-25, CBM-26 and CBM-34.
This work is concentrated on families CBM-20 and CBM-21 of the SBDs. The CBM-20
motif is one of those that have been studied most intensively. It is located almost exclusively at
the C-terminal end of amylolytic enzymes from families GH-13, GH-14 and GH-15 of the
glycoside hydrolases (JANEČEK & ŠEVČÍK, 1999) and its 3-D structure was already determined
(SORIMACHI et al., 1996). On the other hand, there is a lack of information on structure-function
relationships of the CBM-21 motif. It is well known as the N-terminally positioned SBD of
Rhizopus glucoamylase (ASHIKARI et al., 1986). Nowadays many non-amylolytic proteins
(especially from sequenced genomes) have been recognised as possessing segments in their
amino acid sequences that exhibit unambiguous similarities with the experimentally-observed
SBDs of CBM-20 and CBM-21, e.g. laforin (MINASSIAN et al., 2000), genethonin-1 (JANEČEK,
2002) and protein phosphatases (BORK et al., 1998). These facts have stimulated interest in
carrying out a rigorous bioinformatics analysis of the two CBM families.
A relationship between the CBM-20 and CBM-21 SBDs was demonstrated 15 years ago
(SVENSSON et al., 1989). We therefore, in the first step, analyzed the sequences of both families
separately, taking into account the above-mentioned lack of information concerning CBM-21.
Then we attempted to identify the CBM-20 sequence-structural features in the sequences of
CBM-21, in an effort to reveal the residues corresponding to each other in both families. Finally
a sequence alignment was made that served for calculation of the common CBM-20-CBM-21
evolutionary tree. This indicates the possibility of joining the two CBMs into a common clan.
ASHIKARI, T., N AKAMURA, N., TANAKA, Y., … & Y OSHIZUMI, H. 1986. Agric. Biol. Chem. 50: 957-964.
BORK, P., DANDEKAR, T., EISENHABER, F. & HUYNEN, M. 1998. J. Mol. Med. 76: 77-79.
COUTINHO, P.M. & HENRISSAT, B. 1999. http://afmb.cnrs-mrs.fr/cazy/CAZY/.
JANEČEK, Š. 2002. Bioinformatics 18: 1534-1537.
JANEČEK, Š. & ŠEVČÍK, J. 1999. FEBS Lett. 456: 119-125.
MINASSIAN, B.A., I ANZANO, L., MELOCHE, M., … & SCHERER, S.W. 2000. Neurology 55: 341-346.
SORIMACHI, K., J ACKS , A.J., LE GAL-COEFFET, M.F., … & WILLIAMSON, M.P. 1996. J. Mol. Biol. 259:
970-987.
SVENSSON, B., JESPERSEN, H., SIERKS, M.R. & MACGREGOR, E.A. 1989. Biochem. J. 264: 309-311.
72
POSTER - P20
Kinetics of carbon sources utilization by parental
and mutant strains of Bacillus licheniformis for the
production of α-amylase using agricultural byproducts
Irfana MARIAM, Hamad ASHRAF & Saeed Ahmad NAGRA
Institute of Chemistry, Quaid-e-Azam Campus, University of the Punjab, Lahore 54590, Pakistan;
e-mail: [email protected]
The present study is concerned with the optimization and kinetic evaluation of carbon sources
for the production of α-amylase by parental and mutant strains of Bacillus licheniformis. Of all
the carbon sources tested, starch along with the lactose was found to be the best carbon source
for the production of α-amylase. The maximum production of enzyme (3050 U/g/min) was
achieved when the starch at 2%, lactose at 1% were added in the fermentation medium. The
production of the enzyme following growth of the organism was found optimum 48 h after
inoculation by all the bacterial culture. The kinetic study indicated that the values of Qp, Qx and
Yp/x by Bacillus licheniformis GCUCM-30 was highly significant in the presence of starch and
lactose in the fermentation medium and varied significantly than the parental and other mutant
derivatives of Bacillus licheniformis. This mutant gave 14 folds increase in the productivity of
α-amylase than the original parental strain.
73
POSTER - P21
Purification and characterization of recombinant
high pI barley α-glucosidase
Henrik NÆSTED, Kirsten BOJSEN & Birte SVENSSON
Biochemistry and Nutrition Group, BioCentrum-DTU, Technical University of Denmark, Søltofts Plads,
Building 224, DK-2800 Kgs. Lyngby, Denmark; e-mail: [email protected]
α-Glucosidase activity in barley seeds plays a crucial role in embryonic development. The
concerted action of α-glucosidase, α-amylase, α-amylase, and limit dextrinase serves as the
machinery responsible for the supply of glucose as energy source for the developing embryo in
the germinating seed (MACGREGOR & SISSONS, 1994). Recently expression and characterization
of the recombinant full length and fully functional barley high pI α-glucosidase in Pichia
pastoris has been achieved. In order to facilitate protein yield in the mg range, a clone
representing an N-terminal hexa histidine tagged recombinant form of the enzyme was grown
under high cell-density fermentation conditions. This approach enabled a successful protein
expression profile under the highly sensitive methanol utilization phase using a BiotatB 5 L
reactor for the fermentation procedure. The enzyme was purified from 3.5 liter α-glucosidase
containing culture using a four step purification strategy yielding 42 mg of pure enzyme.
Remarkably, the purified enzyme exhibits a higher molecular mass in SDS-PAGE than expected
from the primary structure. The apparent molecular mass of 100 kDa compared to 92 kDa
calculated from the amino acid sequence of the native enzyme indicates a possible posttranslational glycosylation of the recombinant α-glucosidase. Preliminary enzyme kinetic
analysis has demonstrated that the purified α-glucosidase displayed high stability during the 5
day long fermenentation and exhibited a specific activity in the range of the native enzyme
purified from malt (FRANDSEN et al., 2000). The kinetic values Km, Vmax and kcat are determined
to 1.7 mM, 139 nM s-1 and 85 s-1 using maltose as substrate. The presented data illustrate the
first successful production of enzymatically active full length recombinant high pI barley αglucosidase (TIBBOT et al. 1998; FRANDSEN et al., 2000).
This project is funded under the 5th Framework Programme of the European Commission, Contract Reference QLRT-200002400, “New Products from Starch-Derived 1,5-Anhydro-D-Fructose (NEPSA)”.
FRANDSEN, T.P., LOK, F., MIRGORODSKAYA, E., ROEPSTORFF, P. & SVENSSON, B. 2000. Plant Physiol. 123: 275-286.
MACGREGOR, A.W. & SISSONS, M.J. 1994. J. Cereal Sci. 19: 161-169.
TIBBOT, B.K., HENSON, C.Y. & SKADSEN, R.W. 1998. Plant Mol. Biol. 38: 379-391.
74
POSTER - P22
The low in vitro starch digestibility of cooked
legumes is mainly determined by the organisation
in plant tissue, not by antinutritional factors
Morten Munch NIELSEN & Vibeke BARKHOLT
Biochemistry and Nutrition Group, BioCentrum-DTU, Technical University of Denmark, Søltofts Plads,
Building 224, DK-2800 Kgs. Lyngby, Denmark; e-mail: [email protected]
Hydrolytic index (HI) describes the rate of in vitro hydrolysis of starch by pancreatic α-amylase.
Legumes (beans, peas and lentils) are characterised by low values of HI, which is directly
proportional with the glycaemic index. The glycaemic index is a physiological measure that
describes the response in blood glucose in healthy subjects. A low glycaemic index has many
beneficial physiological effects in controlling and preventing various metabolic diseases such as
diabetes mellitus, coronary heart disease and colon cancer (JENKINS et al., 2002).
The high amount of antinutritional factors in legumes has been associated with the low
value of HI. Especially proteinaceous α-amylase inhibitors, hydrolysable and condensed tannins
and phytic acid are reported to inhibit α-amylase in vitro (KNUCKLES & BETSCHART, 1987;
CARMONA et al., 1996). However it is still a matter debate to which extent the legume starch
digestibility is affected by antinutritional factors (THARANATHAN & MAHADEVAMMA, 2003).
Besides antinutritional factors the food structure and the amount of retrograded starch
has an influence on HI. TOVAR et al. (1990) showed that starch granules in boiled legume pulses
are still enclosed within the cell wall, which results in a decrease in HI-value compared to boiled
legume flours.
The purpose of this study was to investigate the effects of the different factors on the
hydrolytic index (HI) in field pea (Pisum sativum). All HI was determined using porcine
pancreas α-amylase on one per cent starch suspensions at pH 7.4.
HI of different mixtures of pea starch, -fibre and -protein from Cosucra, Belgium was
compared to the HI of flour from dehulled peas. The effect of phytate was evaluated by using
pea protein isolate where phytate had been removed using exogenous phytase. Flour from red
kidney beans (Phaseolus vulgaris) was also included in order to evaluate the effect of tannins.
Wheat starch was used as a reference.
The HI of boiled pea starch and boiled pea flour were identical, which indicate that the
extent of disruption of cell wall structure in the raw pea flour was complete.
For all tested samples [legume flours and the mixtures of pea starch, fibre and protein
(+/- phytase treatment)] the HI’s were identical, which suggest that soluble inhibitory factors
have no role on determining the hydrolytic index.
CARMONA, A., BORGUDD, L., BORGES, G. & LEVY-BENSHIMOL, A. 1996. J. Nutr. Biochem. 7: 445-450.
JENKINS, D.J.A., KENDALL, C.W.C., AUGUSTIN, L.S.A., FRANCESCHI, S., HAMIDI, M., MARCHIE, A., JENKINS, A.L. &
AXELSEN, M. 2002. Am. J. Clin. Nutr. 76: 266S-273S.
KNUCKLES, B.E. & BETSCHART , A.A. 1987. J. Food. Sci. 52: 719-721.
THARANATHAN, R.N. & M AHADEVAMMA, S. 2003. Trends Food Sci. Technol. 14: 507-518.
TOVAR, J., BJÖRCK, I.M.E. & ASP, N.G. 1990. J. Agr. Food Chem. 38: 1818-1823.
75
POSTER - P23
DR-AS: a novel amylosucrase from Deinococcus
radiodurans
Gabrielle POTOCKI-VERONESE, Sandra PIZZUT-SERIN, Cécile ALBENNE, Bart A.
VAN DER VEEN, Pierre MONSAN & Magali REMAUD-SIMEON
INSA DGBA, UMR INRA 792, UMR CNRS 5504, 135 avenue de Rangueil, 31077 Toulouse Cedex 4,
France; e-mail: [email protected]
Locus NP_294657.1 (dr-as) identified in Deinococcus radiodurans genome was initially
annotated as an α-amylase encoding gene on the basis of sequence similarities with enzymes
from GH family 13. Sequence alignment analyses suggested that this gene more probably
encodes an amylosucrase. The putative encoded protein (DR-AS) shares 42 % identity with N.
polysaccharea amylosucrase (AS) and presents fragments of sequences or residues thought to be
involved in AS specificity. First, B domains of DR-AS and AS are highly similar. In addition,
DR-AS also reveals a long sequence between putative β-strand 7 and α-helix 7 which could
correspond to AS B’ domain, which turns AS active site into a deep pocket. Finally, conserved
residues Asp133 and Arg518 could form a salt bridge by analogy with AS and be responsible
for the glucosyl unit transfer specificity. To investigate the activity of the putative encoded
protein, gene NP_294657.1 was cloned and expressed in E. coli. When acting on sucrose, the
pure recombinant enzyme was shown to catalyse insoluble amylose-like polymer synthesis
accompanied by side-reactions (sucrose hydrolysis, sucrose isomers and soluble
maltooligosaccharide formation). Kinetic analyses further showed that DR-AS follows a nonMichaelian behaviour toward sucrose substrate and is activated by glycogen like AS. DR-AS is
less stable than AS and more sensitive to TRIS inhibition. This demonstrates that gene
NP_294657.1 encodes an amylosucrase and sequence comparison enabled to propose a foot
print of amylosucrase specificity in family 13.
76
POSTER - P24
Statistical optimization of production medium for
Aspergillus oryzae and properties of its α-amylase
produced in solid cultures
Sumitra RAMACHANDRAN1, Ashok PANDEY1, Viviana NAGY2 & George SZAKACS2
1
Biotechnology Division, Regional Research Laboratory, CSIR, Trivandrum - 95 019, India;
e-mail: [email protected]
Department of Agricultural Chemical Technology, Technical University of Budapst, 1111 Budapest,
Hungary
2
Aspergillus oryzae is an asexual ascomycetous fungus, widely used in the industrial production
of α-amylase. It grows by hyphal extension and branching. Its hyphal mode of growth, good
tolerance to low water activity (aw) and high osmotic pressure conditions make fungi efficient
and competitive in natural microflora for bioconversion of solid substrates. Solid-state
fermentation (SSF) was carried out for the production of the enzyme using three different strains
of Aspergillus oryzae namely A. oryzae NRRL 6270, NRRL 1808, IFO 30103 on various
substrates such groundnut oil cake, sesame oil cake, wheat bran, jack fruit seed powder and
spent brewing grain. Spent brewing grain was found to be a good substrate for A. oryzae NRRL
6270. Statistical optimization using placket burman design and central composite design was
used to optimise the growth medium for the same culture. However, A. oryzae IFO 30103 was
the best producer of α-amylase among the three strains. Enzyme produced was partially purified
and characterized The enzyme showed molecular weight of 68 kDa by SDS-PAGE. Except Mn,
all other metal ions such as Ca, K, Na, Mg were found to be inhibitory for the enzyme activity.
The enzyme was optimally active at 50 oC and pH 5.0.
A.oryzae NRRL 1808 was found to produce considerably high titre of protease, hence
α-amylase produced by the strains could have been inhibited.
PANDEY, A. 2003. Biochem. Eng. J. 13: 81-84.
PANDEY, A., NIGAM, P., SOCCOL, C.R., SINGH, D., SOCCOL, V.T. & MOHAN, R. 2000. Biotechnol. Appl. Biochem. 31: 135152.
RAMACHANDRAN, S., P ATEL, A.K., NAMPOOTHIRI, K.M., CHANDRAN, S., S ZAKACS, G., SOCCOL, C.R. & PANDEY, A. 2004.
Brazilian Arch. Biol. Technol. 47: 309-317.
77
POSTER - P25
New expression system of Pyrococcus woesei
α-amylase in Escherichia coli
Piotr SZWEDA, Beata GRZYBOWSKA & Józef SYNOWIECKI
Department of Food Chemistry, Technology and Biotechnology, Gdansk University of Technology, 80 – 952
Gdansk, Poland; e-mail: [email protected]
An archeon Pyrococcus woesei produces thermostable α-amylase that could be used in the
starch industry. The enzyme has optimum activity at 100 oC, does not require Ca2+ ions for
stability and retains about 60% of maximal activity in pH range 4.5 to 7.0, which is similar to
the natural pH of starch slurry. Production of this enzyme for large–scale is limited by
difficulties of growing of this organism. The aim of our study was construction of expression
system for production of large amounts of Pyrococcus woesei alpha amylase in mesophilic host
Escherichia coli. Gene encoding an enzyme was cloned into pTYB2 vector, and obtained
pTYB2–amyl plasmid was used for expression of the target protein in Escherichia coli
BL21(DE3)pLysS. The recombinant α-amylase contains fusion domains: intein and CBD
(chitin binding domain) at C–terminus. Incorporation of intein and CBD as fusion domains to
the enzyme molecule led to a lower level of inclusion bodies formation as compared with earlier
described expression systems of this protein, in this case they exhibit only 35% of total cell
activity. The productivity of the expression system was very high. From 1 liter of the growth
medium about 190,000 units was obtained as a soluble form and it was much more than for
native producer Pyrococcus woesei (1000 u/L of growth medium) and others expression
systems. Surprisingly zymography revealed that cells of Escherichia coli contain two enzymes
with amylolytic activity. A possible of explanation for this phenomenon is that a cleavage of
fusion protein was caused by 2–mercaptoetanol added to the buffer used for samples preparation
and free α-amylase was released. We did not observe binding the fusion protein to the chitin
beads, and it was impossible to purify it by affinity chromatography. The best way of
purification of the enzyme was thermal precipitation (75 oC for 0.5 h) of cell–free extract. About
75% of the proteins were precipitated and about 70% of initial enzyme activity retained in the
supernatant. The staining of the SDS–PAGE gel revealed disappearing of the band
corresponding to the fusion protein after thermal precipitation of cell–free extract. The most
probably the protein that contains a large (about 50 kDa) nonthermostable domain is precipitated
or it could suggest thermal cleavage of the investigated fusion enzyme. The recombinant
enzyme has maximal activity at pH 5.6 and keeps over 50% of maximal activity in the range of
4.5 to 7.8 that reduce the scale of pH adjustments between steps of starch liquefaction and
saccharification. The enzyme reached highest activity at 95 oC. The half–life of this preparation
in 0.05 M acetate buffer (pH 5.6) at 90 oC and 110 oC was 11 and 3.5 h, respectively and
retained over 24% of residual activity following incubation for 2 h at 110 oC. The main end
product of prolonged starch hydrolysis catalyzed by this enzyme was maltose however small
amounts of glucose and oligosaccharides were also detected. The productivity, high
thermostability and simple and cheap method of purification could be beneficial for large–scale
production of this enzyme.
78
POSTER - P26
Bacterial and archaeal enzymes homologous to
glucoamylase
Takashi TONOZUKA, Kazuhiro ICHIKAWA, Rie UOTSU-TOMITA, Masahiro MIZUNO,
Atsushi NISHIKAWA & Yoshiyuki SAKANO
Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8, Saiwaicho, Fuchu, Tokyo 183-8509, Japan; e-mail: [email protected]
Glucoamylase, classified into GH family 15, is an exohydrolase that releases β-D-glucose from
the non-reducing ends of starch or related oligo- and polysaccharides. Although there are
numerous reports on fungal glucoamylases, relatively little is known about prokaryotic
glucoamylases and related GH family 15 enzymes. We cloned two prokaryotic genes encoding
enzymes whose primary structures are homologous to those of glucoamylases. Their enzymatic
properties are, however, markedly different from typical glucoamylases in spite of their
structural similarities. A glucoamylase from Thermoactinomyces vulgaris R-47 (TGA)
efficiently hydrolyzes small maltooligosacchrides (UOTSU-TOMITA et al., 2001). Also, a
glucodextranase from Arthrobater globiformis I-42 (GDase) efficiently releases β-D-glucose
from the non-reducing end of dextran (MIZUNO et al., 2004). Unlike typical glucoamylases, both
TGA and GDase hydrolyze starch less efficiently. In this presentation, the study of TGA will be
mainly focused.
Enzymatic analyses of TGA showed that, like other glucoamylases, TGA produced β-Dglucose from its substrate. However, TGA hydrolyzed maltooligosaccharides such as
maltotetraose and maltose more efficiently than starch. The primary structure of TGA resembled
a putative glucoamylase from the hyperthermophilic archaeon Methanococcus jannaschii
(MGA), while homologies between TGA and the fungal glucoamylases were low. The
enzymatic properties of recombinant MGA produced in E. coli cells were similar to those of
TGA. Subsite affinities of TGA were determined, and the A1+A2 value was highly positive
whereas A4—A6 values were negative and little affinity was detected at subsites 3 and 7
(ICHIKAWA et al., 2004). The results indicate that TGA is a metabolizing enzyme specific for
small maltooligosaccharides.
UOTSU-TOMITA, R., TONOZUKA, T., SAKAI, H. & SAKANO, Y. 2001. Appl. Microbiol. Biotechnol. 56: 465-473.
MIZUNO, M., TONOZUKA, T., S UZUKI, S., UOTSU-TOMITA, R. KAMITORI, S., NISHIKAWA, A. & SAKANO, Y. 2004. J. Biol.
Chem. 279: 10575-10583.
ICHIKAWA, K., TONOZUKA, T., UOTSU-TOMITA, R., AKEBOSHI, H., NISHIKAWA, A. & S AKANO, Y. 2004. Biosci. Biotechnol.
Biochem. 68: 413-420.
79
POSTER - P27
Thermostability of α-amylase produced by Bacillus
licheniformis and its evaluation as desizer in textile
industry
Ikram-UL-HAQ, Hamad ASHRAF & Javed IQBAL
Biotechnology Research Centre, Department of Botany, Government College, University
Lahore, Lahore 54000, Pakistan; e-mail: [email protected]
α-Amylase (α-1,4-glucan-glucanhydrolase, EC 3.2.1.1), an extracellular enzyme degrades α-14-glucosidic linkages of starch and related substrates in an endo-fashion producing
oligosaccharides including maltose, glucose and α-limit dextrin. The present study is concerned
with the production and optimization of conditions for the stability and activity of α-amylase
from Bacillus licheniformis and its evaluation as desizer in textile industry. The thermostability
of the enzyme is Ca2+ dependent. The thermostability of the enzyme was significantly improved
by increase in the concentration of Ca2+ ions in the fermentation medium. The enzyme was
found to be highly active at 60-70 oC and also gave promising results even at 100 oC as 0.3 M
Ca2+ was added in the fermentation medium. The α-amylase was partially purified with acetone
fractionation and ammonium sulphate precipitation. The partially purified enzyme gave about
25 times more activity than the crude enzyme. The desizing of cotton cloth with partially
purified α-amylase was found to be more promising than that with the crude enzyme because it
takes less time for desizing and 100% desizing was achieved. The end products of the starch
hydrolysis by this enzyme were glucose, maltose and maltotetrose.
80
POSTER - P28
New glucan-acting enzymes in the fungus
Aspergillus niger
1,2
3
3
4
Rachel M. VAN DER KAAIJ , Yuan XIAOLIAN , Arthur RAM , Elena MARTENS ,
4
1,2
1,5
Harrie J. KOOLS , Gert-Jan W. EUVERINK , Marc J.E.C. VAN DER MAAREL &
1,2
Lubbert DIJKHUIZEN
1
Centre for Carbohydrate Bioengineering, TNO-University of Groningen, Haren, The Netherlands;
e-mail: [email protected]
2
Microbial Physiology Research Group, Groningen Biomolecular Sciences and Biotechnology Institute
(GBB), University of Groningen, Kerklaan 30, 9751NN Haren, The Netherlands
3
Institute for Molecular Plant Sciences, Fungal Genetics, University of Leiden, Leiden, The Netherlands
4
Department of Microbiology, Fungal Genomics, Wageningen University, Wageningen, The Netherlands
5
Innovative Ingredients and Products Department, TNO Nutrition and Food Research, Groningen, The
Netherlands
The filamentous fungus Aspergillus niger is extensively used for the industrial production of
enzymes and other products like vitamins and organic acid. It is also an interesting host for
novel enzymes because of its GRAS status. The Dutch life science company DSM recently
made the full genomic sequence of A. niger available to our project, of which the aim is to
disclose the carbohydrate modifying network of this organism. By mining the genome with
sequences of homologous genes, new as well as known members of the glycoside hydrolase
families 13 and 77 were recognised. A previously unknown putative extracellular α-amylase
was identified from the genome and successfully overexpressed in A. niger. Its predicted endoamylase activity was confirmed on a double-blocked maltoheptaose substrate (pNP-G7). Three
other putative extracellular enzymes were found displaying all family 13 features. These
enzymes have a predicted C-terminal glycosyl-phosphatidylinositol (GPI)-anchor for attachment
of the protein to the cell wall or cell membrane. The proteins might be acting as amylases, but
they could also play a role in the formation of α-glucans that are known to be present in the cell
wall. Furthermore, we found 8 new putative α-glucosidases and 2 new putative glucoamylases,
which might also be involved in the extracellular degradation of starch or glycogen. Intracellular
members of the families 13 and 77 may have very different functions compared to extracellular
amylases. Examples are the involvement in the production and degradation of glucan storage
compounds like trehalose, α(1,1)-disaccharide, or glycogen, an α-1,4 glucan with a high
percentage of α(1,6)-branches. It was reported previously that A. niger can produce glycogen
(MATTEY & ALLAN, 1990) but no information is currently available on the genes or enzymes
involved. We identified the three genes encoding the proteins essential for glycogen production
in the genome of A. niger, as well as two genes needed for its degradation. The family 13 and 77
enzymes involved in these processes are glycogen branching and debranching enzyme,
respectively. After this in-depth sequence analysis of the whole genome sequence of A. niger we
conclude that a number of previously unknown genes are present potentially encoding novel
family 13 and 77 proteins. Their predicted activity and in vivo role will have to be confirmed by
overexpression and characterization of the proteins.
MATTEY, M. & ALLAN, A. 1990. Biochemical Society Trans. 18: 1020-1021.
81
POSTER - P29
Heterologous expression and characterization of
α-1,4-glucan lyase of glycosidase family 31
Shukun YU, Karsten, M. KRAGH, Katrine S. LARSEN & Susan MADRID
Danisco Innovation, Danisco A/S, Langebrogade 1, PO Box 17, DK 1001, Copenhagen, Denmark;
e-mail: [email protected]
α-1,4-Glucan lyase (EC 4.2.1.13) is the first enzyme that links EC 3 hydrolases and EC 4 lyases
since it catalyses a lytic reaction just like polysaccharide lyases while on the other hand it shares
amino acid sequence similarity and the same catalytic nucleophile with family 31 glycosidases
(YU et al., 1999; LEE et al., 2002, 2003). Here below is described our endeavour in expressing
this lyase in methylotrophic yeast Hansenula polymorpha and characterization of the
recombinant lyase.
As documented α-1,4-glucan lyase is a single polypeptide enzyme with a MW of
117,030 (YU et al., 1999). In our laboratory more than 10 lyase genes have been cloned and
studied in detail (YU et al., 1997, 1999). One of the lyases of algal origin (YU et al., 1997) has
been chosen to study its expression in the yeast H. polymorpha. The full-length algal gene and
its truncated forms were constructed by PCR and recombinant DNA techniques. These
constructs were transformed into the yeast by electroporation. PCR-screening showed that all
four lyase gene constructs had been integrated into the genome of the yeast. Western blot
analysis revealed that the full length and one of the truncated forms of the lyase gene were
expressed. Activity screening showed that all transformants with the full-length gene showed
glucan lyase activity. The formed anhydrofructose was detected by both the colorimetric method
and by HPLC. A method was developed to recover the intracellularly expressed lyase. It was
found that the detergent LTAB selectively extracted the lyase from the yeast biomass, resulting
in a 95% pure lyase. The purified recombinant lyase was characterized in detail. It was found
that the expressed lyase from the full-length lyase gene was N-terminal 11 amino acid truncated
as shown by both N-terminal sequencing and by MALDI-TOF mass spectrometry. Furthermore
the growth conditions were examined for optimal lyase expression. At 24 °C or 30 °C the total
lyase activity increased by 10-fold in contrast to growth at 37 °C. It was for the first time shown
that such a high MW enzyme could be heterologously expressed in H. polymorpha. The purified
recombinant glucan lyase is identical to wild type glucan lyase in stability, catalytic activity and
other kinetic parameters.
LEE, S.S., Y U, S. & WITHERS, S.G. 2002. J. Am. Chem. Soc. 124: 4948-4949.
LEE, S.S., Y U, S. & WITHERS, S.G. 2003. Biochemistry 42: 13081-1390.
Y U, S., BOJSEN, K., S VENSSON, B. & M ARCUSSEN, J. 1999. Biochim. Biophys. Acta 1433: 1-15.
Y U, S., CHRISTENSEN, T.M.I.E., KRAGH, K.M., BOJSEN, K. & M ARCUSSEN, J. 1997. Biochim. Biophys. Acta 1339: 311-320.
82
POSTER - P30
Relation between amino acid percentage and
optimal temperature of α-amylase
1
1
1
2
Ge-xin ZHANG , Yin-shi PIAO , Jian-yao ZHU , Wei-jiang LI & Wen-bo
3
XU
1
School of Chemical and Material Engineering, Southern Yangtze University, Wuxi, Jiangsu,
China, 214036; e-mail: [email protected]
Key Laboratory of Industrial Biotechnology of Ministry of Education, Southern Yangtze
University, Wuxi, Jiangsu, China, 214036
3
School of Information Technology, Southern Yangtze University, Wuxi, Jiangsu, China, 214036
2
α-Amylase hydrolyzes starch, glycogen, and related polysaccharides by cleaving internal α-1,4glucosidic bonds. Since starch starts being soluble only at 100 oC and above, the majority of
industrial applications of α-amylases require their use at temperatures of up to 110 oC (BENTLEY
& WILLIAMS, 1996), thus necessitating a relentless search for increasingly thermostable
enzymes.
Now, protein-engineering studies of temperature-activity profiles have been initiated
(SVENSSON, 1994). The way depended on two inventions: one is site-directed mutagenesis;
another is bioinformatics. Site-directed mutagenesis put remodeling enzyme into execution in
experiment; bioinformatics give the information how to do. So man must need to know relation
between enzyme’s sequences and their function. We studied previously related dipeptide and
characteristic dipeptide of optimal pH in α-amylase and got some information (ZHANG & LI,
2002).
Downed all sequences, which are relational α-amylase from SWISS-PROT
(http://www.expasy.ch/sprot) (January 2004). After clearing up, found 341 α-amylase
sequences. According their feature table, deleted signal peptide in sequence. Those sequences
consisted of our α-amylase’s bank. On the other hand, gathered a lot of literatures, which are
involved α-amylase’s optimal temperature. If an α-amylase has much optimal temperature
value, we accepted the value according the newest literature. Then searched α-amylase’s
sequences that have optimal temperature in our α-amylase’s bank. Found 63 α-amylases that
have optimal temperature data and amino acid sequences. We called this α-amylase’s
temperature-sequence bank. Computed percent of amino acid every sequences in α-amylase’s
bank. Then multiple linear regression models were used with method of least squares. Follow
formula is the multiple linear regression equation. In equation, single letter of amino acid
expresses percent of amino acid.
Temperature = 463.92077 - 2.48583A - 12.65145C - 6.99591D - 2.90921E - 7.42691F 5.68087G + 8.96338H - 0.09425I - 2.6811K - 3.59764L + 0.52771M - 5.86747N 1.22791P - 12.29561Q - 6.20562R - 5.05828S - 4.03611T - 2.54628V - 8.80071W +
0.09446Y
Predicting α-amylase’s optimal temperature in temperature-sequence bank with this
equation. Its correlation coefficient is 0.95969. The SD is 5.54388.
83
Compared our result with effect of single amino acid 227 substitutions on BLA
thermostability (DECLERCK et al., 2000), the case is best: 17.181; better: 33.921; good : 22.467;
bad: 22.467; worse: 3.084; worst: 0.881.
This study supported by the Science Foundation of Southern Yangtze University.
BENTLEY, I. S. & WILLIAMS, E. C. 1996. In: Industrial Enzymology, 2nd ed.; GODFREY, T. & WEST, S.I. (eds), Stockton
Press, New York, N.Y., pp. 339-357.
DECLERCK, N., MACHIUS, M., WIEGAND, G., HUBER, R. & GAILLARDIN, C. 2000. J. Mol. Biol. 301: 1041-1057.
SVENSSON, B. 1994. Plant Mol. Biol. 25: 141-157.
ZHANG, G.X. & LI, W.J. 2002. Biochem. Biophys. Res. Commun. 299: 647-651.
84
POSTER - P31
Relation between amino acid percentage and
optimal pH of α-amylase
1
1
2
Ge-xin ZHANG , Yin-shi PIAO , Wei-jiang LI & Wen-bo XU
3
1
School of Chemical and Material Engineering, Southern Yangtze University, Wuxi, Jiangsu,
China, 214036; e-mail: [email protected]
Key Laboratory of Industrial Biotechnology of Ministry of Education, Southern Yangtze
University, Wuxi, Jiangsu, China, 214036
3
School of Information Technology, Southern Yangtze University, Wuxi, Jiangsu, China, 214036
2
α-Amylases have found widespread use in industrial processes. But most α-amylase must be
improved for rigorous pH conditions. Protein engineering studies of pH-activity profiles have
been initiated (NIELSEN & BORCHERT, 2000). Remodeling enzyme’s first structure will create a
new enzyme for use. The way depended on two inventions: one is site-directed mutagenesis;
another is bioinformatics. We studied previously related dipeptide and characteristic dipeptide
of optimal pH in alpha-amylase and got some informations (ZHANG & LI, 2002). May we predict
optimal pH of α-amylase by percent of amino acid? How is predicting result?
We downed all sequences, which are relational α-amylase from SWISS-PROT
(http://www.expasy.ch/sprot) (January 2004). After clearing up, we found 341 α-amylase
sequences. According their feature table, we deleted signal peptide in sequence. Those
sequences consisted of our α-amylase’s bank. On the other hand, we gathered a lot of literatures,
which are involved α-amylase’s optimal pH. If an α-amylase has much optimal pH value, we
accepted the value according the newest literature. Then we searched α-amylase’s sequences
that have optimal pH in our α-amylase’s bank. We found 78 α-amylases that have optimal pH
data and amino acid sequences. We called this α-amylase’s pH-sequence bank.
We computed percent of amino acid every sequences in α-amylase’s bank. Then
multiple linear regression models were used with method of least squares. Follow formula is the
multiple linear regression equation. In equation, single letter of amino acid expresses percent of
amino acid.
pH = 16.14129 - 0.05190A + 0.12596C + 0.01285D + 0.01215E + 0.17663F - 0.19464G 0.06694H - 0.67907I + 0.11551K - 0.25012L - 0.10119M - 0.11485N - 0.10562P +
0.00550Q + 0.23175R + 0.00176S - 0.20236T - 0.28327V - 0.11930W - 0.00167Y
Its correlation coefficient is 0.68341. The SD is 0.77695.
This study shows that we may predict optimal pH of α-amylase by percent of amino
acid. The predicting results are reasonable.
NIELSEN, J.E. & BORCHERT, T.V. 2000. Biochim. Biophys. Acta 1543: 253-274.
ZHANG, G.X. & LI, W.J. 2002. Biochem. Biophys. Res. Commun. 299: 647-651.
85
POSTER - P32
Bioinformatics of the family GH-57: conserved
sequence regions, evolutionarily related subfamilies
and SLH-like motifs
Richard ZONA & Štefan JANEČEK
Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84551 Bratislava,
Slovakia; e-mail: [email protected]
Sixty-six amino acid sequences belonging to glycoside hydrolase family GH-57 were collected
using the CAZy server (COUTINHO & HENRISSAT, 1999), Pfam database (BATEMAN et al., 2002)
and BLAST tools (ALTSCHUL et al., 1997). Owing to the extreme sequence heterogeneity of the
GH-57 members, sequence alignments were performed using mainly manual methods. Five
conserved regions were identified, which are postulated to be GH-57 consensus motifs (ZONA et
al., 2004). In the 659 amino acid-long 4-α-glucanotransferase from Thermococcus litoralis,
these motifs correspond to 13_HQP (region I), 76_GQLEIV (region II), 120_WLTERV (region
III), 212_HDDGEKFGVW (region IV), and 350_AQCNDAYWH (region V). The third and
fourth conserved regions contain the catalytic nucleophile, Glu123, and the proton donor,
Asp214, respectively (underlined).
Based on our original sequence alignment (ZONA et al., 2004), the residues Glu291 and
Asp394 were proposed as the nucleophile and proton donor respectively in a GH-57
amylopullulanase from Thermococcus hydrothermalis. Both residues were found to be critical
for the pullulanolytic and amylolytic activities of the amylopullulanase, supporting the
prediction and strongly suggesting that the bifunctionality of the amylopullulanase is determined
by a single catalytic centre (ZONA et al., 2004). Our alignment also revealed that certain GH-57
members do not possess the Glu and the Asp corresponding to the predicted GH-57 catalytic
residues. However, the sequences concerned by this anomaly encode putative proteins for which
no biochemical or enzymatic data is yet available. Finally, the evolutionary trees generated for
GH-57 revealed that the entire family could be divided into several subfamilies that may reflect
the different enzyme specificities.
In the amylopullulanase from Thermococcus hydrothermalis, sequence segments called
the surface-layer homology (SLH) motifs were recognised (ERRA-PUJADA et al., 1999). These
segments were found to contain two and a half typical SLH motifs of the Pfam family PF00395
(BATEMAN et al., 2002). Therefore they were named SLH motif-bearing domains (ERRA-PUJADA
et al., 1999). Interestingly, all these SLH-like motifs identifiable in the sequences of GH-57
members come from the amylopullulanase GH-57 subfamily. Some members possess one SLHlike motif, whereas some of them possess two such motifs. A segment corresponding with the
GH-57 SLH-like motifs was recently found in a GH-15 glucodextranase (MIZUNO et al., 2004).
ALTSCHUL, S.F., STEPHEN, F., MADDEN, T.L., ... & LIPMAN, D.J. 1997. Nucleic Acids Res. 25: 3389-3402.
BATEMAN, A., BIRNEY, E., CERRUTI, L., … & SONNHAMMER, E.L. 2002. Nucleic Acids Res. 30: 276-280.
COUTINHO, P.M. & HENRISSAT, B. 1999. http://afmb.cnrs-mrs.fr/~cazy/CAZY/index.html
ERRA-P UJADA, M., D EBEIRE, P., D UCHIRON, F. & O’DONOHUE, M.J. 1999. J. Bacteriol. 181: 3284-3287.
MIZUNO, M., TONOZUKA, T., SUZUKI , S., … & SAKANO, Y. 2004. J. Biol. Chem. 279: 10575-10583.
ZONA, R., CHANG-PI-HIN, F., O’DONOHUE, M.J. & JANECEK, S. 2004. Eur. J. Biochem. 271: 2863-2872.
86
POSTER - P33
SLH motifs in the members of the α-amylase family:
relationships within the families of glycoside
hydrolases
Richard ZONA & Štefan JANEČEK
Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84551 Bratislava,
Slovakia; e-mail: [email protected]
Many glycoside hydrolases (GH) are exceptionally large proteins consisting of catalytic and
non-catalytic domains. With regard to the non-catalytic domains, much research has been
performed on the carbohydrate-binding modules (CBM), whereas a substantially less attention
has been paid to the surface layer homology (SLH) motifs. The SLH sequences, usually
positioned C-terminally and about 50-60 amino acid residues long, are possibly involved in the
attachment of proteins to the underlying cell wall (LUPAS et al., 1994). The SLH domains mostly
occur in more copies as repeats.
According to the Pfam database (BATEMAN et al., 2002), the SLH module constitutes the
family PF00395. Three amylopullulanases from the α-amylase family GH-13, contain the SLH
modules; each in three copies. Within the CAZy (COUTINHO & HENRISSAT, 1999) in addition to
the α-amylase family GH-13 the typical 50-60 amino acid residues long SLH modules are
present in further at least six GH families (Table 1). Moreover the SLH-like motifs were found
in two more families, GH-15 and GH-57 (Table 1), as the longer SLH motif-bearing domains
containing two and a half typical SLH motifs (ERRA-PUJADA et al., 1999).
Table 1. Glycoside hydrolases containing the SLH motifs.
Family
Clan
EC
Enzyme
SLH module
Catalytic fold
GH-5
GH-10
GH-13
GH-15
GH-16
GH-26
GH-28
GH-57
GH-73
GH-A
GH-A
GH-H
GH-L
GH-B
GH-A
GH-N
---
3.2.1.4; 3.2.1.78
3.2.1.8
3.2.1.1/41
3.2.1.70
3.2.1.73
3.2.1.78
3.2.1.15
3.2.1.1/41
3.2.1.96
endo-1,4-glucanase; β-mannanase
xylanase
amylopullulanase
glucodextranase
lichenase
β-mannanase
polygalacturonase
amylopullulanase
endo-β-N-acetylglucosaminidase
50-60 aa
50-60 aa
50-60 aa
~ 2.5 x module
50-60 aa
50-60 aa
50-60 aa
~ 2.5 x module
50-60 aa
(β/α)8-barrel
(β/α)8-barrel
(β/α)8-barrel
(α/α)6-barrel
β-jelly roll
(β/α)8-barrel
β-helix
(β/α)7-barrel
not known
The evolutionary pictures will be presented that illustrate the relationships among the
individual copies of SLH motifs in the frame of a given enzyme sequence and/or a GH family,
among the SLH motifs originating from the various GH families as well as between the typical
SLH motifs and the longer SLH motif-bearing domains.
BATEMAN, A., BIRNEY, E., CERRUTI, L., … & SONNHAMMER, E.L. 2002. Nucleic Acids Res. 30: 276-280.
COUTINHO, P.M. & HENRISSAT, B. 1999. http://afmb.cnrs-mrs.fr/~cazy/CAZY/index.html
ERRA-P UJADA, M., D EBEIRE, P., D UCHIRON, F. & O’DONOHUE, M.J. 1999. J. Bacteriol. 181: 3284-3287.
LUPAS, A., ENGELHARDT , H., P ETERS, J., S ANTARIUS, U., VOLKER, S. & BAUMEISTER, W. 1994. J. Bacteriol. 176: 12241233.
87
88
2nd Symposium on the Alpha-Amylase Family
PARTICIPANTS AS AUTHORS
89
A
Abou Hachem, M.
Aghajari, N.
Albenne, C.
Ali, S.
Andersen, C.
L13, P1
L2, L3, L13
L19, L24, P2, P23
P3
L23
B
Bardowski, J.
Barends, T.R.M.
Baunsgaard, L.
Beier, L.
Bejar, S.
Borchert, T.V.
Bozonnet, S.
P9
L4, P4
P5, P8
L23
P6
L23
L2, L13, L21, P17
C
Christiansen, C.
Coutinho, P.M.
P8
L1
D
Dijkhuizen, L.
Dijkstra, B.W.
L4, L6, L8, L22, P4, P16, P28
L4, P4
E
Ernst, H.A.
L17
F
Fujii, K.
L5
H
Hamedi, J.
Haser, R.
Hashim, S.O.
Hassani-nasab, A.
Horváthová, V.
P12
L2, L3, L13
P13
P12
P11
J
Janeček, Š.
Jensen, M.T.
P11, P14, P18, P19, P32, P33
L2, L13, P17
K
Kandra, L.
Kaper, T.
Khajeh, K.
L7, L28
L4, L22, P4
L9, P10, P15
90
Kimura, A.
Kralj, S.
Kramhøft, B.
Kuriki, T.
L14
L6, P16
L13, P17
L5, L32
L
Lohi, H.
Lo Leggio, L.
L25
L17
M
MacGregor, E.A.
Machovič, M.
MacRae, E.A.
Mariam, I.
Mizuno, M.
Monsan, P.
L26, P14, P19
P18, P19
L30
P3, P20
L15, P26
L19, L24, P2, P23
N
Naderi-Manesh, H.
Næsted, H.
Nielsen, M.M.
Nordberg Karlsson, E.
L9, P10, P15
P21
P22
L29
O
O'Donohue, M.J.
L18
P
Park, H.-S.
Park, K.-H.
Patel, B.
Potocki-Veronese, G.
P7
L20, P7
L27
L19, P2, P23
R
Ramachandran, N.
Ramachandran, S.
Ramasubbu, N.
Ravaud, S.
Robyt, J.F.
Rodríguez-Sanoja, R.
L11
P24
L7
L3
L31
L12
S
Sajedi, R.H.
Skov, L.
Svensson, B.
Szweda, P.
L9
L24, P2
L2, L13, L17, L21, P1, P8, P14, P17, P19, P21
P25
91
Ševčík, J.
L16
T
Tonozuka, T.
Turner, P.
L15, P26
L29
U
ul-Haq, I.
P3, P27
V
van der Kaaij, R.M.
van der Maarel, M.J.E.C.
van der Veen, B.A.
Viksø-Nielsen, A.
P28
L4, L6, L22, P28
L19, L24, P2, P23
L23
Y
Yang, S.-J.
Yu, S.
P7
L17, P29
Z
Zhang, G.-x.
Zona, R.
L10, P30, P31
P19, P32, P33
92
2nd Symposium on the Alpha-Amylase Family
LIST OF PARTICIPANTS
93
Abou Hachem, Maher
Biochemistry and Nutrition Group, BioCentrum-DTU, Technical University of Denmark, Søltofts
Plads - Building 224, DK-2800 Kgs. Lyngby, Denmark
E-mail: [email protected]
Aghajari, Nushin
Institut de Biologie et Chimie des Protéines UMR 5086, Laboratoire de BioCristallographie, CNRS
and Université Claude Bernard Lyon I, 7 Passage du Vercors, 69367 Lyon cedex 07, France
E-mail: [email protected]
Albenne, Cécile
Centre de Bioingénierie Gilbert Durand, UMR CNRS 5504, UMR INRA 792, INSA DGBA, 135,
avenue de Rangueil, 31 077 Toulouse Cedex 4, France
E-mail: [email protected]
Ali, Sikander
Biotechnology Research Centre, Department of Botany, Government College, University Lahore,
Lahore 54000, Pakistan
E-mail: [email protected]
Andersen, Carsten
Novozymes A/S, Krogshoejvej, DK-2880 Bagsvaerd, Denmark
E-mail: [email protected]
Bardowski, Jacek
Institute of Biochemistry and Biophysics of Polish Academy of Sciences, Pawinskiego 5a, 02-106
Warsaw, Poland
E-mail: [email protected]
Bardowska, Urszula
Accompanying person (Bardowski, Jacek)
Barends, Thomas R.M.
Laboratory of Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen,
The Netherlands
E-mail: [email protected]
Baunsgaard, Lone
Center for Molecular Plant Physiology (PlaCe), Plant Biochemistry Laboratory, Department of
Plant Biology, The Royal Veterinary and Agricultural University, DK-1871 Frederiksberg C,
Copenhagen, Denmark
E-mail: [email protected]
Beier, Lars
Novozymes A/S, Krogshoejvej, DK-2880 Bagsvaerd, Denmark
E-mail: [email protected]
Bejar, Samir
Laboratoire d’Enzymes et de Métabolites des Procaryotes, Centre de Biotechnologie de Sfax, BP
"K", 3038 Sfax Tunisie
E-mail: [email protected]
Borchert, Torben V.
Novozymes A/S, Krogshoejvej, DK-2880 Bagsvaerd, Denmark
E-mail: [email protected]
94
Bozonnet, Sophie
Biochemistry and Nutrition Group, BioCentrum-DTU, Technical University of Denmark, Søltofts
Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark
E-mail: [email protected]
Christiansen, Camilla
Center for Molecular Plant Physiology (PlaCe), Plant Biochemistry Laboratory, Department of
Plant Biology, The Royal Veterinary and Agricultural University, DK-1871 Frederiksberg C,
Copenhagen, Denmark
E-mail: [email protected]
Coutinho, Pedro M.
UMR6098, AFMB / CNRS / Universités Aix-Marseille I et II, 31 chemin Joseph Aiguier, 13402
Marseille cedex 20, France
E-mail: [email protected]
Dijkhuizen, Lubbert
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB),
Kerklaan 30, 9751 NN Haren, The Netherlands
E-mail: [email protected]
Dijkstra, Bauke W.
Laboratory of Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen,
The Netherlands
E-mail: [email protected]
Ernst, Heidi A.
Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen
Ø, Denmark
E-mail: [email protected]
Fujii, Kazutoshi
Biochemical Research Laboratory, Ezaki Glico Co., Ltd., 4-6-5 Utajima, Nishiyodogawa-ku, Osaka
555-8502, Japan
E-mail: [email protected]
Hamedi, Javad
Microbiology Division, Department of Biology, Faculty of Science, University of Tehran, P.O. Box:
14155-6455, Tehran, Iran
E-mail: [email protected]
Haser, Richard
Laboratoire de Bio-Cristallographie, Institut de Biologie et Chimie des Protéines, UMR 5086CNRS/UCBL, 7, Passage du Vercors, F-69367 Lyon cedex 07, France
E-mail: [email protected]
Hashim, Suhaila O.
Department of Biotechnology, Lund University, Box 124, SE-221 00 Lund, Sweden
E-mail: [email protected]
Hassani-nasab, Azam
Microbiology Division, Department of Biology, Faculty of Science, University of Tehran, P.O. Box:
14155-6455, Tehran, Iran
E-mail: [email protected]
95
Horváthová, Viera
Department of Biotechnologies, Faculty of Natural Sciences, University of SS Cyril and Methodius,
SK-91700 Trnava, Slovakia
E-mail: [email protected]
Im, Hee-Hyuck
Accompanying person (Park, Kwan-Hwa)
Janeček, Štefan
Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84251
Bratislava, Slovakia
E-mail: [email protected]
Jensen, Morten Tovborg
Novozymes A/S, Krogshoejvej, DK-2880 Bagsvaerd, Denmark
E-mail: [email protected]
Kandra, Lili
Institute of Biochemistry, Faculty of Sciences, University of Debrecen, H-4010 Debrecen, PO Box
55, Hungary
E-mail: [email protected]
Kaper, Thijs
Laboratory of Microbiology, University of Groningen, Kerklaan 30, 9751 NN, Haren, The
Netherlands
E-mail: [email protected]
Khajeh, Khosro
Department of Biochemistry and Biophysics, Faculty of Science, Tarbiat Modarres University, P.
O. Box 14115-175, Tehran, Iran
E-mail: [email protected]
Kim, Jung-Woo
56-1 Sillim-dong, Kwanak-gu, 200-8101 Seoul National University; South Korea
E-mail: [email protected]
Kimura, Atsuo
Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
E-mail: [email protected]
Kralj, Slavko
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB),
University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands
E-mail: [email protected]
Kramhøft, Birte
Biochemistry and Nutrition Group, BioCentrum-DTU, Technical University of Denmark, Søltofts
Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark
E-mail: [email protected]
Kuriki, Takashi
Biochemical Research Laboratory, Ezaki Glico Co., Ltd., 4-6-5 Utajima, Nishiyodogawa-ku, Osaka
555-8502, Japan
E-mail: [email protected]
96
Lohi, Hannes
The Hospital for Sick Children and Department of Molecular and Medical Genetics, the University
of Toronto, Toronto, Canada
E-mail: [email protected]
Lo Leggio, Leila
Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen
Ø, Denmark
E-mail: [email protected]
MacGregor, E. Ann
2 Nicklaus Green, Livingston, West Lothian, EH54 8RX, U.K.
E-mail: [email protected]
Machovič, Martin
Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84251
Bratislava, Slovakia
E-mail: [email protected]
MacRae, Elspeth A.
HortResearch, Mt Albert Research Centre, Private Bag 92169, Auckland, New Zealand
E-mail: [email protected]
Mariam, Irfana
Institute of Chemistry, Quaid-e-Azam Campus, University of the Punjab, Lahore 54590, Pakistan
E-mail: [email protected]
Mizuno, Masahiro
United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology,
3-5-8, Saiwai-cho, Fuchu, Tokyo 183-8509, Japan
E-mail: [email protected]
Monsan, Pierre
Centre de Bioingénierie Gilbert Durand, UMR CNRS 5504, UMR INRA 792, INSA DGBA, 135,
avenue de Rangueil, 31 077 Toulouse Cedex 4, France
E-mail: [email protected]
Naderi-Manesh, Hossein
Department of Biochemistry and Biophysics, Faculty of Science, Tarbiat Modarres University, P.
O. Box 14115-175, Tehran, Iran
E-mail: [email protected]
Næsted, Henrik
Biochemistry and Nutrition Group, BioCentrum-DTU, Technical University of Denmark, Søltofts
Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark
E-mail: [email protected]
Nielsen, Morten M.
Biochemistry and Nutrition Group, BioCentrum-DTU, Technical University of Denmark, Søltofts
Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark
E-mail: [email protected]
Nordberg Karlsson, Eva
Department of Biotechnology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden
E-mail: [email protected]
97
O'Donohue, Michael J.
UMR FARE, Institut National de la Recherche Agronomique, 8, rue Gabriel Voisin, BP 316, 51688
cedex 02, France
E-mail: [email protected]
Paës, Gabriel
UMR FARE, Institut National de la Recherche Agronomique, 8, rue Gabriel Voisin, BP 316, 51688
cedex 02, France
E-mail: [email protected]
Park, Hye-Sun
56-1 Sillim-dong, Kwanak-gu, 200-8101 Seoul National University; South Korea
E-mail: [email protected]
Park, Jong-Tae
56-1 Sillim-dong, Kwanak-gu, 200-8101 Seoul National University; South Korea
E-mail: [email protected]
Park, Kwan-Hwa
Department of Food Science & Technology, College of Agriculture and Life Sciences, Seoul
National University, Suwon 441-744, Korea
E-mail: [email protected]
Patel, Bharat
Microbial Diversity and Discovery Group, School of Biomolecular and Biomedical Sciences,
Faculty of Scienc, Griffith University, Brisbane 4111, Queensland, Australia
E-mail: [email protected]
Potocki-Veronese, Gabrielle
INSA DGBA, UMR INRA 792, UMR CNRS 5504, 135 avenue de Rangueil, 31077 Toulouse
Cedex 4, France
E-mail: [email protected]
Preiss, Jack
Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing,
MI 48824, USA
E-mail: [email protected]
Preiss, Karen Sue
Accompanying person (Preiss, Jack)
Ramachandran, Nivetha
Institute for Wine Biotechnology, Victoria street, J.H. Neethling building, Stellenbosch University,
Stellenbosch 7600 South Africa
E-mail: [email protected]
Ramachandran, Sumitra
Biotechnology Division, Regional Research Laboratory, CSIR, Trivandrum - 95 019, India
E-mail: [email protected]
Ramasubbu, Narayanan
Department of Oral Biology, University of Medicine and Dentistry of New Jersey, Newark, NJ
07103 USA
E-mail: [email protected]
98
Ravaud, Stéphanie
Laboratoire de BioCristallographie, IBCP UMR 5086, CNRS UCBL, IFR128 “Biosciences LyonGerland”, 7 passage du Vercors, 69367 Lyon Cedex 07, France
E-mail: [email protected]
Robyt, John F.
Laboratory of Carbohydrate Chemistry and Enzymology, Department of Biochemistry, Biophysics,
and Molecular Biology, 4252 Molecular Biology Bldg., Iowa State University, Ames, IA 50011, USA
E-mail: [email protected]
Robyt, Lois
Accompanying person (Robyt, John F.)
Rodríguez-Sanoja, Romina
Depto. de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, UNAM,
Ciudad Universitaria, C.P. 04510, A.P. 70228, México, D.F.
E-mail: [email protected]
Sajedi, Reza H.
Department of Biochemistry and Biophysics, Faculty of Science, Tarbiat Modarres University, P.
O. Box 14115-175, Tehran, Iran
E-mail: [email protected]
Skov, Lars
Biostructural Research, Department of Medicinal Chemistry, The Danish University of
Pharmaceutical Sciences, Universitetsparken 2, DK 2100 Copenhagen, Denmark
E-mail: [email protected]
Svensson, Birte
Biochemistry and Nutrition Group, BioCentrum-DTU, Technical University of Denmark, Søltofts
Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark
E-mail: [email protected]
Szweda, Piotr
Department of Food Chemistry, Technology and Biotechnology, Gdansk University of Technology,
80 – 952 Gdansk, Poland
E-mail: [email protected]
Ševčík, Jozef
Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84251
Bratislava, Slovakia
E-mail: [email protected]
Tams, Jeppe Wegener
Novozymes A/S, Novo Allé, Byg. 6B3, 98.1., DK-2880 Bagsvaerd, Denmark
E-mail: [email protected]
Tonozuka, Haruko
Accompanying person (Tonozuka, Takashi)
Tonozuka, Takashi
Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8,
Saiwai-cho, Fuchu, Tokyo 183-8509, Japan
E-mail: [email protected]
99
Turner, Pernilla
Department of Biotechnology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden
E-mail: [email protected],
ul-Haq, Ikram
Biotechnology Research Centre, Department of Botany, Government College, University Lahore,
Lahore 54000, Pakistan
E-mail: [email protected]
van der Kaaij, Rachel M.
Centre for Carbohydrate Bioengineering, TNO-University of Groningen, Haren, The Netherlands
E-mail: [email protected]
van der Maarel, Marc J.E.C.
Centre for Carbohydrate Bioengineering TNO-RUG, P.O. Box 14, 9750 AA Haren, The
Netherlands
E-mail: [email protected]
van der Veen, Bart A.
Centre de Bioingénierie Gilbert Durand, UMR CNRS 5504, UMR INRA 792, INSA, 135 avenue de
Rangueil, 31077 Toulouse Cedex 4, France
E-mail: [email protected]
van der Veen, Anna
Accompanying person (van der Veen, Bart A.)
Viksø-Nielsen, Anders
Novozymes A/S, Starch & Brewing, Laurentsvej 55, 8G2.06, 2880 Bagsvaerd, Denmark
E-mail: [email protected]
Yang, Sung-Jae
56-1 Sillim-dong, Kwanak-gu, 200-8101 Seoul National University; South Korea
E-mail: [email protected]
Yu, Shukun
Danisco, Edwin Rahrs Vej 38, DK-8220 Brabrand, Denmark
E-mail: [email protected]
Zhang, Ge-xin
School of Chemical and Material Engineering, Southern Yangtze University, Wuxi,
Jiangsu, China, 214036
E-mail: [email protected]
Zona, Richard
Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84251
Bratislava, Slovakia
E-mail: [email protected]
100