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ISSN 2342-5423
ISBN 978-951-51-2451-7
21/2016
Helsinki 2016
QIAO SHI Synthesis and Structural Characterization of Glucooligosaccharides and Dextran from Weissella confusa Dextransucrases
Recent Publications in this Series
dissertationes schola doctoralis scientiae circumiectalis,
alimentariae, biologicae. universitatis helsinkiensis
QIAO SHI
Synthesis and Structural Characterization of
Glucooligosaccharides and Dextran from
Weissella confusa Dextransucrases
DEPARTMENT OF FOOD AND ENVIRONMENTAL SCIENCES
FACULTY OF AGRICULTURE AND FORESTRY
DOCTORAL PROGRAMME IN FOOD CHAIN AND HEALTH
UNIVERSITY OF HELSINKI
21/2016
Department of Food and Environmental Sciences
Faculty of Agriculture and Forestry
University of Helsinki
Doctoral Programme in Food Chain and Health
Doctoral School in Environmental, Food and Biological Sciences
Synthesis and structural characterization of
glucooligosaccharides and dextran from
Weissella confusa dextransucrases
Qiao Shi
Doctoral Thesis
To be presented, with the permission of the Faculty of Agriculture and
Forestry, University of Helsinki, for public examination in Auditorium 2,
Infocenter Korona (Viikinkaari 11), on September 29th, 2016,
at 12 o’clock noon.
Helsinki 2016
Custos
Professor Maija Tenkanen
Department of Food and Environmental Sciences
University of Helsinki, Finland
Supervisor
Professor Maija Tenkanen
Department of Food and Environmental Sciences
University of Helsinki, Finland
Reviewers
Dr. Gregory L. Côté
Agricultural Research Service
United States Department of Agriculture, USA
Professor Markus Linder
School of Chemical Technology
Aalto University, Finland
Opponent
Professor Henk Schols
Laboratory of Food Chemistry
Wageningen University, The Netherlands
Cover picture: Representation of a dextransucrase (based on Protein Data Bank no.
3HZ3) and its oligosaccharide products, made by Qiao Shi.
ISBN 978-951-51-2451-7 (Paperback)
ISBN 978-951-51-2452-4 (PDF)
ISSN 2342-5423 (Print)
ISSN 2342-5431 (Online)
Unigrafia
Helsinki 2016
ABSTRACT
Many lactic acid bacteria are able to synthesize dextrans from sucrose by
dextransucrases. Weissella confusa strains have attracted increasing attention due to
their production of texture-modifying dextrans during food fermentation. Potential
prebiotic oligosaccharides have also been produced by dextransucrases in the presence
of sucrose and acceptor sugars. However, only few W. confusa dextransucrases have
been previously studied, and the reactions of Weissella dextransucrases to synthesize
oligosaccharides have not been investigated. In this thesis, two W. confusa
dextransucrases from efficient dextran-producers were studied, and their products were
structurally characterized because this information is essential for a better usability of
these enzymes and corresponding bacteria in food and health applications.
The biochemical and kinetic properties of one of the W. confusa dextransucrases were
studied. Two activity assays were compared to determine the kinetic parameters. A
sucrose radioisotope assay gave a KM of 14.7 mM and a Vmax of 8.2 µmol/(mg∙min),
whereas a Nelson-Somogyi assay gave values of 13.0 mM and 19.9 µmol/(mg∙min),
respectively. The dextrans from the two W. confusa dextransucrases were found by, e.g.,
NMR analysis to consist of mainly α-(1→6) linkages and 3% α-(1→3) branches, of
which some were elongated. A high-performance size-exclusion chromatography
analysis of the dextrans revealed high molar masses of 107‒108 g/mol.
Weissella dextransucrases were also studied with acceptor sugars for the synthesis of
glucooligosaccharides. The most efficient acceptor was maltose, followed by isomaltose,
maltotriose, and nigerose, which formed series of glucooligosaccharides by the further
elongation of intermediate acceptor products. The products derived from maltose formed
two homologous series, with one series being predominant and the other being minor.
The major maltose product series were linear isomaltooligosaccharides (IMOs) with
reducing-end maltose units, as identified by multistage mass spectrometry (MSn)
analysis. The minor maltose series were revealed by an NMR analysis of the isolated
minor trisaccharide product to bear a novel branched structure. These products contained
an α-(1→2)-linked glucosyl residue on the reducing residue of the linear IMOs. These
structures have not been previously obtained by a dextransucrase. They probably formed
by the attachment of a single-unit branch to linear IMOs.
For the acceptor analogs lactose and cellobiose, their main acceptor products were
identified by NMR analysis to be branched trisaccharides, with a glucosyl residue α(1→2) linked to the acceptor’s reducing end. Surprisingly, a side product, isomelezitose
1
(6Fru-α-Glcp-sucrose), was produced when using lactose as an acceptor. The synthesis
of this nonreducing trisaccharide by a dextransucrase was reported here for the first time.
Linear IMOs with a degree of polymerization ≥3.3 serve as prebiotics for their resistance
to digestion. However, industrial IMO production often leads to a high portion of
unwanted digestible sugars. The dextransucrase reaction in the presence of the efficient
acceptor maltose was demonstrated here as a promising alternative synthesis process to
control IMO size distribution by varying the sucrose/maltose ratio. The effects of
substrate concentrations (0.15–1 M) and dextransucrase dosage (1–10 U/g sucrose) on
the IMO yield and profile were modeled. High sucrose (1 M) and medium maltose (0.5
M) concentrations were found to be optimal for the synthesis of long-chain IMOs, with
366 g/L of total IMOs attained.
2
ACKNOWLEDGEMENTS
This thesis was carried out at the Department of Food and Environmental Sciences,
Faculty of Agriculture and Forestry, University of Helsinki. The work was financially
supported by the Finnish Graduate School on Applied Bioscience: Bioengineering, Food
& Nutrition, Environment (ABS Graduate School), the Academy of Finland via project
“Molecular and functional characterization of dextran production in Weissella spp. ‒
Superior dextran producers for cereal applications (WISEDextran)” and the Finnish
Food and Drink Industries' Federation (ETL).
I am deeply indebted to my supervisor Professor Maija Tenkanen. She provided me with
the great opportunity to pursue a doctoral degree in her research group and has offered
me insightful scientific guidance, advice, and encouragement throughout the years. Her
open-mindedness, curiosity, and enthusiasm towards science have inspired me. It was
also a privilege for me to work with Docent Liisa Virkki, who introduced me to NMR
spectral interpretation. I feel grateful to Professor Vieno Piironen and Research
Professor Kristiina Kruus for participating in my follow-up group during my thesis work.
I also want to express my sincere gratitude to Dr. Gregory L. Côté and Professor Markus
Linder for their pre-examination of this thesis.
I owe my gratitude to Docent Hannu Maaheimo, Assistant Professor Kati Katina, Docent
Päivi Tuomainen, Minna Juvonen, Dr. Ndegwa Henry Maina, Kaj-Roger Hurme, Dr.
Sanna Koutaniemi, and Dr. Leena Pitkänen, who gave me invaluable help in their fields
of expertise, as well as Yaxi Hou, who assisted me with laboratory work. I also thank
the project collaborators Dr. Riikka Juvonen, Ilkka Kajala, and Dr. Antti Nyyssölä from
the VTT Technical Research Centre of Finland Ltd. and Professor Arun Goyal and
Shraddha Shukla from the Indian Institute of Technology Guwahati (IITG). This thesis
could not have been completed without their fruitful collaborations.
I am fortunate to have been surrounded by friendly and supportive colleagues along the
journey, including Sun-Li Chong, Taru Rautavesi, Miikka Olin, Kirsi Mikkonen,
Susanna Heikkinen, Kirsti Parikka, Minna Malmberg, Laura Huikko, Paula Hirsilä,
Abdul Ghafar, Suvi Alakalhunmaa, Bhawani Chamlagain, Yan Xu, and Yu Wang, to
name a few. I truly appreciate the joyful time I shared with all of the nice people from
the department, especially those in D building.
Last but not least, my warmest gratitude goes to my dearest family, Xiaoguang Shi,
Wenxiu Zhu, and Ling Zou, for being my rock and having faith in me! I cannot thank
them enough!
3
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following publications:
I.
Kajala I, Shi Q, Nyyssölä A, Maina NH, Hou Y, Katina K, Tenkanen M, Juvonen
R. 2015. Cloning and characterization of a Weissella confusa dextransucrase and
its application in high fibre baking. PLoS ONE 10:e0116418.
II.
Shukla S, Shi Q, Maina NH, Juvonen M, Tenkanen M, Goyal A. 2014. Weissella
confusa Cab3 dextransucrase: properties and in vitro synthesis of dextran and
glucooligosaccharides. Carbohydr. Polym. 101:554-64.
III.
Shi Q, Juvonen M, Hou Y, Kajala I, Nyyssölä A, Maina NH, Maaheimo H, Virkki
L, Tenkanen M. 2016. Lactose- and cellobiose-derived branched trisaccharides
and a sucrose-containing trisaccharide produced by acceptor reactions of
Weissella confusa dextransucrase. Food Chem. 190:226-36.
IV.
Shi Q, Hou Y, Juvonen M, Tuomainen P, Kajala I, Shukla S, Goyal A, Maaheimo
H, Katina K, Tenkanen M. 2016. Optimization of isomaltooligosaccharide size
distribution by acceptor reaction of Weissella confusa dextransucrase and
characterization of novel α-(1→2)-branched isomaltooligosaccharides. J. Agric.
Food Chem. 64:3276-86.
The author’s contribution
I.
Shi Q planned and performed the experiments related to dextransucrase and
dextran characterization. She interpreted the results and prepared that part of the
manuscript.
II.
Shi Q was responsible for producing and characterizing glucooligosaccharides.
She participated in planning and performing the related experiments, interpreted
the results, and prepared the manuscript, including the introduction and parts
related to glucooligosaccharide work and most of the dextran characterization.
III.
Shi Q participated in planning the study and was responsible for all of the
experimental work and result interpretation, except for the MS analysis. She
prepared the manuscript except for the MS part and was the corresponding author.
4
IV.
Shi Q was co-responsible for the experimental work and results interpretation
related to optimization. She planned and performed most of the experiments and
interpreted the results regarding novel oligosaccharides. She prepared the
manuscript and was the corresponding author.
Other related publications of the author:
Juvonen R, Honkapää K, Maina NH, Shi Q, Viljanen K, Maaheimo H, Virkki L,
Tenkanen M, Lantto R. 2015. The impact of fermentation with exopolysaccharide
producing lactic acid bacteria on rheological, chemical and sensory properties of pureed
carrots (Daucus carota L.). Int. J. Food Microbiol. 207:109-18.
Kajala I, Mäkelä J, Coda R, Shukla S, Shi Q, Maina NH, Juvonen R, Ekholm P, Goyal
A, Tenkanen M, Katina K. 2016. Rye bran as fermentation matrix boosts in situ dextran
production by Weissella confusa compared to wheat bran. Appl. Microbiol. Biotechnol.
100:3499-510.
5
ABBREVIATIONS
BIMi (i≥3)
Cel3
COSY
D2 O
DMSO
DNS
DP
DQFCOSY
DSR-S
EPS
ESI-IT-MSn
GH
GLOSs
HePS
HMBC
HoPS
HPAEC-PAD
HPSEC
HSQC
IMOs
LAB
Lac3
LIMi (i≥4)
NMR
NR3
PEG
Q2
R2
TLC
TOCSY
WcCab3-DSR
WcE392-rDSR
maltose-derived branched isomaltooligosaccharides of
DPs≥3
cellobiose-derived trisaccharide
homonuclear correlation spectroscopy
deuterium oxide
dimethyl sulfoxide
3,5-dinitrosalicylic acid
degree of polymerization
double-quantum-filtered COSY
dextransucrase from Leuconostoc mesenteroides NRRL B512F
exopolysaccharides
electrospray ionization multistage ion trap mass
spectrometry
glycoside hydrolase
glucooligosaccharides
heteropolysaccharides
heteronuclear multiple-bond correlation spectroscopy
homopolysaccharides
high-performance anion-exchange chromatography with
pulsed amperometric detection
high-performance size-exclusion chromatography
heteronuclear single-quantum coherence spectroscopy
isomaltooligosaccharides
lactic acid bacteria
lactose-derived trisaccharide
maltose-derived linear isomaltooligosaccharides of DPs≥4
nuclear magnetic resonance
nonreducing sucrose-containing trisaccharide
polyethylene glycol
the coefficient of prediction in regression analysis
the coefficient of determination in regression analysis
thin-layer chromatography
total correlation spectroscopy
dextransucrase from W. confusa Cab3
recombinant dextransucrase from W. confusa VTT E-90392
6
TABLE OF CONTENTS
Abstract ........................................................................................................................... 1
Acknowledgements......................................................................................................... 3
List of original publications ............................................................................................ 4
Abbreviations .................................................................................................................. 6
1. Introduction................................................................................................................. 9
2. Review of the literature ............................................................................................ 11
2.1 Carbohydrates from lactic acid bacteria ............................................................. 11
2.2 Glucansucrases ................................................................................................... 12
2.2.1 Catalytic mechanism of glucansucrases ....................................................... 12
2.2.2 Production and characterization of dextransucrases ..................................... 15
2.3 Carbohydrates synthesized by dextransucrases .................................................. 16
2.3.1 Substrate and product specificity of dextransucrases ................................... 16
2.3.2 Functional oligosaccharides from dextransucrases ...................................... 19
2.3.3 Characterization of dextransucrase-synthesized carbohydrates ................... 21
2.4 Weissella spp. and Weissella dextransucrases .................................................... 23
3. Aim of the study ....................................................................................................... 26
4. Materials and methods .............................................................................................. 27
4.1 Dextransucrase preparations and commercial enzymes ..................................... 27
4.2 Biochemical characterization of WcE392-rDSR (I) ........................................... 27
4.3 Analysis of monosaccharides and oligosaccharides ........................................... 28
4.4 Synthesis, isolation, and molar mass characterization of dextrans (I and II) ..... 29
4.5 Acceptor reactions and isolation of oligosaccharides (II‒ IV) ........................... 29
4.6 Structural analysis of dextrans and oligosaccharides ......................................... 31
4.6.1 Enzymatic hydrolysis of dextrans and oligosaccharides (II and III) ............ 31
4.6.2 NMR spectroscopy analysis of dextrans and oligosaccharides (I‒IV) ......... 31
4.6.3 MSn analysis of oligosaccharides (II‒IV) ..................................................... 32
4.7 Experimental design for IMO synthesis (IV) ..................................................... 32
5. Results....................................................................................................................... 34
5.1 Characterization of WcE392-rDSR (I) ............................................................... 34
7
5.2 Characterization of Weissella dextrans (I and II) ................................................ 35
5.3 Characterization of the oligosaccharides produced by dextransucrases.............. 38
5.3.1 Acceptor selectivity (III) ............................................................................... 38
5.3.2 Principal maltose product series (II) ............................................................. 38
5.3.3 Minor maltose product series (IV) ................................................................ 41
5.3.4 Lactose- and cellobiose-derived branched trisaccharides (III)...................... 42
5.3.5 Novel sucrose-containing trisaccharide (III) ................................................. 46
5.3.6 Applicability of MSn analysis in the characterization of dextransucrasesynthesized GLOSs (III and IV) ................................................................... 48
5.4 Optimization of IMO synthesis by response surface modeling (IV)................... 52
6. Discussion ................................................................................................................. 57
6.1 Properties of Weissella dextransucrases and dextrans (I and II) ......................... 57
6.2 Specificity of Weissella dextransucrases in acceptor reactions ........................... 58
6.2.1 Comparison with commercial Lc. mesenteroides dextransucrase (III and
IV) ................................................................................................................. 58
6.2.2 Specificity to form an α-(1→2) branch (III and IV) ..................................... 60
6.2.3 Novel sucrose-containing trisaccharide (III) ................................................. 61
6.3 Potential of dextransucrases in prebiotic GLOS synthesis .................................. 62
6.3.1 Synthesis of prebiotic IMOs from maltose (IV)............................................ 62
6.3.2 Synthesis of other GLOSs (III) ..................................................................... 63
7. Conclusion ................................................................................................................. 65
References ..................................................................................................................... 67
8
1. INTRODUCTION
The lactic acid fermentation of food has been practiced worldwide for thousands of years
for improved preservability and flavors. Moreover, the oligo- and polysaccharides
secreted by lactic acid bacteria (LAB) can enhance the textural and nutritional properties
of the fermented foods (Seo DM et al., 2007; Welman 2009; Di Cagno et al., 2013; Cho
et al., 2014). The exopolysaccharides (EPS) produced in situ by LAB can avoid being
declared on food labels, because the LAB with a history of safe use have, for example,
GRAS (generally recognized as safe) or QPS (qualified presumption of safety) status in
the United States and the European Union, respectively (Waldherr and Vogel 2009).
Such EPS offer an attractive solution for producing additive-free foods, which are
increasingly pursued by today’s consumers. EPS-producing cultures have been applied
in fermented milks, vegetables, and fruits and in cheese and sourdoughs, where EPS
exert technological advantages, such as controlling syneresis; enhancing the thickness,
creaminess, and smoothness of yoghurt; and improving the dough rheology, bread
volume, and shelf life of sourdough breads (Di Cagno et al., 2006; Schwab et al., 2008;
Welman 2009; Katina et al., 2009; Galle et al., 2012). In addition to sensory benefits,
there is accumulated evidence on the health benefits of oligo- and polysaccharides from
LAB, including prebiotic, cholesterol-lowing, immunomodulatory, and anticarcinogenic effects (Schwab et al., 2008; Welman 2009; Ryan et al., 2015).
In addition to the in situ synthesis of oligo- and polysaccharides in fermented foods,
LAB have been used to produce α-glucans of industrial interest (Zannini et al., 2015;
Miao et al., 2016). For example, dextran produced by Leuconostoc mesenteroides was
one of the first biopolymers produced on an industrial scale and has various applications
in food, medicine, and separation technology as a food thickener, blood plasma expander,
and size-exclusion chromatography matrix, respectively. (Waldherr and Vogel 2009;
Zannini et al., 2015). Dextran was approved by the European Commission for use as a
novel food ingredient in bakery products for improved softness, crumb texture, and loaf
volume (Zannini et al., 2015). LAB produce α-glucans extracellularly via sucrose-acting
α-transglucosylases, e.g., dextransucrases. These enzymes are promising biosynthetic
tools because, unlike Leloir type-glycosyltransferases, they do not require energy-rich
sugar nucleotides as glycosyl donor substrates and can readily synthesize α-glucans from
a sole substrate sucrose (Monsan et al., 2010). In addition to polymers, these enzymes
can also synthesize, e.g., α-glucooligosaccharides (GLOSs) in the presence of a wide
variety of acceptor sugars. These enzymes of LAB have high regio- and
stereospecificities compared to those of chemical approaches and have been used to
synthesize structurally diverse carbohydrates (Leemhuis et al., 2013; Vuillemin et al.,
2016). For example, Leuconostoc dextransucrases have been used to produce various
9
prebiotic GLOSs, e.g., isomaltooligosaccharides (IMOs) (Goffin et al., 2011; RuizMatute et al., 2011; García-Cayuela et al., 2014).
Weissella spp. are LAB frequently found in plant materials and fermented foods (Fusco
et al., 2015). They have drawn attention in recent years as efficient dextran producers
for food applications. In situ-produced Weissella dextrans improve the shelf life, volume,
and softness of sourdough breads and the texture of fermented vegetable foods (Katina
et al., 2009; Galle et al., 2010; Juvonen et al., 2015). Weissella spp. have an advantage
over many Leuconostoc starters in that they do not lead to over-acidification in foods.
To facilitate the future use of this genus and its dextransucrases, it is important to
understand the structures and possible functions of its products. To date, only few
dextransucrases from W. confusa and W. cibaria have been characterized (Bounaix et
al., 2010; Amari et al., 2012; Bejar et al., 2013; Rao and Goyal 2013), and no Weissella
dextransucrase has been examined as a synthetic tool for, e.g., potential prebiotic GLOSs.
In this thesis, the dextransucrases from two efficient dextran-producing W. confusa
strains were studied, with one dextransucrase biochemically characterized (article I).
Their dextran (articles I and II) and GLOS products (articles II‒IV) were structurally
analyzed, focusing on GLOSs produced in the presence of various acceptor sugars. The
reaction with the acceptor maltose was explored to synthesize prebiotic IMOs (article
IV).
10
2. REVIEW OF THE LITERATURE
2.1 Carbohydrates from lactic acid bacteria
Several LAB produce polysaccharides that are components of the cell wall or are
released from the cell. The latter can be divided into capsular polysaccharides (CPS) and
EPS (Zannini et al., 2015). Whereas CPS are permanently attached to the cell surface in
the form of capsules, EPS are secreted into the environment (Ruas-Madiedo et al., 2009).
EPS are involved in biofilm formation and have various physiological functions, such
as in surface adhesion and the protection of bacterial cells from environmental stress
(Zannini et al., 2015). EPS can be divided into two groups based on the mechanism of
biosynthesis and the precursors required (Boels et al., 2001). One group comprises
homopolysaccharides (HoPS), namely α-glucans and β-fructans, which are synthesized
extracellularly by glucansucrases (glycoside hydrolase family 70) and fructansucrases
(glycoside hydrolase family 68), respectively. Both enzymes use sucrose as a substrate,
while the latter can also use, e.g., raffinose to synthesize β-fructan in the absence of
sucrose. The other group includes β-glucans (HoPS) and heteropolysaccharides (HePS).
Their synthesis requires sugar nucleotide precursors and several enzymes that are
involved in central carbon metabolism and in the synthesis of some cell-wall
components (Zannini et al., 2015).
Sucrose-derived HoPS are synthesized extracellularly by the action of glycansucrases of
certain LAB, such as Leuconostoc, Lactobacillus, Streptococcus, and Weissella (Zannini
et al., 2015). One bacterium may harbor multiple glucansucrase and fructansucrase
genes (Olvera et al., 2007; Malik et al., 2009; Malang et al., 2015). When a strain secretes
multiple glucansucrases, these enzymes may act jointly to synthesize a hybrid polymer
that is different from that produced by purified enzyme (Côté and Skory 2015). The
hydrolysis of the glycosidic linkage of sucrose provides these glycansucrases with the
energy to form a new glycosidic linkage in corresponding HoPS (Zannini et al., 2015).
Because the biosynthesis of these HoPS is less energy demanding than that of HePS,
they can be produced in larger amounts. According to the type of glucosidic linkages, αglucans are classified into dextrans, mutans, alternans, and reuterans (Leemhuis et al.,
2013). Dextrans contain mainly α-(1→6) linkages and α-(1→2), α-(1→3), or α-(1→4)
branch linkages, mutans mainly α-(1→3) linkages, reuterans mainly α-(1→4) linkages,
and alternans contain alternating α-(1→6) and α-(1→3) linkages (Naessens et al., 2005;
Bounaix et al., 2009). β-Fructans are categorized into levans, with mainly β-(2→6)
linkages, and inulins, with mainly β-(2→1) linkages (Zannini et al., 2015). In addition
to polymeric glucans and fructans, LAB can also synthesize oligosaccharides, such as
11
isomaltooligosaccharides and fructooligosaccharides. The size and yield of carbohydrate
products depend on the strain and fermentation conditions (Jeanes et al., 1954; Schwab
et al., 2008; Anwar et al., 2010; Malang et al., 2015).
2.2 Glucansucrases
Because of the high interest in the carbohydrates of LAB origin for food and non-food
applications, their synthesis machineries have been extensively explored, and a large
number of synthesizing enzymes have been identified and characterized. Glucansucrases
(EC 2.4.1.5) are produced extracellularly by LAB to synthesize α-glucans (Leemhuis et
al., 2013). They are named based on the α-glucans that they synthesize, i.e.,
dextransucrases, mutansucrases, alternansucrases and reuteransucrases. Although
glucansucrases are transferases with respect to their activity, they are structurally close
to glycoside hydrolases and are classified as part of the glycoside hydrolase family 70
(GH70), which contains mainly glucansucrases (Leemhuis et al., 2013). The recently
characterized 4,6-α-glucanotransferases, which are inactive on sucrose but convert
starch or starch hydrolysates into isomalto/maltopolysaccharides, constitute a new
subfamily of GH70 (Bai et al., 2015). Moreover, by sequencing the genomes of the
producers of original α-glucans, a cluster of branching sucrases specialized in dextran
grafting through α-(1→2) or α-(1→3) linkages have been recently discovered (Passerini
et al., 2015; Vuillemin et al., 2016). GH70 enzymes have been found almost exclusively
in LAB according to the entries in the Carbohydrate-Active Enzymes (CAZy) database
(Lombard et al., 2014). So far, the encoding genes of more than three hundred GH70
enzymes have been cloned and sequenced, and the three-dimensional structures of four
truncated enzymes have been solved (Vujicic-Zagar et al., 2010; Ito et al., 2011; Brison
et al., 2012; Pijning et al., 2012). The GH70 enzymes are mechanistically, structurally,
and evolutionary closely related to GH13 (α-amylases) and GH77 (amylomaltases)
enzymes. The three families containing a catalytic (β/α)8-barrel domain form the GH-H
clan (Stam et al., 2006).
2.2.1 Catalytic mechanism of glucansucrases
The catalysis of glucansucrases is proposed to be a two-step process, starting with the
cleavage of the substrate sucrose and followed by the formation of a covalent β-glucosylenzyme intermediate and the release of a free fructose (Figure 1). In the second step, the
glucosyl moiety is transferred to an acceptor, retaining the α-anomeric configuration
(Leemhuis et al., 2013). Hydrolysis takes place when a water molecule acts as the
acceptor. In the presence of suitable low-molar-mass molecules, e.g., maltose,
12
glucansucrases catalyze so-called acceptor reactions, where the glucosyl residue from
sucrose is diverted from α-glucan formation and transferred onto the small molecule
instead (Figure 1). Di- and oligosaccharides are formed when carbohydrates act as the
acceptor molecules, whereas glucoconjugates are formed in the presence of noncarbohydrate compounds (Monsan et al., 2010).
H2O
hydrolysis
fructose
R-OH
acceptor
reaction
covalent glucosylenzyme intermediate
dextran
synthesis
Figure 1. Types of reactions catalyzed by dextransucrases.
The three-dimensional structures of truncated glucansucrases revealed that they exhibit
a U-fold shape, organized into five (A, B, C, IV, and V) domains (Vujicic-Zagar et al.,
2010; Ito et al., 2011; Brison et al., 2012; Pijning et al., 2012). Except for domain C at
the base of the U-fold, other domains are formed by discontiguous N- and C-terminal
segments of the polypeptide, as illustrated by the glucansucrase of Lactobacillus reuteri
180 with its N-terminal end (~740 residues) truncated (Figure 2). Domains A, B, and C
resemble family GH13 enzymes, whereas domains IV and V are unique in GH70
enzymes. The catalytic domain A adopts a (β/α)8-barrel fold and harbors a catalytic triad,
which is composed of two aspartates (acting separately as a nucleophile and a transitionstate stabilizer) and one glutamate (acting as an acid/base catalyst) (Leemhuis et al.,
2013). The acceptor binding site is shaped by residues from domains A and B.
Mutagenesis studies have shown that the interplay of these residues determines how a
growing glucan chain interacts with the acceptor binding site (which hydroxyl group of
a glucosyl residue is capable of attacking the glucosyl-enzyme intermediate to form a
glycosidic linkage) and thus the proportion of specific linkages in the glucan synthesized
13
(Irague et al., 2013; Leemhuis et al., 2013; Meng et al., 2015a; Meng et al., 2015c).
Domain V is part of the glucan binding domain consisting of N- and C-terminal
segments, while domain IV may provide a hinge that brings the glucan-bound domain
V toward or away from the catalytic site to facilitate the synthesis (Leemhuis et al., 2013;
Meng et al., 2015b).
Figure 2. Crystal structure of an N-terminus-truncated dextransucrase from Lactobacillus reuteri
180 and schematic presentation of the U-fold shape of the polypeptide chain (adapted from
Vujicic-Zagar et al., 2010).
Moulis and others (2006b) proposed a semi-processive mechanism for the
polymerization of glucans. There is a lag-phase before glucan synthesis starts, when
sucrose and glucose (produced by hydrolysis) act as initiators of polymerization.
Glucose is the preferred initiator when present in sufficient amounts. Elongation occurs
through the non-processive (the product is released after each elongation step of the
acceptor) attachment of glucosyl moieties to the nonreducing end of the initially formed
products. The longer oligosaccharides are better acceptors than are sucrose, fructose,
and glucose, ensuring that the glucosyl moieties are used to synthesize glucans instead
of large amounts of oligosaccharides. Once a glucan chain reaches a minimal size, it
may remain attached to the enzyme, and the elongation becomes processive. The glucanbinding domain is involved in the processive mechanism, as shown by a gradual loss of
elongation efficiency (a decrease in the ratio of high-/low-molar-mass population) as the
domain is progressively deleted (Moulis et al., 2006b).
14
2.2.2 Production and characterization of dextransucrases
Among LAB, the expression of the dextransucrase-encoding gene is usually sucroseinducible in wild-type strains of Leuconostoc spp. but constitutive in, e.g., Streptococcus
spp. and some Lactobacillus strains (Naessens et al., 2005; Bounaix et al., 2010). The
constitutive expression of the glucansucrase-encoding gene is suggested to be
advantageous in ecosystems with fluctuating sucrose concentrations, such as the oral
cavity (Leemhuis et al., 2013). Dextransucrase production is affected by temperature,
aeration and medium composition, and methods such as response surface methodology
have been used to optimize these culture conditions (Purama and Goyal 2008a). Native
dextransucrases from different LAB have been purified by, e.g., salt, alcohol
precipitation, polyethylene glycol (PEG) fractionation, ultrafiltration, and
chromatography (Naessens et al., 2005; Purama and Goyal 2008b). PEG fractionation
can separate dextransucrases from contaminating enzymes, such as levansucrase;
however, dextran remains in the preparation (Majumder et al., 2007). Because the
induced production of dextransucrase leads to the concomitant production of dextran,
which can envelop the enzyme and hinder the enzyme characterization (Moulis et al.,
2006a), it is desired to produce dextransucrases by the constitutive mutants or
heterologous hosts, where the expression of the dextransucrase-encoding gene does not
require sucrose induction and therefore no dextran is produced concomitantly. The
dextran present, however, stabilizes dextransucrase, and so does PEG and Tween 80
(Majumder et al., 2007). The presence of Ca2+ is essential for dextransucrase activity
(Naessens et al., 2005). The Ca2+-binding site is located at the interface between domains
A and B (Figure 2). The absence of Ca2+ may lead to a compromised substrate binding
and thus a decreased activity (Vujicic-Zagar et al., 2010). Dextransucrases normally
have a pH optimum of 5.0‒5.5 and a Michaelis-Menten constant (KM) for sucrose of
approximately 12‒16 mM (Naessens et al., 2005).
Several assays have been used for dextransucrase activity measurement (Vettori et al.,
2011). They can be divided into two groups. One group is based on the measurement of
the fructose released, which represents sucrose consumption (from both hydrolytic and
transferase reactions). The hydrolytic activity, which is usually low, can be determined
by quantifying the free glucose released. Reducing-value assays are based on an increase
in the reducing value in the reaction mixture (mostly resulting from the fructose released
in dextran formation) and are normally used for dextransucrase activity assays, although
the dextran product itself and small amounts of side products, i.e., leucrose (α-D-Glcp(1→5)-D-Frup, an acceptor product of fructose), glucose and fructose resulting from
hydrolysis, also contribute to the increased reducing value. The commonly used
reducing-value assays include the 3,5-dinitrosalicylic acid (DNS) method (Sumner and
15
Howell 1935) and the Nelson-Somogyi method (Nelson 1944; Somogyi 1945). The
other assay group is based on the quantification of dextran product by, e.g., its weight.
The 14C-sucrose radioisotope method has also been used (Vettori et al., 2011; Côté and
Skory 2012) and is based on the incorporation of 14C-labeled glucose from 14C-Ulabeled sucrose to the growing dextran chain. The second group provides a more
accurate measurement of dextransucrase activity (Germaine et al., 1974; Vettori et al.,
2011).
2.3 Carbohydrates synthesized by dextransucrases
Glucansucrases are useful synthetic tools for various carbohydrates and glucoconjugates
because of their high regioselectivity in glucosyl transfer reactions, relaxed specificity
toward a variety of acceptors, and low cost of the donor substrate sucrose (Seibel et al.,
2010). Moreover, glucansucrases have been used to improve the flavor, water solubility,
and oxidative stability of various compounds for food, cosmetic, and therapeutic
applications (Côté et al., 2009; Kim et al., 2012; Liang et al., 2016).
2.3.1 Substrate and product specificity of dextransucrases
Although glucansucrases are highly specific towards sucrose, certain other
carbohydrates can also serve as glycosyl donors (Jung and Mayer 1981; Seto et al., 2008;
Timm et al., 2013). Especially, the transfer of a different glycosyl residue than a glucosyl
one indicates broader applications of glucansucrases in carbohydrate bioconversion. For
example, carbonyl-group-containing dextran was synthesized using keto-sucrose at C-2
by dextransucrase (Seto et al., 2008). Similarly, a reuteransucrase was used to transfer
an allosyl (differs in C3 configuration from glucosyl) group to several acceptors with αD-allopyranosyl β-D-fructofuranoside as a donor substrate (Timm et al., 2013).
The binding kinetics between Lc. mesenteroides dextransucrase and immobilized
glucose, maltose, and dextran were determined using a quartz crystal microbalance
(Nihira et al., 2011; Fang et al., 2015). The dissociation constants for glucose, maltose,
and dextran were 64, 35, and 18 nM, respectively, indicating an increasing order of
specificity between the enzyme and these acceptors. The effectiveness of different
acceptors was compared for Lc. mesenteroides dextransucrases at a sucrose/acceptor
ratio of 1:1 (Robyt and Eklund 1983; Kothari and Goyal 2013). Among the acceptor
sugars tested in both studies, maltose and, to a lesser extent, isomaltose were the best
acceptors; D-glucose, lactose, and cellobiose were moderately effective; and raffinose,
D-galactose, and D-xylose were poor acceptors. The more effective acceptors can
16
generally be further glucosylated into a series of oligosaccharides and suppress dextran
formation to a higher degree (Robyt and Eklund 1983; Kothari and Goyal 2013).
Dextransucrases are able to form mainly α-(1→6) linkages and, to a lesser extent, α(1→2), α-(1→3), or α-(1→4) linkages (Leemhuis et al., 2013). It has been proposed that
the linkage specificity depends on the orientation, in which the acceptor is guided toward
the reaction center and is therefore determined by the conformation of the acceptor
binding cleft (Leemhuis et al., 2013). Amino acid residues forming acceptor binding
subsites affect dextransucrase specificity, as mutations of targeted residues were shown
to alter the linkage composition of the product (Brison et al., 2012; Leemhuis et al., 2013;
Meng et al., 2015c). Many dextransucrases catalyze the formation of more than one type
of glucosidic linkage, requiring different binding modes of an acceptor. Thus, the
acceptor binding cleft needs to be wide enough to accommodate the acceptor in different
orientations. For example, the structure of a truncated dextransucrase from Lactobacillus
reuteri 180, which forms both α-(1→6) and α-(1→3) linkages, shows a rather wide
binding cleft. However, a unique branching sucrase derived from Leuconostoc citreum
NRRL B-1299 (formerly known as Leuconostoc mesenteroides NRRL B-1299)
dextransucrase only has α-(1→2) branching activity, and correspondingly, its binding
cleft is partially blocked, allowing only one acceptor binding mode (Brison et al., 2012;
Leemhuis et al., 2013).
In acceptor reactions, the product pattern for a given acceptor depends on dextransucrase
specificity. Maltose is the most studied acceptor due to its high effectiveness, and the
patterns of oligosaccharide products of maltose acceptor reactions corresponded to the
structure of the dextran produced by the same strain (Côté and Leathers 2005). For
example, the typical dextransucrases that predominantly synthesize α-(1→6) linkages,
represented by the commercially used Leuconostoc mesenteroides NRRL B-512F
dextransucrase (DSR-S), produce homologous oligosaccharides in the presence of
maltose (Robyt and Eklund 1983). The initial product, panose (α-D-Glcp-(1→6)-α-DGlcp-(1→4)-D-Glcp), is elongated by the successive attachment of glucosyl residues to
the 6-OH of the nonreducing end glucose, forming linear isomaltooligosaccharides
(IMOs), as illustrated by series A in Figure 3. For Lc. citreum NRRL B-1299
dextransucrase synthesizing α-(1→2)-branched dextran, however, the maltose acceptor
products are three homologous IMO series, with the major series constituting linear
IMOs of series A (Figure 3). The two minor series (marked B and C in Figure 3) have
α-(1→6)-linked main chains with a single-unit branch α-(1→2) attached to the terminal
and the penultimate α-(1→6)-linked residue of the nonreducing end, respectively (Dols
et al., 1998).
17
Figure 3. Oligosaccharide series (named A, B, and C) produced in the maltose acceptor reaction
of dextransucrases. Series A are produced by Lc. mesenteroides NRRL B-512F and Lc. citreum
NRRL B-1299 dextransucrases, whereas series B and C are only produced by B-1299
dextransucrase (adapted from Dols et al., 1998).
In addition to this thesis work, GLOS products from different acceptors (Table 1) have
only been characterized for DSR-S from Lc. mesenteroides B-512F (Yamauchi and
Ohwada 1969; Robyt 1995; Demuth et al., 2002; Côté et al., 2009; Díez-Municio et al.,
2012a; Barea-Alvarez et al., 2014; Côté et al., 2014). The type of linkage that is formed
is a function of the acceptor structure. Surprisingly, α-(1→2)-linkage is formed on
lactose and cellobiose, even though this enzyme is known to synthesize α-(1→6) and α(1→3) linkages in dextran. In addition to 2Glc-α-D-Glcp-lactose, an unknown
trisaccharide has been reported for the lactose acceptor reaction (Díez-Municio et al.,
2012b). Controversial results have been reported regarding the main cellobiose product.
Instead of 21-α-D-Glcp-cellobiose, the enzyme from a dextransucrase-hyper-producing
Lc. mesenteroides B-512F constitutive mutant was reported to produce 22-α-D-Glcpcellobiose (Kim et al., 2010). However, both the wild-type and mutant enzymes formed
a minor trisaccharide: linear 62-α-D-Glcp-cellobiose (Argüello Morales et al., 2001; Kim
et al., 2010).
18
Table 1. Acceptor products formed by DSR-S from Lc. mesenteroides NRRL B-512F.
Acceptor
First acceptor producta
Reference
Maltose (α-D-Glcp-(1→4)-D-Glcp)
Panose*
Robyt 1995
Isomaltose (α-D-Glcp-(1→6)-D-Glcp)
Isomaltotriose*
"
Nigerose (α-D-Glcp-(1→3)-D-Glcp)
6 -α-D-Glcp-nigerose*
"
Methyl α-D-glucopyranoside
D-Glucose
Lactose (β-D-Galp-(1→4)-D-Glcp)
Cellobiose (β-D-Glcp-(1→4)-D-Glcp)
D-Fructose
Methyl α-isomaltoside*
Isomaltose*
2Glc-α-D-Glcp-lactose
21-α-D-Glcp-cellobiose*
Leucrose
(α-D-Glcp-(1→5)-D-Frup)
Isomaltulose
(α-D-Glcp-(1→6)-D-Fruf)
α-D-Glcp-(1→1)-β-D-Galf
63-α-D-Glcp-maltotriose*
61-α-D-Glcp-maltotriose
64-α-D-Glcp-maltotetraose*
61-α-D-Glcp-maltotetraose
62-α-D-Glcp-gentiobiose
α-D-Glcp-(1→6)-D-Manp
α-D-Glcp-(1→1)-β-DManp
4Gal-α-D-Glcp-raffinose
"
"
"
"
"
"
"
Isomaltulose (α-D-Glcp-(1→6)-D-Fruf)
62-α-D-Glcp-isomaltulose*
Barea-Alvarez et al., 2014
Lactulose (β-D-Galp-(1→4)-D-Fruf)
β-D-Galp-(1→4)-β-D-Fruf(2→1)-α-D-Glcp
α-D-Glcp-(1→4)-β-D-Fruf(2→1)-α-D-Glcp
Díez-Municio et al., 2012b
D-Galactose
Maltotriose
Maltotetraose
Gentiobiose (β-D-Glcp-(1→6)-D-Glcp)
D-Mannose
Raffinose
(α-D-Galp-(1→6)-α-D-Glcp(1→2)-β-D-Fruf)
Maltulose (α-D-Glcp-(1→4)-D-Fruf)
2
"
Yamauchi and Ohwada 1969
Côté et al., 2014
Robyt 1995
Côté et al., 2009
Demuth et al., 2002
The starred product is the first member of a homologous series with α-isomaltooligosaccharides attached to the acceptor.
a
2.3.2 Functional oligosaccharides from dextransucrases
Glucansucrase-catalyzed synthesis provides access to a large range of bioactive
glucooligosaccharides (GLOSs), particularly non-digestible oligosaccharides with
prebiotic effects (Monsan et al., 2010). The use of prebiotics is gaining popularity
because, compared to probiotics, they are resistant to the aggressive gastro-intestinal
environment and cheaper and easier to incorporate into the diet (Goffin et al., 2011; Al19
Sheraji et al., 2013). Prebiotics are currently defined as the dietary components that
selectively stimulate the growth and/or activity(ies) of one or a limited number of
microbial genus(era)/species in the gut microbiota and thus confer health benefits to the
host (Roberfroid et al., 2010). The acceptor reactions of glucansucrases, especially
dextransucrases, from Leuconostoc spp. have been used to synthesize functional GLOSs
(Chung and Day 2002; Seo DM et al., 2007; Holt et al., 2008; Goffin et al., 2011; Kothari
and Goyal 2015). The GLOSs derived from maltose, lactose, lactulose, and cellobiose
by DSR-S from Lc. mesenteroides NRRL B-512F have shown prebiotic potential in vitro
and for maltose products also in vivo (Argüello Morales et al., 2001; Ruiz-Matute et al.,
2011; Díez-Municio et al., 2012b; García-Cayuela et al., 2014). GLOS products of
Leuconostoc dextransucrases have also shown other health benefits, e.g., preventing the
installation of type 2 diabetes, preventing dental caries, and inhibiting cancer cells (Kim
et al., 2010; Monsan et al., 2010; Kothari and Goyal 2015).
Among the GLOSs from dextransucrases, maltose-derived isomaltooligosaccharides
(IMOs) represent the most commercialized prebiotic products. In particular, the IMOs
containing α-(1→2)-linkages from Lc. citreum NRRL B-1299 dextransucrase are
marketed as prebiotics for their low digestibility and produced on an industrial scale of
40 metric tons/year (Plou et al., 2002). The linear IMOs with higher degrees of
polymerization (DPs) also demonstrated prebiotic effects in humans and animals (Goffin
et al., 2011; Yen et al., 2011; Ketabi et al., 2011). In fact, IMOs of DP 3.3 were fermented
in the large intestine of rats, while IMOs of DP 2.7 were mainly digested and absorbed
in the small intestine, without reaching the large intestine (Iwaya et al., 2012). However,
commercial IMOs usually contain a high proportion of digestible carbohydrates, which
decreases their functionality as prebiotics (Hu et al., 2013). The industrial IMO
production includes the enzymatic hydrolysis of starch into high maltose syrup, followed
by the action of α-transglucosidase, which catalyzes the transfer of the nonreducing
glucosyl residue of maltose to the 6-OH group of glucose (released by hydrolysis), the
nonreducing glucosyl unit of maltose or any α-glucooligosaccharide present in the
solution (Goffin et al., 2011). Isomaltose, panose and isomaltotriose are the main
components in the IMOs thus produced. The dextransucrase acceptor reaction holds
promise for the size-controlled synthesis of IMOs by varying concentrations of sucrose
(donor) and maltose (acceptor). With an increase in the sucrose/maltose ratio, the DP of
both the major and the longest products increased (Su and Robyt 1993; Lee et al., 1997;
Lee et al., 2008). However, detailed studies are needed to select the optimal synthesis
conditions for IMOs of higher DP.
20
2.3.3 Characterization of dextransucrase-synthesized carbohydrates
Knowledge of the structure of dextransucrase-synthesized products is essential for the
application of enzymes and the dextran and oligosaccharide products. Typical strategies
for the structural characterization of dextrans and oligosaccharides are presented in
Figure 4. Extracellular water-soluble glucans can simply be purified by the solvent
(normally ethanol) precipitation of the cell-free culture and dialysis against water when
no severe interfering compound, e.g., protein is present (Ruas-Madiedo and de los
Reyes-Gavilán 2005). The molar mass of dextrans can be determined by, e.g.,
ultracentrifugation, high-performance size-exclusion chromatography (HPSEC), and
asymmetric flow field-flow fractionation (AsFlFFF) (Irague et al., 2012; Maina et al.,
2014). HPSEC is widely used to analyze the molar mass distribution based on the
separation of molecules by hydrodynamic radius (Pitkänen 2011). Weight-average
molar mass (Mw) can be determined absolutely by HPSEC using universal calibration
(applying refractive index and viscosity detectors) or static light scattering detection. A
wide range of Mw (106‒109 g/mol) has been reported for native dextrans; however,
discrepancy can arise from the different analysis methods applied (Maina 2012). DMSO
was reported to be the solvent of choice for dextran Mw determination by HPSEC, as
aggregate formation was less prevalent in DMSO than in aqueous solution. Moreover,
the degradation of dextrans due to shear forces during HPSEC analysis was not
significant in DMSO (Maina et al., 2014). High-molar-mass dextrans with low-branched
structures have applications in food industries as hydrocolloids (Lacaze et al., 2007;
Waldherr and Vogel 2009).
The monosaccharide compositions of polysaccharides can be analyzed after total
depolymerization by acid methanolysis or acid hydrolysis (Leemhuis et al., 2013). The
monomers released are analyzed by gas chromatography (GC) or high-performance
liquid chromatography (HPLC). High-performance anion-exchange chromatography
with pulsed amperometric detection (HPAEC-PAD) is one of the most frequently used
techniques for mono- and oligosaccharide analysis. It exempts derivatization and affords
sensitive detection. Carbohydrates are readily separated by HPAEC under alkaline
conditions, where retention increases with decreasing pKa (Corradini et al., 2012). For
structural elucidation, large α-glucans often need to be degraded into the constituting
oligosaccharides by partial acid hydrolysis or specific enzymes. The fragments are then
analyzed by, e.g., thin-layer chromatography, HPLC, or NMR spectroscopy, and the
structure and abundance of these building blocks are used to build a composite polymer
structure (Remaud-Slmeon et al., 1994; van Leeuwen et al., 2008b; Vettori et al., 2012).
Enzyme-aided methods are useful for fingerprinting the type of branch linkages in
different dextrans (Sawai et al., 1978; Maina 2012). One method involves the specific
21
hydrolysis of dextran with a mixture of dextranase and α-glucosidase. The
chromatographic profiles of enzyme-resistant oligosaccharides vary depending on the
branch points of dextran and can therefore be used as structural markers (Maina 2012).
Glucansucrase or LAB
Glucan
Partial hydrolysis
Profiling by e.g.
HPAEC-PAD
Mw determination
by e.g. HPSEC
Detailed information
by NMR spectroscopy
Monosaccharide
composition
Oligosaccharides
Isolated
oligosaccharides
Linkage sequence
by ESI-MSn
Linkage ratio by
methylation
Figure 4. Commonly used strategies and techniques for the structural analysis of products from
glucansucrases.
The position of linkages between monosaccharide units in oligo- and polysaccharide can
be determined by methylation analysis. In this method, all of the free hydroxyl groups
in a carbohydrate are tagged by methylation prior to hydrolysis, after which the
monomers are reduced and derivatized for gas chromatography-mass spectrometry (GCMS) analysis (Naessens et al., 2005). Nuclear magnetic resonance (NMR) spectroscopy
is the most powerful technique for the structure elucidation of oligosaccharides and has
been intensively used for characterizing glucansucrase-synthesized carbohydrates (Côté
and Skory 2012; Díez-Municio et al., 2012a; Barea-Alvarez et al., 2014). It can provide
all of the information about the type of constituent monosaccharides, including ring size
and anomeric configuration, and the sequence of glycosidic linkages (Bubb 2003).
Combinations of two-dimensional 1H and 13C techniques have been developed, such as
homonuclear (1H-1H) COSY and TOCSY experiments for proton chemical shifts of each
residue and heteronuclear (1H-13C) HSQC and HMBC experiments for substitution
information (Bubb 2003). The primary structures of α-glucans have been characterized
based on the 1H NMR structural-reporter-group concept (van Leeuwen et al., 2008a; van
Leeuwen et al., 2008b).
22
Multistage mass spectrometry (MSn) has proven valuable in the sequential analysis of
the glycosidic linkages in oligosaccharides (Usui et al., 2009; Mischnick 2012; Maina
et al., 2013). Matrix-assisted laser desorption/ionization (MALDI) and electrospray
ionization (ESI) are the main ionization techniques used, with the latter coupling MS
with liquid chromatography (Harvey 2012). In negative ion MSn, C-ions (Figure 5) are
predominantly produced due to glycosidic cleavage, whereas isomeric C- and Y-ions
are produced in positive ion MSn. Negative ion MSn is preferably used for the structural
analysis of an underivatized linear oligosaccharide (Maina et al., 2013). In each MSn
stage, the cross-ring cleavages at the reducing end residue of the isolated C-ion are
diagnostic of the reducing end linkage. The linkage sequence of an unknown
oligosaccharide can be deduced by comparing its fragmentation behaviors in MSn
against a library of model compounds (Mischnick 2012). Unlike NMR spectroscopy
analysis, which requires purified carbohydrates, MS coupled with liquid
chromatography provides an integrated strategy for the rapid characterization of
oligosaccharides in mixtures (Wuhrer et al., 2009).
Figure 5. Nomenclature of fragment ions of cellotriose according to Domon and Costello (1988).
2.4 Weissella spp. and Weissella dextransucrases
The genus Weissella was proposed in 1993 after the reclassification of some
Leuconostoc-like bacteria (Collins et al., 1993). Weissella are Gram-positive, catalasenegative, non-endospore-forming, coccoid or rod-shaped heterofermentative LAB from
the family Leuconostocaceae (Fusco et al., 2015). Dextran production is a phenotypic
criterion for the identification of bacteria in this genus. Weissella strains have been found
in a wide range of habitats, such as plants and vegetables and the gastrointestinal tract
of animals and humans (Fusco et al., 2015). Some strains of W. confusa and W. cibaria
species have been suggested to be probiotics (Ayeni et al., 2011; Lee et al., 2012).
Although W. confusa has rarely been described as a human opportunistic pathogen,
infections usually occur in patients with immunosuppression or underlying diseases
(Fusco et al., 2015).
23
Weissella spp. are among the most frequently occurring LAB in raw and spontaneously
fermented cereals, vegetables, and fruits. They have adapted to the specific plant matrix
and usually lead to desired and predictable changes in the shelf life, nutritional and
sensory properties of the matrix (Di Cagno et al., 2013). In a study to modify the texture
of pureed carrots by fermentation with EPS-producing LAB, dextran-producing W.
confusa VTT E-90392, which was initially isolated from a sour carrot mash, conferred
the highest viscosity and desirable sensory profile (Juvonen et al., 2015). The highly
nutritious barley malt extract was fermented by W. cibaria MG1, resulting in a
functional alcohol-free beverage with desirable textural properties (Zannini et al., 2013).
Sourdough is a cocktail of LAB and yeasts originating from wheat or rye flour and is
used traditionally as a leavening agent in baking (Di Cagno et al., 2006). As common
sourdough isolates, the closely related W. confusa and W. cibaria are promising in
starting sourdoughs (Di Cagno et al., 2006; Katina et al., 2009; Galle et al., 2010). In
wheat sourdough, W. confusa VTT E-90392 synthesized a high yield of dextran, whereas
the commercial dextran-producing Leuconostoc mesenteroides NRRL B-512F formed
almost none under the same conditions. Unlike many Leuconostoc spp., Weissella spp.
produce dextran without extensive acidification, as they do not convert fructose into
mannitol, with concomitant acetate production (Galle et al., 2010). Therefore, the
technological benefits of Weissella dextran are not overridden by high acidity in
sourdough bread baking. The in situ-produced dextran improved the moisture retention,
dough rheology, bread volume and crumb softness and retarded staling (Di Cagno et al.,
2006; Schwab et al., 2008; Katina et al., 2009; Galle et al., 2010; Galle et al., 2012).
Moreover, W. confusa and W. cibaria also produced a significant amount of IMOs in
sourdoughs and thus provided extra health benefits (Schwab et al., 2008; Katina et al.,
2009; Galle et al., 2010). The amounts of IMOs and dextran in a W. confusa VTT E90392 sourdough were estimated to be 4.0% and 1.8% (both dry weight), respectively,
produced from 10% (dry weight) added sucrose (Maina 2012). The supplementation of
sucrose for Weissella dextran production also leads to free fructose production in
sourdough (5% fructose produced from 10% added sucrose), which does not comply to
the general diet advised to reduce sugar consumption. However, fructose leads to a lower
blood glucose increase compared to sucrose or glucose (European Food Safety Authority
(EFSA) 2011) and thus has a relatively small health implication.
Dextransucrases from Weissella spp. may also find various uses, e.g., to modify food
structure without concomitant acidification or for safety considerations compared to
whole-cell fermentation. The crude dextransucrase from pear-derived Weissella sp.
TN610 induced the solidification of sucrose-supplemented milk and was suggested as a
promising, safe food additive (Bejar et al., 2013). Several W. cibaria and W. confusa
24
isolates from sourdoughs were found to produce constitutive soluble dextransucrases, as
the enzyme activities were always higher when they grew on glucose than on sucrose
(Bounaix et al., 2010). However, for W. confusa Cab3 isolated from fermented cabbage,
a strain related to this thesis work, dextransucrase production was boosted with sucrose
(Shukla and Goyal 2011). In addition to this thesis, the biochemical and kinetic
properties of dextransucrases from two other Weissella strains have been recently
studied, with one being a native enzyme and the other being a recombinant one (Amari
et al., 2012; Rao and Goyal 2013). The gene encoding the latter (of strain LBAE C39-2)
was cloned and expressed in E. coli. An amino acid sequence alignment indicated that
Weissella dextransucrases form a distinct phylogenetic group within glucansucrases
(Amari et al., 2012). The two enzymes exhibited similar molar masses (approximately
180 kDa), pH optima (pH 5.4), thermal stabilities (inactivated rapidly above 35 °C), and
Michaelis-Menten kinetic parameters (KM and Vmax values of 8.6–13 mM and 20–27.5
µmol/(mg∙min), respectively), which were also comparable to the properties reported
for Leuconostoc dextransucrases (Naessens et al., 2005; Côté and Skory 2012). Of note,
the activity and thus the kinetic values of both enzymes were measured by conventional
reducing-value assays.
Dextrans from Weissella spp. share highly similar structures, normally consisting of
predominantly α-(1→6) linkages and only a few α-(1→3) branch linkages (<5%)
(Bounaix et al., 2009; Maina et al., 2011; Ahmed et al., 2012; Bejar et al., 2013).
Weissella dextrans show high molar masses of >106 g/mol (Ahmed et al., 2012; Maina
et al., 2014). In fact, the technological benefits of dextrans in sourdoughs are attributed
to their low degree of branching and high molar mass (Lacaze et al., 2007). Other than
the GLOSs formed in the presence of maltose (Katina et al., 2009), the acceptor products
of Weissella dextransucrases have not yet been investigated. Weissella spp. may form,
simultaneously with dextrans, byproducts from, e.g., lactose acceptor reaction when
endogenous sugars are present in food matrices. Furthermore, the enzymes may provide
a new opportunity for the biosynthesis of GLOSs of industrial interest via acceptor
reactions.
25
3. AIM OF THE STUDY
The overall aim of the thesis was to study the function and applicability of Weissella
dextransucrases, especially in the synthesis of glucooligosaccharides. Dextransucrases
from two efficient dextran-producing W. confusa strains were studied, namely, a native
enzyme from W. confusa Cab3 (WcCab3-DSR) and a recombinant one from W. confusa
VTT E-90392 (WcE392-rDSR), as no W. confusa dextransucrase had been studied in
detail by the time that this work started.
The specific objectives were to:
·
study the biochemical and kinetic properties of WcE392-rDSR and compare a
reducing-value assay with a 14C-sucrose radioisotope assay to determine these
parameters (article I)
·
structurally characterize the dextrans synthesized by the enzymes (articles I and
II)
·
explore the selectivity of Weissella dextransucrase towards various acceptor
sugars and structurally characterize the selected GLOSs (articles II‒IV)
·
apply Weissella dextransucrase for the synthesis of prebiotic IMOs (article IV)
26
4. MATERIALS AND METHODS
4.1 Dextransucrase preparations and commercial enzymes
Sources of Weissella dextransucrases and other enzymes used in this work are presented
in Table 2. The Weissella preparations are described in articles I and II. Briefly, the gene
encoding dextransucrase of W. confusa VTT E-90392 was cloned into Lactococcus
lactis NZ9800. The resultant recombinant enzyme WcE392-rDSR was purified by
nickel affinity chromatography or partially purified (the crude form) by ultrafiltering the
culture supernatant (article I). W. confusa Cab3 was previously isolated from fermented
cabbage, and WcCab3-DSR was partially purified by PEG fractionation (article II).
Table 2. Dextransucrase preparations and commercial enzymes used in the study.
Type
Name
Source
Article
Dextransucrase
Purified recombinant dextransucrase from
W. confusa E-90392 (WcE392-rDSR)
A crude preparation of WcE392-rDSR
Native dextransucrase from W. confusa
Cab3 (WcCab3-DSR)
Commercial dextransucrase from
Leuconostoc mesenteroides
Chaetomium erraticum dextranase
Aspergillus niger α-glucosidase
(E-TRNGL)
Bacillus stearothermophilus αglucosidase (E-TSAGL)
A. niger β-galactosidase
Agrobacterium sp. β-glucosidase
Yeast invertase (β-fructosidase)
VTT Technical Research
Centre of Finland Ltd.
VTT
Indian Institute of
Technology Guwahati
Sigma-Aldrich, MO
I
Sigma-Aldrich
Megazyme, Ireland
II
II, III
Megazyme
II
Megazyme
Megazyme
Megazyme
III
III
III
Hydrolytic
enzyme
I, III, IV
II, IV
IV
4.2 Biochemical characterization of WcE392-rDSR (I)
The protein concentration of the WcE392-rDSR preparation was determined by the DC
Protein Assay Kit (Bio-Rad, CA) with bovine serum albumin as the standard. The
activity of WcE392-rDSR was assayed by the 14C-sucrose radioisotope method and the
Nelson-Somogyi method (Nelson 1944; Somogyi 1945; Côté and Skory 2012). One
activity unit was defined as the amount of enzyme that catalyzed the incorporation of 1
27
µmol of D-glucose into dextran and the formation of 1 µmol of reducing sugar in 1 min,
respectively.
The Nelson-Somogyi method was used to determine the pH optimum, the effect of
temperature on the activity, and the thermal stability of WcE392-rDSR. Kinetic
parameters were determined by both the 14C-sucrose radioisotope and Nelson-Somogyi
methods, with the sucrose concentration ranging from 3 to 146 mM. The initial velocity
data were analyzed by nonlinear regression using GraphPad Prism 6 (GraphPad, CA).
4.3 Analysis of monosaccharides and oligosaccharides
HPAEC-PAD was used to analyze mono- and oligosaccharides. Prior to analysis, the
samples were filtered through a 10 kDa Amicon Ultra-0.5 centrifugal filter (Millipore,
MA) or a 0.45 μm membrane (Pall Corporation, NY). The instrument (unless otherwise
specified) was equipped with a CarboPac PA-100 column (250 × 4 mm, i.d, Dionex,
CA), a Decade detector (Antec Leyden, The Netherlands), a Waters 717 autosampler,
and two Waters 515 pumps. For a qualitative analysis of the oligosaccharide profiles,
the elution was performed with 100 mM NaOH (15 min), followed by a gradient to 120
mM Na-acetate in 100 mM NaOH (20 min) with a flow rate of 1 ml/min.
For the quantification of mono- and oligosaccharides, the same system was run by
another elution method, starting with 75 mM of NaOH (8 min) and followed by a
gradient to 100 mM of Na-acetate in 75 mM of NaOH (27 min) with a flow rate of 1
ml/min. In the case of the co-elution of lactose and sucrose, another HPAEC-PAD
system was used in article III. Glucose, fructose, galactose, maltose, sucrose, lactose,
cellobiose (Merck, Germany), leucrose (Santa Cruz Biotechnology, CA), isomaltose,
isomaltotriose, panose (TCI Europe nv, Belgium) and kojibiose (Carbosynth, UK) were
used as standards. Panose was also used as a standard for the quantification of total IMO
products (by integrating the total peak area) and individual IMOs of DP4‒6 in article IV,
as no reference compounds were available. Maltooligosaccharides of DP3‒6 (Sigma–
Aldrich, MO) were used in article IV to study the effect of DP on the elution time and
detector response factor for members of a homologous series. Oligosaccharides in
isolated fractions in article III were also separated by thin-layer chromatography (TLC)
(Côté and Leathers 2005) to confirm that the peaks detected by HPAEC-PAD consisted
of single compounds.
28
The total carbohydrate concentration in oligosaccharide and dextran solutions (in
articles III and IV) was determined by the phenol-sulfuric acid method (Dubois et al.,
1956).
4.4 Synthesis, isolation, and molar mass characterization of dextrans (I
and II)
Dextrans were synthesized in vitro by incubating 1.06 U/ml (Nelson-Somogyi units
unless otherwise specified) of WcE392-rDSR or WcCab3-DSR in 20 mM Na-acetate
buffer, pH 5.4, containing 2 mM CaCl2 and 146 mM sucrose at 35 °C for 24 h. After the
addition of two or three volumes of ethanol, the solution was centrifuged at 10,000 g
and 4 °C for 15 min. The re-dissolved dextran pellet was precipitated and re-dissolved
again. Dextrans were produced on agars by the native host W. confusa VTT E-90392 or
the heterologous host L. lactis and extracted according to Maina et al. (2008). The
dextrans were freeze-dried for characterization. The selective enzymatic hydrolysis and
NMR spectroscopy analysis of dextrans are described in following sections.
The molar mass of dextrans was determined with a high-performance size-exclusion
chromatography (HPSEC) multidetector system (Maina et al., 2014). Dextran solutions
of 2.0 mg/mL were prepared in dimethylsulfoxide (DMSO) with 10 mM LiBr. The
solutions were kept at room temperature for at least four days for dissolution and then
filtered using a 0.45 μm membrane filter before analysis. The system was equipped with
two analytical columns (exclusion limit >2 × 106) and a guard column. The HPSEC data
were processed with the Viscotek OmniSEC 4.5 software. A dη/dc value of 72 µL/g for
dextran in DMSO-based eluent was used (Basedow and Ebert 1979).
4.5 Acceptor reactions and isolation of oligosaccharides (II‒ IV)
To compare the specificity of WcE392-rDSR, WcCab3-DSR, and the commercial Lc.
mesenteroides dextransucrase, maltose acceptor reactions were carried out under similar
conditions (Table 3). WcE392-rDSR was also tested with several other di- and
trisaccharides as acceptors (Table 3). The reactions were conducted at 30 °C in 0.2 ml
of a 20 mM Na-acetate buffer (pH 5.4) containing 2 mM of CaCl2 for 24 h. The enzyme
action was terminated by incubation in a boiling water bath for 10 min. A
sucrose:acceptor molar ratio of 4 was used, except for the lactose reaction, where a
second major product was promoted with an increasing sucrose:lactose ratio. In article
IV, panose and a mixture of maltose trisaccharide products of a total carbohydrate
29
concentration of 1 mM were studied as acceptors in reactions using 5 mM of sucrose
and 5.5 U/g sucrose of WcE392-rDSR at room temperature for 24 h.
Table 3. Acceptor information and the initial concentrations of sucrose (Suc) and the acceptor
(Acc) in WcE392-rDSR-catalyzed (10 U/g of sucrose) reactions.
Acceptor
Structure
Supplier
Maltose
Lactose
Cellobiose
Laminaribiose
Nigerose
Melibiose
Isomaltose
Maltotriose
α-D-Glcp-(1→4)-D-Glcp
β-D-Galp-(1→4)-D-Glcp
β-D-Glcp-(1→4)-D-Glcp
β-D-Glcp-(1→3)-D-Glcp
α-D-Glcp-(1→3)-D-Glcp
α-D-Galp-(1→6)-D-Glcp
α-D-Glcp-(1→6)-D-Glcp
α-D-Glcp-(1→4)-α-DGlcp-(1→4)-D-Glcp
α-D-Glcp-(1→4)-[α-DGlcp-(1→6)]-D-Glcp
Merck, Germany
150
"
150
"
50
Megazyme, Ireland
50
Sigma-Aldrich, MO
50
"
50
TCI Europe nv, Belgium 25
Sigma-Aldrich
50
4
6.7
4
4
4
4
4
4
Carbosynth, UK
4
Forkosea
a
Acc
(mM)
50
Suc:Acc
molar ratio
Name according to Hansen et al. (2008).
A common series of oligosaccharides were synthesized by WcE392-rDSR and WcCab3DSR in the presence of maltose, as exhibited by their similar HPAEC-PAD profiles.
Thus, only the products from WcCab3-DSR were taken for structural characterization.
The synthesis was carried out in 100 ml using 292 mM sucrose, 146 mM maltose, and a
WcCab3-DSR dosage of 10 U/g sucrose. Moreover, to characterize the two main
products (named Lac3 and NR3) observed in the WcE392-rDSR lactose acceptor
reaction, the synthesis was carried out in 50 ml. The major product of the cellobiose
acceptor reaction (named Cel3) was also structurally characterized for comparison with
the product derived from analogous lactose. The cellobiose acceptor reaction was scaled
up to 50 ml.
After the reaction was stopped, two or three volumes of ethanol (Etax A, Altia
Corporation, Finland) were added to the reaction mixture, which was then centrifuged
to remove polymeric dextran. The supernatant containing fructose, residual acceptor,
and acceptor products was concentrated using a rotatory vacuum evaporator (Heidolph
Laborota 4001 Digital, Sigma-Aldrich, MO). Prior to injection into a Biogel P2 column
(5 × 95 cm; Bio-Rad, Hercules, CA), the concentrate was filtered through a 0.45 μm
membrane filter. Water was the eluent at a flow rate ≤0.7 ml/min. The oligosaccharides
30
in the collected fractions were analyzed by HPAEC-PAD. The fractions containing the
highest proportion of products of interest were used for a subsequent structural analysis.
4.6 Structural analysis of dextrans and oligosaccharides
4.6.1 Enzymatic hydrolysis of dextrans and oligosaccharides (II and III)
Dextransucrase products were selectively digested with specific enzymes for structural
information. The hydrolysis reactions are presented in Table 4. After enzyme
inactivation in a boiling water bath for 10 min, the digests were analyzed by HPAECPAD and compared to controls without enzyme treatment.
Table 4. Enzymatic hydrolysis conditions for dextran and oligosaccharide characterization.
Analyte
Enzyme dosagesa
Conditionb
Dextran
0.6 U/mg of C. erraticum dextranase and
0.06 U/mg of A. niger α-glucosidase
0.3 U/mg of C. erraticum dextranase or
0.04 U/mg of B. stearothermophilus α-glucosidase
25 U/mg of A. niger α-glucosidase or
50 U/mg of A. niger β-galactosidase
25 U/mg of A. niger α-glucosidase or
75 U/mg of Agrobacterium sp. β-glucosidase
25 U/mg of A. niger α-glucosidase,
200 U/mg of dextranase,
or 100 U/mg of yeast invertase
30 °C, 48 h
Maltose-derived
product series
Lactose-derived
trisaccharide (Lac3)
Cellobiose-derived
trisaccharide (Cel3)
Nonreducing
trisaccharide (NR3)
"
overnight at
30 °C
"
"
a
Enzyme dosages reference the amount of dextran, total carbohydrate in dextran-removed
maltose acceptor reaction mixture, or total carbohydrate in isolated product fractions for Lac3,
Cel3 and NR3.
b
Reactions were performed in Na-acetate or Na-citrate buffer at 20‒50 mM and pH 5.0‒5.5.
4.6.2 NMR spectroscopy analysis of dextrans and oligosaccharides (I‒IV)
NMR spectra were recorded with a 600 MHz Bruker Avance III NMR spectrometer
(Bruker BioSpin, Germany). The dextran (10 mg/ml) and isolated trisaccharides (>0.3
mg/ml) were exchanged with D2O (MagniSolv, Merck, Germany) and placed in NMR
tubes (Wilmad NMR tubes, 5 mm, ultra-imperial grade, Sigma-Aldrich, MO). The 1H
and the corresponding 2D (multiplicity-edited HSQC, DQFCOSY, TOCSY, and HMBC)
NMR experiments were performed at a temperature at which the residual water signal
did not affect the signals of the analyte. Topspin v.3.2 (Bruker, Germany) software was
31
used to process and analyze the NMR spectra. The chemical shifts of 1H (δH) and 13C
(δC) were referenced to acetone (δH 2.225 and δC 31.55, respectively).
4.6.3 MSn analysis of oligosaccharides (II‒IV)
Electrospray ionization multistage ion trap mass spectrometry (ESI-IT-MSn) was used
for the molar mass determination and characterization of the oligosaccharides isolated
by gel filtration. The analysis was carried out in negative or positive mode ([M+Cl]- and
[M+Li]+, respectively) using the Agilent XCT Plus Ion Trap mass spectrometer with an
electrospray ion source (Agilent Technologies, CA) or a Bruker Esquire LC quadrupole
ion trap mass spectrometer with an electrospray ion source (Bruker Daltonik GmbH,
Germany). A 1–10 µl sample (~0.5 mg/ml) was diluted in 200–400 µl of methanolwater-formic acid (50:49:1, v:v:v), and 0.5–1 µl of 10 mg/ml ammonium chloride or
lithium acetate was added for adduct ion formation. The samples were introduced to the
ion source with a direct infusion syringe pump at a flow rate of 5 µl/min. MS spectra
were processed with LC/MSD Trap Software (Bruker Daltonik GmbH). Cellotriose
(Megazyme) and panose (TCI Europe nv) were used as model compounds for the
characterization of Cel3 and BIM3 (the minor trisaccharide obtained in the maltose
acceptor reaction), respectively.
The maltose and lactose acceptor reactions were further carried out using [UL-13C6glc]
sucrose (Omicron Biochemicals Inc. IN) so that the GLOS products were 13C6glc-labeled
for ESI-IT-MSn analysis. The maltose reaction was conducted by incubating 0.3 U of
commercial Lc. mesenteroides dextransucrase in 0.5 ml of 20 mM Na-acetate buffer, pH
5.4, containing 0.1 M 13C6glc sucrose and 0.2 M maltose at 30 °C for 48 h. The GLOS
products were isolated by gel filtration. According to TLC analysis, the fraction
containing solely DP5 was selected for MSn analysis as [M+Cl]- and [M+Li]+ ions.
13C glc sucrose was also used to synthesize the novel trisaccharides (Lac3 and NR3)
6
identified in the lactose reaction mixture. The product mixture obtained was analyzed
by MSn to characterize the fragmentation patterns of the two labeled products (article
III).
4.7 Experimental design for IMO synthesis (IV)
The concentrations of sucrose, maltose, and WcE392-rDSR were selected as three
independent variables in the experimental design. Preliminary experiments were carried
out to select the following ranges of factors: sucrose (0.15–1 M), maltose (0.15–1 M),
and dextransucrase in relation to sucrose concentration (1–10 U/g sucrose). An
32
experimental series consisting of 18 experiments was designed to model the effects of
the factors on IMO synthesis. The reactions were conducted in 20 mM Na-acetate buffer,
pH 5.4, containing 2 mM CaCl2 at 30 °C for 24 h.
The yields of individual IMOs of DP3‒6, the yield of total IMOs, and the percent of
consumed maltose were each modelled by a quadratic regression model. Modde 9.0
(Umetrics AB, Sweden) was used to analyze the results by multiple linear regression
(MLR) or partial least squares (PLS) method and to generate contour plots. The validity
of the regression models was evaluated by the coefficient of determination (R2) and the
coefficient of prediction (Q2).
33
5. RESULTS
The dextransucrases from two efficient dextran-producing Weissella confusa strains (W.
confusa Cab3 isolated at IITG and W. confusa E-90392 from VTT Culture Collection)
were the subjects of this study. In the beginning, the dextransucrase from native W.
confusa E-90392 was produced and partially purified by PEG fractionation. However,
the yield was low and subsequent purification attempts failed to remove the
concomitantly produced dextran from the enzyme preparation (unpublished). Because
the presence of dextran is known to affect the enzyme characteristics, dextran-free
enzyme was obtained by cloning and expressing the dextransucrase gene in Lactococcus
lactis with a nisin-inducible expression system (others’ work). The recombinant W.
confusa E-90392 dextransucrase (WcE392-rDSR) was used in biochemical and kinetic
studies. Moreover, the dextransucrase from the other strain, W. confusa Cab3 (WcCab3DSR), was obtained with better yield by PEG fractionation and was used to synthesize
dextran and GLOSs in parallel with WcE392-rDSR to compare their product specificity.
5.1 Characterization of WcE392-rDSR (I)
The specific activity of the nickel-affinity-purified WcE392-rDSR was 7.2 ± 0.3 U/mg
and 18.9 ± 0.6 U/mg, as determined by the 14C-sucrose radioisotope assay and the
Nelson-Somogyi assay, respectively. According to the HPAEC-PAD analysis of the
reaction mixture after sucrose depletion, glucose and leucrose accounted for only 2%
and 5% (w/w) of the total mono- and disaccharides. Fructose represented the remaining
93%, indicating that the transferase reaction synthesizing dextran comprises the main
proportion of the WcE392-rDSR activity.
The Nelson-Somogyi assay was used to determine the effects of pH and temperature on
the activity. The optimal pH for WcE392-rDSR activity was observed at pH 5.4 in 20 M
Na-acetate buffer (Figure 6A). The rate of dextran synthesis increased with higher
temperatures up to 35 °C, after which it decreased (Figure 6A), as shown in the activity
assay (15 min) conducted at different temperatures. The thermal stability of WcE392rDSR was studied at 25, 35 and 40 °C in the presence or absence of glycerol up to 24 h
(Figure 6B). The enzyme stability decreased sharply when the temperature increased
from 35 °C (half-life of 26.3 h) to 40 °C (half-life of 7.4 h). Glycerol enhanced the
stability at each of the three temperatures; for example, it increased the enzyme half-life
at 35 °C from 26.3 to 37.5 h.
34
WcE392-rDSR exhibited Michaelis-Menten kinetics with a KM of 14.7 mM, a Vmax of
8.2 µmol/(mg∙min) and a kcat of 23.2 1/s, determined using the sucrose radioisotope assay.
The sucrose radioisotope assay and the Nelson-Somogyi assay gave similar KM values,
whereas values approximately 2.5 times higher were obtained for Vmax, kcat and kcat/KM
with the Nelson-Somogyi method (Table 5).
Table 5. Specific activities and kinetics of WcE392-rDSR (Table 5 in article I).
Assay
Activity
(U/mg)
KM
(mM)
Vmax
kcat
[µmol/(mg∙min)] (1/s)
kcat/KM
[1/(M∙s)]
14
7.2 ± 0.3
14.7 ± 2.3
8.2 ± 0.4
23.2 ± 1.1
(1.6 ± 0.3) × 103
18.9 ± 0.6
13.0 ± 1.1
19.9 ± 0.6
56.4 ± 1.7
(4.3 ± 0.5) × 103
C-Sucrose
radioisotope
Nelson-Somogyi
B
A
Figure 6. (A) Effects of pH and temperature on WcE392-rDSR activity. The effect of pH was
assayed in 20 mM Na-acetate buffer (pH 4‒6) or in 20 mM Tris-HCl buffer (pH 6.5‒7.0), and
the effect of temperature in 20 mM Na-acetate buffer, pH 5.4. Error bars represent the standard
errors of the mean (n=3). (B) The thermal stability of WcE392-rDSR in the presence and absence
of 0.5% (v/v) glycerol after incubation for 1, 2, 5 and 24 h. (Adapted from Figures 3 and 4 in
article I.)
5.2 Characterization of Weissella dextrans (I and II)
The dextrans synthesized in vitro by the two Weissella dextransucrases were isolated by
ethanol precipitation and lyophilized for enzyme-aided branch-type fingerprinting,
NMR spectroscopy and HPSEC analysis. For the fingerprinting of branch type, the
35
dextrans were hydrolyzed with a mixture of dextranase and α-glucosidase. The resulting
resistant oligosaccharides exhibited a similar HPAEC-PAD profile (Figure 7A for
WcCab3-DSR dextran, whereas that for WcE392-rDSR dextran is not shown) to those
of dextran extracted from W. confusa VTT E-90392 culture (Maina et al., 2011). Based
on the structure identified earlier for the in vivo-synthesized dextran (Maina et al., 2011),
the enzymatically produced dextrans were shown to contain α-(1→3)-linked branches,
with some branches elongated.
WcCab3-DSR dextran (Figure 7B) had a similar 1H NMR spectrum to that of the
dextrans produced by WcE392-rDSR and its heterologous host L. lactis (Figure S3 in
article I). Based on the relative intensities of the anomeric proton signals at 4.97 and
5.32 ppm, the ratio of α-(1→6) to α-(1→3) linkages was 97:3. The remaining proton
resonances at 3.97, 3.91, 3.77, 3.73, 3.58, and 3.52 ppm were assigned to H-6b, H-5, H6a, H-3, H-2, and H-4 of the α-(1→6)-linked glucopyranosyl residues, respectively. In
the edited HSQC NMR spectrum of WcCab3-DSR dextran (Figure 7C), a cross peak of
4.18→71.6 ppm was assigned to H-5→C-5 of the elongated branch point residue [(1→6)-α-D-Glcp-(1→3)-] (Maina et al., 2011), confirming the presence of branches
with more than one glucosyl unit.
The dextrans that were enzymatically synthesized and those produced in vivo by native
W. confusa VTT E-90392 and heterologous host L. lactis were analyzed by HPSEC. The
molar masses were obtained using DMSO with 10 mM LiBr as the solvent because
dextrans are prone to aggregation in aqueous solutions (Maina et al., 2014). One major
peak was observed with both refractive index (RI) and light scattering detectors (HPSEC
chromatograms not shown). The molar masses of dextrans were in the range of 1.8 to
8.5 × 107 g/mol.
36
Figure 7. (A) HPAEC-PAD chromatograms showing resistant oligosaccharides from WcCab3DSR dextran after dextranase and α-glucosidase treatment. 1H (B) and edited HSQC (C) NMR
spectra of WcCab3-DSR dextran. The spectra were recorded at 600 MHz in D2O at 50 °C. Peaks
in the NMR spectra were referenced to internal acetone (δH 2.225 ppm and δC 31.55 ppm).
(Adapted from Figures 3 and 4 in article II.)
37
5.3
Characterization
dextransucrases
of
the
oligosaccharides
produced
by
5.3.1 Acceptor selectivity (III)
The action of WcE392-rDSR on several glucose-containing di- and trisaccharides was
evaluated. In all of the reactions, a large amount of fructose was released, and dextran
was produced. Judging by the product peak area in the HPAEC-PAD chromatograms
(Figure S1 in article III), the more effective acceptors among those tested were maltose,
isomaltose, maltotriose, and nigerose. A series of products were detected in these
reaction mixtures, indicating that the acceptor products could be further elongated.
Lactose and cellobiose showed moderate effectiveness (section 5.3.4), whereas
melibiose, laminaribiose, and forkose were poor acceptors (data not shown) for
WcE392-rDSR.
5.3.2 Principal maltose product series (II)
The HPAEC-PAD profile of the glucooligosaccharides (GLOSs) produced in the
presence of maltose was similar between WcCab3-DSR, WcE392-rDSR, and the
commercial Lc. mesenteroides dextransucrase. According to the HPAEC-PAD
chromatogram of WcCab3-DSR products (Figure 8A, those of WcE392-rDSR and Lc.
mesenteroides dextransucrase are not shown), a number of GLOSs were produced, with
some being predominant and others being minor. After treatment with endo-acting
dextranase, most GLOSs eluting after panose were degraded into their building blocks,
i.e., glucose, isomaltose, isomaltotriose, and panose (Figure 8B), indicating that they
have consecutive α-(1→6) linked glucosyl residues attached to the nonreducing end of
the maltose and therefore belong to isomaltooligosaccharides (IMOs). As expected, the
GLOSs were resistant to the hydrolysis of an α-glucosidase exo-acting on α-(1→4)
linkages from the nonreducing end (article II).
The GLOSs that were produced in the WcCab3-DSR maltose reaction were isolated
according to DP by gel filtration (Figures 8C‒F). A HPAEC-PAD and MS analysis of
the fractions showed that for each principal IMO product, there was a minor product of
the same DP. The minor products eluted earlier than did the corresponding major
products (Figures 8C‒F) and seemed to constitute another series. The HPAEC retention
times of the two product series were plotted versus their DP (Figure S4 in article IV).
The plot for the minor series exhibited a similar slope to that of the principal IMOs but
distinct from that of the α-(1→4)-linked maltooligosaccharides. For GLOSs growing via
the same linkage, the slope (representing the peak interval between adjacent members
38
in the series) appeared to depend on the type of linkage. The HPAEC mobility result
indicated that the two series observed in the maltose reaction mixture were IMOs. This
was confirmed by the susceptibility of higher DP products of both series to dextranase
(Figure 8B). Because a minor DP4 product coeluted with panose in HPAEC, the
dextranase hydrolysate was further fractionated by gel filtration (data not shown). An
HPAEC-PAD analysis of the isolated DP4 fractions showed that the minor DP4
remained after dextranase hydrolysis. The members in principal series, supposedly
linear, were named after panose, LIM4 (DP4), LIM5 (DP5), and so on, while the minor
IMOs were suspected to be branched and named BIM3 (DP3), BIM4 (DP4), and so on.
Fru
DP4‒DP12
Mal
Pan
Glc
A
IM2
B
IM3
LIM6
C
BIM6
LIM5
D
BIM5
m/z 863
LIM4
E
BIM4
m/z 701
Pan
F
BIM3
0.00
5.00
10.00
15.00
20.00
25.00
m/z 539
30.00
35.00
40.00 min
Figure 8. HPAEC-PAD profiles of (A and B) acceptor products synthesized by WcCab3-DSR
from 5% (w/v) maltose and 10% (w/v) sucrose: (A) unhydrolyzed; (B) hydrolyzed by dextranase;
and (C‒F) selected fractions of isolated products by gel filtration. The m/z of DP3‒5 as [M+Cl]ions are shown. The principal product series are named Pan and LIM4‒6 and the minor series
BIM3‒6. Labeled peaks are: Glc, glucose; Fru, fructose; IM2, isomaltose; IM3, isomaltotriose;
Mal, maltose; and Pan, panose. (Adapted from Figure 4 and Figure 2 in articles I and IV,
respectively.)
To confirm the proposed structures for the principal series, negative ion MSn was used
to analyze isolated principal products of DP3‒5 ([M+Cl]- ions of m/z at 539, 701, and
863, respectively). The diagnostic fragment ions at m/z 425 and 383 (loss of 78 and 120
Da, respectively) and m/z 281, 251, and 221 (loss of 60, 90 and 120 Da, respectively) in
39
the MS2 spectrum of DP3 (not shown) confirmed the α-(1→4) linkage at its reducing
end and the α-(1→6) linkage at its nonreducing end, i.e., panose (Maina et al., 2013).
Similarly, in the MS2 spectrum of the [M+Cl]- ion of DP4 (Figure 9A), fragment ions at
m/z 587 and 545 (loss of 78 Da and 120 Da, respectively) indicated the presence of an
α-(1→4) linkage at the reducing end. The ions at m/z 443, 413, and 383 (loss of 60, 90,
and 120 Da, respectively) in the MS3 spectrum, starting with the ion at m/z 503, indicated
the subsequent α-(1→6) linkage (Figure 9B). The α-(1→6) linkage at the nonreducing
end was confirmed with the MS3 spectrum of the ion at m/z 341 (Figure 9C), which
contained fragment ions at m/z 281, 251, and 221 (loss of 60, 90, and 120 Da,
respectively). The MSn spectra of DP5 showed a similar trend (Figure 3S in article I):
an α-(1→4) linkage at its reducing end and the remaining linkages being α-(1→6). These
findings confirmed that the principal maltose products are homologous IMOs that
contain maltose at the reducing end.
Intens.
x10 6
A
C3
503
DP4 -MS2
1.0
0,4A
3
383
0.8
0.6
[M-H]665
C2
341
0,2A
3
0,2A
4-H2O
587
443
0.4
0,3A
0.2
2,4A
B3
485
3
413
[M+Cl]701
4
545
0.0
B
-MS3 m/z 503
443
8000
383
6000
413
4000
2000
179
503
323
281
485
0
5000
C
-MS3 m/z 341
4000
C1
179
3000
C2
341
2000
0,4A
1000
0,2A
2
281
221
B1
161
2
B2
323
0,3A
2
251
0
100
200
2
300
400
3
500
-
600
700
m/z
Figure 9. MS (A) and MS (B and C) spectra of the [M+Cl] ion of the principal maltose product
of DP4 (LIM4). The MS3 spectra in B and C are for the fragment ions at m/z 503 and 341,
respectively. (Adapted from Figure 5 in article I.)
40
5.3.3 Minor maltose product series (IV)
Because BIM3 of the minor series eluted slightly earlier than did panose in gel filtration,
few fractions collected before the elution of panose contained enriched BIM3 and were
selected for NMR spectroscopy and ESI-IT-MSn (section 5.3.6) analysis. The structure
of BIM3 was determined by a set of NMR experiments using a fraction containing an
approximately equimolar mixture of panose and BIM3. Pure panose was analyzed in
parallel so that the signals from panose could be subtracted from those of the mixture,
and the remaining signals were interpreted. Compared to the 1D 1H spectrum of panose,
five doublets in the anomeric proton region were found for BIM3 (Figure 10). The
integrals of the anomeric protons indicated that the trisaccharide was an anomeric
mixture, with the resonances of one nonreducing residue affected by mutarotation
equilibrium. The proton signals of each spin system were assigned using TOCSY and
DQFCOSY spectra (Figure S5 in article IV). The signals at δH 5.45 (d J1-2 3.4 Hz) and
δH 4.81 (d J1-2 8.0 Hz) were assigned to the α and β anomeric protons of the reducing
glucopyranosyl residue, respectively. The signal at δH 5.49 (d J1-2 3.5 Hz) corresponded
to the anomeric proton of the α-(1→4)-linked glucopyranosyl residue in maltose. The
other two anomeric signals at δH 5.10 (d J1-2 3.6 Hz) and δH 5.41 (d J1-2 3.9 Hz) were
assigned to a terminal α-(1→2)-linked glucopyranosyl residue in α and β anomers,
respectively (Figure 10).
Figure 10. 1D 1H NMR spectra of (A) pure panose and (B) a mixture containing equimolar
amounts of panose and BIM3. The spectra were measured at 600 MHz in D2O at 11 °C. The
structure of BIM3 is illustrated. (Adapted from Figure 4 in article IV.)
41
The downfield shift of C2 signals of the reducing residue in the edited HSQC spectrum
(Figure 4C and Table S3 in article IV) indicated that the third glucosyl residue was
attached to the C2 of maltose-reducing end, forming a branched structure. Direct
evidence for the α-(1→2) linkage was observed in the HMBC (Figure 4D in article IV)
correlation peaks: between GlcI-C2 (δC 76.76) and GlcIII-H1 (δH 5.10) and between
GlcIII-C1 (δC 97.41) and GlcI-H2 (δH 3.67) for the α-anomer and between GlcI-C2 (δC
79.52) and GlcIII-H1(δH 5.41) and between GlcIII-C1 (δC 98.83) and GlcI-H2 (δH 3.41)
for the β-anomer. Thus, BIM3 was identified as α-D-Glcp-(1→2)-[α-D-Glcp-(1→4)]D-Glcp, also known as centose.
To understand the structures and synthesis mechanism of the higher homologues of the
minor series, BIM3 (in a fraction containing an equal molarity of BIM3 and panose) was
tested as an acceptor. The HPAEC-PAD analysis before and after the reaction showed
that BIM3 did not act as an acceptor (data not shown). Thus, the higher DP minor
products contained a single-unit α-(1→2)-branch at the reducing end, and additional α(1→6)-linked residues in the main chain, as the α-(1→2)-linked branch was not able to
elongate further. Moreover, the minor series were proposed to be synthesized through
direct branch formation on an elongated linear IMO rather than through the elongation
of the main chain after branch formation. This mechanism was supported by the product
pattern observed using panose as a sole acceptor. A trace of BIM5 was formed (Figure
S6 in article IV) most probably by branch formation on LIM4. The branch may be
formed on panose as well, although the resulting BIM4 could not be directly detected in
the HPAEC analysis (as it was not well separated from panose).
5.3.4 Lactose- and cellobiose-derived branched trisaccharides (III)
The major products observed in the reaction mixtures of lactose and cellobiose (Figure
11A and 11B) were selected for structural analysis to determine whether the same type
of linkage was formed on the two analogs by Weissella dextransucrase. The products
derived from lactose and cellobiose were referred to as Lac3 and Cel3, respectively, as
they were later found to be trisaccharides. Moreover, in the lactose reactions with
varying sucrose and enzyme concentrations (Figure S2 in article III), a second product
was promoted when both concentrations increased. This product was also selected for
characterization and named NR3 (later found to be a nonreducing trisaccharide, Figure
11A). The two products from the WcE392-rDSR lactose reaction mixture and one from
the cellobiose reaction were isolated by gel filtration (Figure 11C). The selected Lac3
and Cel3 fractions contained negligible amounts of impurities, whereas the NR3 fraction
contained a minor portion of Lac3, in addition to other impurities. The three product
42
fractions were subjected to an ESI-IT-MS2 and NMR spectroscopy analysis and
enzymatic hydrolysis.
Lac3
C
Fraction Lac3
Isolated from lactose
product mixture
NR3
Fraction NR3
Cel3
Fraction Cel3
DP3#
Isolated from cellobiose
product mixture
Cel
5.00
10.00
15.00
20.00
25.00
30.00
35.00 min
Figure 11. HPAEC-PAD profiles of (A and B) acceptor product mixtures of WcE392-rDSR (10
U/g sucrose) with (A) 150 mM lactose and 1 M sucrose and (B) 50 mM cellobiose and 200 mM
sucrose and those of (C) selected product fractions isolated by gel filtration in order of elution.
The insets show the zoomed-in chromatograms of the product areas. Labeled peaks are: Glc,
glucose; Fru, fructose; Leu, leucrose; Lac, lactose; Cel, cellobiose. The peak DP3# was
presumed to be NR3. (Modified from Figure 1 in article III.)
43
The three isolated products were preliminarily analyzed by negative ion MS2. The three
products ([M+Cl]- ions) exhibited an m/z value of 539 and were therefore trisaccharides.
The MS2 spectra of [M+Cl]- ions of Lac3 and Cel3 were similar (Figure 12A), indicating
similar structures and thus fragmentation patterns. The relative abundance of the
fragment ions resulting from the cross-ring cleavage of the reducing-end residue was
different from that of cellotriose (data not shown) and other linear oligosaccharides
containing a β-(1→4)-linked reducing-end hexopyranose (Maina et al., 2013). This
suggests that Lac3 and Cel3 probably have a rare branched structure. The absence of
cross-ring fragment ions in the MS2 spectrum of the [M+Cl]- ion of NR3 (Figure 12A,
the ion at m/z 425 probably resulted from Lac3 present in the fraction) indicated that
NR3 is possibly a nonreducing trisaccharide.
The structures of Lac3 and Cel3 were determined by a set of NMR experiments. Their
anomeric proton regions in 1D 1H spectra (Figure 12B) were similar. Both showed six
anomeric proton signals representing two sets of resonances due to the effect of
mutarotation equilibrium on the two other residues. For example, the signals of Cel3 at
δH 5.44 (d J1-2 3.5 Hz) and δH 4.81 (d J1-2 8.0 Hz) corresponded to α- and β-anomeric
protons of the reducing glucopyranosyl residue, respectively. The signals at δH 4.52 (d
J1-2 7.8 Hz) and δH 4.51 (d J1-2 7.9 Hz) corresponded to the anomeric proton of the β(1→4)-linked glucopyranosyl residue. The last two anomeric signals at δH 5.10 and δH
5.37 (both d J1-2 3.8 Hz) were from an α-(1→2)-linked glucopyranosyl residue. Evidence
for α-(1→2)-linkage in Cel3 was observed in the HMBC (Figure S3 in article III)
correlation peaks: between GlcI-C2 (δC 76.84) and GlcIII-H1 (δH 5.10) and between
GlcIII-C1 (δC 97.70) and GlcI-H2 (δH 3.67) for the α-anomer and between GlcI-C2 (δC
79.60) and GlcIII-H1(δH 5.37) and between GlcIII-C1 (δC 99.20) and GlcI-H2 (δH 3.41)
for the β-anomer. Thus, Cel3 was identified as α-D-Glcp-(1→2)-[β-D-Glcp-(1→4)]-DGlcp (21-α-Glcp-cellobiose). Similarly, Lac3 was identified as α-D-Glcp-(1→2)-[β-DGalp-(1→4)]-D-Glcp (2Glc-α-Glcp-lactose). Thus, both Lac3 and Cel3 contain a
glucosyl residue that is α-(1→2) linked to the reducing end of the acceptor disaccharide
(lactose and cellobiose, respectively).
44
A
B
Intens.
6
x10
6
425
Cel3
β-GlcII(β)
4
503
Hex
2
-
179
485
221
341
263
β-GlcII(α)
α-GlcIII(β)
[M-H][M+Cl]
539
α-GlcIII(α)
α-GlcI
Glc II
Glc I
β-GlcI
467
Glc III
0
x106
425
β-Gal(β)
1.5
Lac3
1.0
[M-H]
503
[M+Cl]
539
485
Hex
0.5
179
263
341
383
221
α-GlcII(β)
-
α-GlcI
β-Gal(α)
α-GlcII(α)
Gal
β-GlcI
Glc I
467
x107
3
Glc II
-
[M-H]
503
α-GlcI
2
α-GlcII
NR3
Glc I
Glc II
1
[M+Cl] 539
425
100
150
200
250
300
350
400
450
500
m/z
Fru
Figure 12. MS2 spectra of [M+Cl]- ions (A) and 1D 1H NMR spectra (B) of isolated Cel3, Lac3, and NR3. The NMR spectra were measured at 600 MHz in
D2O at 16 ºC. The β-anomeric signals of the reducing-end residue of Lac3 and Cel3 were partially suppressed with the residual water signal. (Adapted from
Figures 2 and S6‒8 in article III.)
45
Glc
Lac3
Lac
Glc
Fraction Lac3
NR3
Fraction NR3
Suc Fru
+ α-glucosidase
Lac3
Cel3
Glc
Cel
Lac3
Gal
Glc
Koj
Glc
Koj
Fraction Cel3
Fraction Lac3 + β-galactosidase
Cel3
15.00
25.00
Fraction Cel3 + β-glucosidase
45.00 min
35.00
Figure 13. HPAEC-PAD profiles of isolated product fractions before (dashed lines) and after
(solid lines) specific enzymatic digestion. The labeled peaks were identified based on their
retention time against standards: Glc, glucose; Fru, fructose; Lac, lactose; Cel, cellobiose; Gal,
galactose; Suc, sucrose; and Koj, kojibiose. (Adapted from Figure 3 in article III.)
The product fractions of Lac3 and Cel3 were selectively digested with specific enzymes
and then analyzed by HPAEC-PAD (Figure 13). The α-(1→2) linkage was hydrolyzed
by α-glucosidase, generating glucose and the corresponding acceptor (lactose and
cellobiose, respectively). The β-(1→4) linkage in Lac3 was hydrolyzed by βgalactosidase, leading to the formation of galactose and kojibiose (α-D-Glcp-(1→2)-DGlcp), as expected. The glucose that formed concomitantly was due to the side reaction
of the β-galactosidase preparation on the α-(1→2)-linked glucosyl residue, as
manifested by the reaction of the β-galactosidase preparation on Cel3 and pure kojibiose
(data not shown). Although Cel3 was not very susceptible to hydrolysis by the βglucosidase used, a small amount of it was converted to glucose and kojibiose. The
above results further support the notion that Lac3 and Cel3 contain a glucosyl unit that
is α-(1→2) linked to the reducing end of their acceptors.
5.3.5 Novel sucrose-containing trisaccharide (III)
Under the conditions selected for the lactose acceptor reaction, a second product denoted
by NR3 (Figure 11A) was obtained. The 1D 1H spectrum of the isolated NR3 (Figure
12B) was different from that of Lac3 and Cel3. Apart from the signals from the
impurities (mainly Lac3) in isolated NR3, there were two anomeric protons at δH 5.42
(d J1-2 3.9 Hz) and δH 4.96 (d J1-2 3.7 Hz), both with an α configuration (Figure 12B).
The presence of only two anomeric protons for a trisaccharide indicated that one of its
46
residues has a quaternary anomeric carbon and no corresponding anomeric proton.
Because sucrose was the only sugar in the reaction system with this feature, NR3 was
inferred to contain sucrose, which was supported by comparing its 13C assignments with
those of sucrose (Table S3 in article III). The third residue exhibited resonances typical
of a terminal α-(1→6)-linked α-glucopyranosyl unit, which was found attached to the
fructofuranosyl residue of sucrose, as manifested by the downfield shift of
fructofuranosyl C6 signal in the edited HSQC. The linkage was also confirmed by
correlation peaks in the HMBC spectrum (Figure S5 in article III) between Fru-C2 (δC
104.71) and GlcI-H1 (δH 5.42), Fru-C6 (δC 70.08) and GlcII-H1 (δH 4.96), and GlcII-C1
(δC 99.58) and Fru-H6 (δH 4.06 and 3.76). NR3 was consequently identified as an α-DGlcp-(1→2)-[α-D-Glcp-(1→6)]-β-D-Fruf
(6Fru-α-Glcp-sucrose
/
2Fru-α-Glcpisomaltulose), also called isomelezitose.
NR3 was hydrolyzed to glucose, fructose, and sucrose by α-glucosidase (Figure 13). The
release of glucose and sucrose is in line with the proposed structure, whereas fructose
seems to result from the further hydrolysis of sucrose. NR3 was resistant to dextranase
(data not shown), which requires two consecutive α-(1→6)-linked glucosyl units for its
function (Maina et al., 2011). NR3 was not hydrolyzed by invertase, whereas raffinose
(α-D-Galp-(1→6)-α-D-Glcp-(1→2)-β-D-Fruf) was hydrolyzed to fructose and
melibiose (α-D-Galp-(1→6)-D-Glcp) (data not shown). This agrees with the structure
of NR3 having a glucosyl unit, which is α-(1→6) linked to the fructosyl residue, whereas
the galactosyl unit in raffinose is α-(1→6) linked to the glucosyl residue of sucrose.
In the product mixture of cellobiose acceptor reaction, NR3, if present, could not be
directly detected by HPAEC-PAD due to its coelution with cellobiose. However, the
analysis of the product fractions isolated by gel filtration (Figure 11C) showed the
presence of an unknown product, probably a trisaccharide according to its elution
behavior in gel filtration. This product was presumed to be NR3 based on its HPAEC
retention time. Moreover, a product with a similar HPAEC retention time to NR3 was
found in the product mixtures of all the tested acceptors except for maltose and
isomaltose (Figure S1 in article III and others unpublished). However, higher dilution
was used for the product mixtures of the two effective acceptors and thus the possibly
formed NR3 could not be detected. These observations indicated the formation of NR3
as a side product from endogenous sugars in dextransucrase-catalyzed reactions.
47
5.3.6 Applicability of MSn analysis in the
dextransucrase-synthesized GLOSs (III and IV)
characterization
of
13C glc
6
sucrose (6 carbon atoms in the glucosyl residue are 13C-labeled, while those in
the fructosyl residue are unlabeled) was used for the synthesis of GLOSs, the MSn
analysis of which provided information on the monosaccharide composition of unknown
GLOSs. In the beginning of the characterization of the minor products in the maltose
acceptor reaction, they were first suspected to be fructose-containing GLOSs, as
dextransucrases are known to use fructose and sucrose as acceptors (Moulis et al., 2006b;
Seo E et al., 2007). The reaction was thus carried out with 13C6glc sucrose, as fructosecontaining GLOSs (with one unlabeled residue and the remaining residues labeled) can
be differentiated from the major maltose-based GLOSs (with two unlabeled residues).
Therefore, if the minor series contain fructose, their m/z will be 6 plus that of the major
maltose-based product of the same DP. However, when analyzing the two products of
DP5 isolated from the maltose reaction mixture, only a single m/z signal was detected
that corresponded to a pentaose with 2 unlabeled residues (data not shown). As the minor
DP5 product was present in an appreciable amount in the isolated DP5 mixture (Figure
8D), the result indicated that the minor series probably also contained maltose rather
than fructose. Unfortunately, the negative and positive MSn spectra of the labeled DP5
product mixture (data not shown) did not show any diagnostic fragment ions other than
those known to be formed from the major LIM5, which was later explained by the
fragmentation patterns of the minor products.
The use of 13C6glc sucrose was also useful in the MSn characterization of the two novel
structures (Lac3 and NR3) from lactose acceptor reaction after their structural
elucidation by NMR spectroscopy analysis. Attempts to characterize unlabeled Lac3
were not successful due to the presence of isomeric impurity hypothesized to be a
fructose-containing linear structure, the presence of which was later confirmed (article
III). To solve this problem, synthesis was carried out again with 13C6glc sucrose. Because
the m/z of resultant Lac3 and NR3 differed by 6, which enabled their separate
fragmentation in MSn, the reaction mixture was directly analyzed without fractionation.
The GLOSs composition and their sugar components were reflected in the MS spectrum
of the labeled product mixture (Figure 14). Notably, the GLOSs containing two
unlabeled hexose units (lactose-derived) were found in only DP3 and 4 and were not
further elongated. Because of the addition of a labeled glucosyl branch in Lac3, the
differentiation of the two nonreducing end residues was made possible, enabling a
thorough MSn characterization of its fragmentation behavior (not within the scope of
this thesis). In short, the combination of both positive and negative ionization techniques
was beneficial for the analysis of rare oligosaccharides containing a 2,4-O-disubstituted
48
reducing end residue. The negative ion MS2 spectra of Lac3 and Cel3 showed an intense
signal corresponding to a loss of 60 Da+H2O. In positive ion MS2 spectra, the cross-ring
ions resulting from a loss of <162 Da were not observed (others’ results in article III).
These results provide reference MSn spectral data for such rare structures.
Figure 14. MS spectrum ([M+Cl]-) of the 13C6glc-labeled oligosaccharide mixture from the
WcE392-rDSR lactose reaction. In the spectra, the number of * shows that of the 13C6glc-labeled
residues in the compound. Solid circles symbolize the labeled glucosyl residues transferred from
sucrose, open circles represent unlabeled residues in the acceptor lactose, and triangles are
unlabeled fructosyl residues. The inset shows the HPAEC-PAD chromatogram of the same
mixture. (Adapted from Figure S9 in article III.)
The MSn fragmentation behavior of Lac3 was useful for the subsequent preliminary
characterization of BIM3 by MSn. A mixture of panose and BIM3 with a ratio of ~7:3
was used for MSn analysis. It was analyzed in parallel with pure panose; thus, the
difference in their fragment ion profiles indicated the behavior of BIM3 in the mixture.
The fragmentation pattern of panose (Maina et al., 2013) is illustrated in Figure 15I. In
the negative MS2 spectrum of the [M+Cl]- ions in the mixture (Figure 15II-A), the
presence of BIM3 resulted in an increase in cross-ring fragment ions, such as m/z 425,
263, and 221, compared to pure panose (Figure 15I-A). The profile of the fragment ions
originating from BIM3 resembled that from Lac3 and Cel3 (paper III), which contained
a 2,4-disubstituted reducing residue.
In positive ionization, the MS2 spectrum of the [M+Li]+ ion of panose was generally
similar to that of the mixture with BIM3 (Figure 15I-B and II-B, respectively). However,
the fragment ions at m/z 391 and 451 in the spectrum of the mixture were lower in
intensity compared to those in the spectrum of panose, indicating a lack of such fragment
ions from BIM3. Similarly, cross-ring cleavage ions that formed by the loss of <162 Da
(corresponding to ions at m/z 391 and 451) in the MS2 spectrum of the [M+Li]+ ion were
also absent from Lac3 (article III). When the Y2 and C2 ions at m/z 349 were further
fragmented at the MS3 stage, a marked increase in cross-ring fragment ion at m/z 229
49
was observed in the spectrum of the mixture (Figure 15II-C) compared to the
corresponding spectrum of panose (Figure 15I-C). The cross-ring fragment ion at m/z
229 (loss of 120 Da) was previously reported to be diagnostic of a (1→2) linkage (article
III). The above evidence indicates that BIM3 contains a (1→2)-linked hexopyranosyl
residue on the reducing residue of maltose. The analysis of BIM3 based on negative and
positive MSn spectral data of the reference compound Lac3 demonstrated the potential
of MSn analysis in the structural characterization of rare oligosaccharides, even in
impure samples.
50
Figure 15. MSn spectra of (I) pure panose and (II) a fraction containing panose and BIM3 at a
ratio of ~7:3. (A) Negative ion MS2 of [M+Cl]- m/z 539, (B) positive ion MS2 of [M+Li]+ m/z
511, and (C) positive ion MS3 for fragment ions at m/z 349. The fragmentation pathways of
panose are illustrated in section I. The downward arrows show the intensity changes of the
diagnostic fragment ions due to the presence of BIM3 (Figure 3 in article IV).
51
5.4 Optimization of IMO synthesis by response surface modeling (IV)
The IMO product profile (mainly principal linear IMOs) was systematically studied as
a function of sucrose and maltose concentrations and WcE392-rDSR dosage. The
preliminary experiments showed that dextran synthesis was largely suppressed in the
experimental region (Table S1 in article IV), within which a central composite design
was carried out (Table 6). The yield of individual IMOs of DP3‒6 and the yield of total
IMOs and the percent of consumed maltose were examined as responses after 24 h of
reaction (Table 6). The branched minor products were not quantified; however, the
content of BIM3 was affected in a similar way as was the content of panose (data not
shown). According to a carbohydrate analysis of the product mixtures (Table S2 in
article IV and Table 6), the substrates consumed and the products formed were roughly
balanced. The IMO profile varied between the experimental points, and the four
replicates at the center point of the design showed good reproducibility (Table 6 and
Figure 16). A quadratic equation was used to model each response by excluding
irrelevant terms to obtain the highest coefficients of determination (R2) and prediction
(Q2) possible. The relatively high R2 and Q2 values (Table 7) indicated that the models
fit well with the experimental data and could be used to predict the behavior of responses
within the experimental region.
52
Table 6. The central composite experimental design and response data for modeling the effects
of reaction conditions on IMO synthesis by WcE392-rDSR (Table 1 in article IV).
Run
Maltose
(mol/L)
Sucrose
(mol/L)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
0.15
1
0.15
1
0.15
1
0.15
1
0.15
1
0.575
0.575
0.575
0.575
0.575
0.575
0.575
0.575
0.15
0.15
1
1
0.15
0.15
1
1
0.575
0.575
0.15
1
0.575
0.575
0.575
0.575
0.575
0.575
Enzyme
(U/g
sucrose)
1
1
1
1
10
10
10
10
5.5
5.5
5.5
5.5
1
10
5.5
5.5
5.5
5.5
Panose LIM4
(g/L)
(g/L)
LIM5
(g/L)
LIM6 Total
(g/L) IMOsa
(g/L)
10.39b
19.33
3.89
120.10
13.45
47.87
3.86
120.55
5.96
91.66
37.91
45.70
48.53
43.40
47.62
49.39
47.50
49.00
7.69
39.67
31.20
15.63
2.58
25.98
80.15
33.83
37.78
6.58
97.11
40.06
45.95
56.26
55.05
58.39
58.82
1.46
31.35
3.90
4.50
31.19
19.00
27.96
5.92
0.64
46.89
8.82
14.28
17.00
15.82
18.23
17.83
a
17.24
5.03
22.33
105.46
22.90
23.38
13.18
138.99
19.30
98.77
31.57
98.17
79.17
62.11
79.08
83.00
81.13
84.61
45.69
25.89
162.84
287.16
70.01
82.98
204.43
408.68
154.46
265.03
89.98
365.89
199.51
191.76
229.36
240.15
236.31
243.86
Consumed
maltose (%)
36.71
20.55
86.25
38.15
37.03
28.50
94.19
50.19
84.20
40.37
23.02
64.74
39.77
59.17
49.84
46.69
49.96
49.26
Total IMOs represents all of the IMO products, including DP>6 members of principal linear
series and members of minor branched series.
b
Samples were analyzed twice, and the average values are presented. A hyphen (-) denotes that
the value was below the lower limit of quantification.
53
Fru
Suc
Mal
Pan
LIM4
Run 1
Run 2
LIM5
LIM6
Run 3
Run 4
Run 5
Run 6
Run 7
Run 8
Run 9
Run 10
Run 11
Run 12
Run 13
Run 14
Run 15−18
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
min
Figure 16. HPAEC-PAD profiles of acceptor product mixtures in experimental design. The
conditions of each run are shown in Table 6. Labeled peaks are: Fru, fructose; Suc, sucrose; Mal,
maltose; and Pan and LIM4−6, the major IMO products in the DP range of 3–6. The
chromatograms are under the same intensity scale, and the samples were diluted by the same
amount. (Figure S2 in article IV.)
54
Table 7. Effects of factors expressed as their corresponding coefficients obtained by modeling
for IMO yield (with respect to DP) and maltose consumption in WcE392-rDSR-catalyzed
maltose acceptor reaction (Table 2 in article IV).
Responses
Regression equationsa
Coefficients of
determination (R2)
and prediction (Q2)
Panose (g/L)
LIM4 (g/L)
44.78 + 36.20M + 16.52S + 2.69E + 23.69M∙S
78.46 + 27.67M + 27.80S + 3.13E - 20.26M2
- 14.43S2 + 27.58M∙S
55.09 + 2.22M + 18.58S + 3.97E - 10.09M2
- 7.49E2 + 4.90M∙S + 4.22M∙E
18.79 - 5.19M + 9.64S + 1.80E - 4.32E2
- 2.48M∙S + 0.88S∙E
236.07 + 42.46M + 111.45S + 23.68E - 27.90M2
- 45.86E2 + 41.93M∙S + 14.09M∙E
50.16 - 16.06M + 18.77S + 4.77E + 8.99M2
- 9.42S2 - 8.43M∙S
R2=0.95, Q2=0.89
R2=0.95, Q2=0.82
LIM5 (g/L)
LIM6 (g/L)
Total IMOs (g/L)
Consumed
maltose (%)
R2=0.85, Q2=0.60
R2=0.85, Q2=0.68
R2=0.97, Q2=0.87
R2=0.97, Q2=0.90
a
M, initial maltose concentration (mol/L); S, initial sucrose concentration (mol/L); E, WcE392rDSR concentration (U/g sucrose).
For each response, three two-dimensional contour plots with maltose and sucrose
concentrations as the two axes (Figure S3 in article IV) were used to illustrate the models,
and each plot corresponded to a different enzyme dosage. Because the enzyme dosage
in relation to the sucrose concentration had a minor effect on the responses, contour plots
at an enzyme dosage of 5.5 U/g sucrose were chosen to represent the effects of the two
main variables in Figure 17. The yield of each IMO of DP3−6 and the total IMOs, as
well as the percent of consumed maltose, increased by increasing the initial sucrose
concentration, whereas the initial maltose concentration exhibited varying effects on the
production of each IMO (Figure 17). The maximal production of panose, LIM4, and
total IMOs was attained when maltose and sucrose were both at the highest
concentrations tested. The production of LIM5 and LIM6, however, was favored at the
medium and lowest maltose concentrations, respectively, while maintaining the highest
sucrose concentration. The maltose utilization ratio was influenced positively by sucrose
input and negatively by maltose input. Aiming for a good yield of total IMOs and a
product profile towards higher DP, run 12 employing a medium enzyme load (5.5 U/g
sucrose) and medium maltose (0.575 M) and the highest sucrose (1 M) concentrations
was considered close to the optimum. The run led to sucrose depletion and a 65%
55
consumption of maltose. A yield of 366 g/L of total IMOs was attained, with a weighted
average DP of >4.5 (taking into account the amounts of DP3−6, not higher DP).
Figure 17. Contour plots showing the influence of the initial sucrose and maltose concentrations
in the WcE392-rDSR acceptor reaction on the yields (g/L) of (A) panose, (B) LIM4, (C) LIM5,
(D) LIM6, (E) total IMOs, and (F) the consumed maltose (%). The reactions were conducted
with an enzyme dosage of 5.5 U/g sucrose for 24 h (Figure 1 in article IV).
56
6. DISCUSSION
6.1 Properties of Weissella dextransucrases and dextrans (I and II)
Biochemical characterization showed that WcE392-rDSR was affected similarly to other
reported Weissella dextransucrases by pH and temperature (Kang et al., 2009; Shukla
and Goyal 2011; Amari et al., 2012; Bejar et al., 2013; Rao and Goyal 2013). It should
be pointed out that in the study of the effect of pH on WcE392-rDSR activity, the TrisHCl buffer used for pH 6.5‒7.0 contained Tris, which may have inhibited the enzyme
activity (Côté and Robyt 1982). Thus, the decrease of activity in this pH range may have
resulted from the inhibiting effect of Tris, in addition to that of pH per se. However, for
the purpose of determining the pH optimum, the experiments were not repeated with
another buffer of pH 6.5‒7.0, given that the enzyme activity at pH 6.0 already decreased
drastically in Na-acetate buffer. The quick inactivation above 40 °C and the stabilizing
effect of glycerol were observed for both WcCab3-DSR (others’ results in article II) and
WcE392-rDSR. It should be noted that WcCab3-DSR was produced by sucrose
induction and partially purified by PEG fractionation and thus was bound to dextran
(Majumder et al., 2007), whereas WcE392-rDSR was produced in glucose-containing
medium and devoid of bound dextran. The half-lives of WcCab3-DSR and WcE392rDSR of similar protein concentrations at ~30 °C without any additive were 10.3 h and
26.3 h, respectively, despite the fact that the former was dextran bound and dextran is
known to stabilize dextransucrase. In a recent study of the recombinant dextransucrase
from W. confusa Cab3, the addition of dextran could increase the enzyme activity by at
most 14% (Shukla et al., 2016). The stabilizing and activating effect of dextran on
Weissella dextransucrases thus seems to be minor.
The kinetics for a recombinant dextransucrase from W. confusa LBAE C39-2 and a
native one from W. cibaria JAG8 have been reported to follow Michaelis-Menten
kinetics, with KM and Vmax values of 8.6 mM and 20 µmol/(mg∙min), respectively, for
the former, and 13 mM and 27.5 µmol/(mg∙min), respectively, for the latter (Amari et
al., 2012; Rao and Goyal 2013). The kinetics parameters of the C39-2 dextransucrase
were determined by a 3,5-dinitrosalicylic acid (DNS) assay (Amari et al., 2012), which
has been suggested to overestimate the dextransucrase activity due to the significant
over-oxidation of dextran and other carbohydrates in the sample, thus giving invalid
enzyme assay and kinetics results (Vettori et al., 2011; McIntyre et al., 2013). According
to Vettori et al. (2011), the DNS method generates a 5.8-fold higher activity than the
14C-sucrose radioisotope method. However, it is not known how accurate is the NelsonSomogyi assay, the other commonly used reducing-value assay for dextransucrase
57
activity and kinetics measurements. It was thus compared with the 14C-sucrose
radioisotope assay in this study. The Nelson-Somogyi method gave an approximately
2.5-fold higher activity and Vmax than did the 14C-sucrose radioisotope method, while the
KM values were similar regardless of the method used. Because small amounts of glucose
and leucrose formed as side products, the higher values produced by the NelsonSomogyi method seemed to be due to the oxidation of several residues down the dextran
chain other than the reducing residue. As determined by Nelson-Somogyi method, the
kinetic values for WcE392-rDSR (KM of 13.0 mM and Vmax of 19.9 µmol/(mg∙min))
were similar to those of W. cibaria JAG8 dextransucrase (KM of 13 mM and Vmax of 27.5
µmol/(mg∙min)) (Rao and Goyal 2013). The WcE392-rDSR KM obtained by the 14Csucrose radioisotope method (14.7 mM) was comparable to that of a glucansucrase from
Leuconostoc mesenteroides NRRL B-1118 (12 mM) using the same method (Côté and
Skory 2012). In this thesis, the Vmax (8.2 µmol/(mg∙min)) determined by the 14C-sucrose
radioisotope assay was reported for the first time for a glucansucrase.
The dextran produced in vitro by WcE392-rDSR exhibited high similarity to that
produced in vivo by native and heterologous hosts as well as that by the other enzyme
WcCab3-DSR, as characterized by 1H NMR spectroscopy and HPSEC. The Weissella
dextrans showed an α-(1→6) to α-(1→3) linkage ratio of 97:3, being slightly less
branched than the dextran from Lc. mesenteroides NRRL B-512F (with a ratio of 96:4),
which was analyzed comparatively (Maina et al., 2008; Bounaix et al., 2009). The molar
masses reported for Weissella dextrans ranged between 106 and 107 g/mol (Amari et al.,
2012; Maina et al., 2014). Similarly high molar masses (107‒108 g/mol) were obtained
in this study (articles I and II) by HPSEC. Significantly high molar masses (108 g/mol)
were reported for dextrans synthesized in vitro with Lc. mesenteroides B-512F
dextransucrase mutants applying the asymmetrical flow field flow fractionation
technique (Irague et al., 2012). The high molar mass and low branching make Weissella
dextrans attractive hydrocolloids for food applications, such as in sourdough baking
(Lacaze et al., 2007).
6.2 Specificity of Weissella dextransucrases in acceptor reactions
6.2.1 Comparison with commercial Lc. mesenteroides dextransucrase (III
and IV)
Before this thesis work, the dextransucrase acceptor reactions for GLOS synthesis were
only investigated for DSR-S from Lc. mesenteroides B-512F. WcE392-rDSR was
similar to DSR-S in its selectivity towards the acceptors tested (in decreasing order):
58
maltose, isomaltose, nigerose, lactose, cellobiose, and melibiose (Robyt and Eklund
1983). The unusual acceptors tested in this thesis work, laminaribiose and forkose,
showed poor effectiveness for WcE392-rDSR. Moreover, the product patterns of
WcE392-rDSR were also similar to those of DSR-S based on the product profiles from
maltose, isomaltose, maltotriose, nigerose, lactose, and cellobiose (Robyt and Eklund
1983; Fu and Robyt 1990).
In the presence of lactose or cellobiose, both WcE392-rDSR and DSR-S form mainly a
trisaccharide with a glucosyl residue α-(1→2) linked to the acceptor’s reducing end
(Robyt 1995). In their maltose acceptor reaction, a principal series of linear IMOs are
synthesized from further elongation on panose. Moreover, a number of previously
unknown minor products were also observed in the reactions of both enzymes. In this
thesis, the minor trisaccharide from WcE392-rDSR was identified as centose with a
glucosyl residue α-(1→2) linked to the maltose-reducing end. However, a trace amount
of forkose (α-D-Glcp-(1→4)-[α-D-Glcp-(1→6)]-D-Glcp) has been reported to be
formed by DSR-S from Lc. mesenteroides B-512F (Fu and Robyt 1990). To determine
whether the minor trisaccharide derived from maltose varied with the enzyme source,
the commercial Lc. mesenteroides enzyme (results not shown) was compared to
WcE392-rDSR and WcCab3-DSR in maltose reaction. Their product profiles were
similar in HPAEC-PAD analysis, showing that the ability to synthesize centose (instead
of forkose) and branched IMOs from maltose is shared by these dextransucrases. The
finding was consistent with the use of maltose acceptor product patterns to classify
glucansucrases of different specificities (Côté and Leathers 2005). For dextransucrases
producing typical dextrans (with predominantly α-(1→6) linkages and a low degree of
α-(1→3) branching), their GLOS products from maltose showed a unique pattern,
although this was based on only principal IMO series detected in TLC analysis (Côté
and Leathers 2005). This thesis demonstrates that the dextransucrases highly specific for
α-(1→6) linkage formation also produce the same minor IMO series.
The shared specificity between the two Weissella dextransucrases is consistent with the
close resemblance in their sequences (93% amino acid sequence identity; GenBank:
AKE50934 and AHU88292). The amino acid substitutions are unlikely to cause drastic
differences in their catalytic properties (others’ results in article I). According to the
latest constructed phylogenetic tree of GH70 enzymes (Vuillemin et al., 2016),
WcE392-rDSR and W. confusa LBAE C39-2 dextransucrase (with 100% amino acid
identity to WcCab3-DSR) cluster together and form a branch with a subgroup of two W.
cibaria dextransucrases and a Lactobacillus fermentum dextransucrase. Although DSRS from Lc. mesenteroides NRRL B-512F is separated from Weissella dextransucrases,
and its sequence (GenBank: AAD10952) bears only 52% amino acid similarity to the
59
two Weissella dextransucrases studied, they seem to belong to a phylogenetic group of
typical dextransucrases producing predominantly α-(1→6) linkages. This corresponds
to the similar selectivity and specificity observed for the three dextransucrases.
6.2.2 Specificity to form an α-(1→2) branch (III and IV)
A typical dextransucrase DSR-S was shown to form an α-(1→2)-branched trisaccharide
from β-(1→4)-linked lactose or cellobiose but form an α-(1→6) linkage on the
nonreducing end of α-linked acceptors (Robyt 1995). However, this thesis reports for
the first time that α-linked maltose can be α-(1→2) branched as well and form centose
via typical dextransucrases. The higher DP homologues were proposed to be IMOs
carrying a single-unit α-(1→2) branch at the reducing end. This correlates with an earlier
observation that the α-(1→2) branch added to a maltose derivative, whose C-6 of the
nonreducing end was blocked, could not grow further (Bhattacharjee and Mayer 1993).
Such branched products have not been previously described from the acceptor reaction
of an enzyme of GH family 70. Dextransucrase from Lc. citreum NRRL B-1299 and its
truncated form GBD-CD2 (a branching sucrase) are able to form α-(1→2) branches on
α-(1→6)-linked residues in GLOSs (Dols et al., 1998; Brison et al., 2013). Nevertheless,
in the α-(1→2)-branched IMOs formed by typical dextransucrases, the branch is located
on the reducing-end residue.
Based on the observation that centose did not serve as an acceptor, which ruled out the
possibility of main chain elongation after branch formation, the higher DP homologues
were proposed to be formed through direct branch formation on an elongated linear IMO.
In this thesis, the dextransucrases have been shown to form an α-(1→2) branch not only
on lactose and cellobiose but also on maltose, panose and higher linear IMOs. Thus,
these acceptor sugars interact with the enzymes so that the C-2 of the reducing residue
can be oriented toward the active site and be linked to the C-1 of the glucosyl-enzyme
intermediate. The resultant branched GLOSs, however, were expectedly no longer
accommodated in the enzyme acceptor site to form a regular α-(1→6) linkage at the
nonreducing end. This complies with the fact that the branched trisaccharides derived
from maltose (this study) and cellobiose (Ruiz-Matute et al., 2011) do not elongate
further. GLOSs of only DP3 and DP4 were observed to contain lactose under the
synthesis with 13C6glc-labeled sucrose (Figure 14). Although other lactose-derived
GLOSs were too low in amount to be characterized in this and previous studies (DíezMunicio et al., 2012b), a portion of lactose is expected to be elongated linearly via α(1→6) linkages, explaining the presence of the 13C6glc-labeled lactose-containing DP4
product. In the isolation of Lac3 or Cel3 by gel filtration, they were found coeluting
closely with a small amount of another product (not shown), which may be the linear
60
trisaccharide with an α-(1→6)-linked glucosyl residue. It is interesting to note that
although typical dextransucrases form α-(1→3) branches in dextran, no α-(1→3) linkage
has been found in GLOSs. The recognition of α-(1→2)-branch linkage specificity sheds
light on a new interaction mode between acceptors and the enzyme active site. Enzyme
structure-guided mutations may alter dextransucrase specificity to produce a higher
portion of α-(1→2)-branched GLOSs for novel applications.
6.2.3 Novel sucrose-containing trisaccharide (III)
In the WcE392-rDSR acceptor reaction in the presence of lactose, the production of
sucrose-containing isomelezitose (NR3) by a dextransucrase was described for the first
time. It was independent from the exogenous acceptor and was promoted by higher
concentrations of sucrose and dextransucrase. Previously, an unknown trisaccharide
product was noticed in the lactose reaction mixture of Lc. mesenteroides B-512F
dextransucrase, which may have been isomelezitose (Díez-Municio et al., 2012b). This
product may have also been formed in the presence of cellobiose and other acceptors
tested. However, the reaction rate for isomelezitose formation is much lower compared
to that for effective acceptors to form IMO.
Isomelezitose was previously obtained from the fructose acceptor reaction of an
alternansucrase, where two possible pathways were proposed for its synthesis (Côté et
al., 2008). One involves glucosyl transfer to the 2-OH of the reducing residue of
isomaltulose (α-D-Glcp-(1→6)-D-Fruf, an acceptor product of fructose), resulting in the
formation of a sucrose-type (β-D-Fruf-(2→1)-α-D-Glcp) linkage. The other pathway
involves the transfer of a glucosyl residue to the 6-OH of the fructofuranosyl residue of
another sucrose molecule. In this thesis, isomelezitose was hypothesized to be formed
by the first pathway i.e., as an acceptor product of isomaltulose. Some supportive
evidence has been reported. First, a sucrose-type linkage is formed on lactulose (β-DGalp-(1→4)-D-Fruf) and maltulose (α-D-Glcp-(1→4)-D-Fruf) by Lc. mesenteroides
dextransucrase (Demuth et al., 2002; Díez-Municio et al., 2012a), indicating that a
similar sucrose-type linkage could be formed on isomaltulose. Second, a major
imbalance existed between the consumption of isomaltulose and the yield of
isomaltulose-derived IMOs according to the data published on the isomaltulose acceptor
reaction of Lc. mesenteroides dextransucrase (Barea-Alvarez et al., 2014), indicating
that other compounds may have also been produced from isomaltulose. Third, if sucrose
acts as an acceptor, a glucosyl residue is added to the sucrose-glucosyl moiety (Moulis
et al., 2006b; Dobruchowska et al., 2013) and not on the fructosyl moiety to form
isomelezitose. To test the hypothesis, an acceptor reaction with isomaltulose and 13C6glclabeled sucrose can be carried out. According to the hypothesis, the two glucosyl
61
residues in the isomelezitose thus synthesized can be differentiated in MSn analysis. By
reference to the fragmentation pattern of isomelezitose reported in article III, one should
be able to prove its origin from isomaltulose.
Other sucrose analogs, β-D-Galp-(1→4)-β-D-Fruf-(2→1)-α-D-Glcp (lactulosucrose)
and α-D-Glcp-(1→4)-β-D-Fruf-(2→1)-α-D-Glcp, derived from lactulose and maltulose,
respectively, have been considered donor substrates for dextransucrase after the
consumption of sucrose (Demuth et al., 2002; Díez-Municio et al., 2012a). Additional
study is needed to test the possible capacity of isomelezitose to act as a donor substrate.
Because isomelezitose derives from endogenous sugars in acceptor reactions of both
alternansucrase (Côté et al., 2008) and dextransucrase, it may widely occur as a side
product among glucansucrases.
6.3 Potential of dextransucrases in prebiotic GLOS synthesis
6.3.1 Synthesis of prebiotic IMOs from maltose (IV)
The dextransucrase maltose acceptor reaction represents a promising synthetic pathway
for prebiotic IMOs because of the cost-effectiveness and the abundance of the substrates
used (sucrose and maltose), as well as its ability to control the size distribution of IMOs
(Su and Robyt 1993; Lee et al., 2008). Compared to classical IMO production via a
fungal α-transglucosidase on high-maltose syrup, which preferentially produces low-DP
IMOs (Goffin et al., 2011; Hu et al., 2013), the dextransucrase-catalyzed reaction can be
tailored for the synthesis of higher-DP IMOs. A slight increase in DP (from 2.7 to 3.3)
endowed IMOs with non-digestibility for their function as prebiotics (Iwaya et al., 2012).
Moreover, in the presence of the effective acceptor maltose, dextran synthesis can be
largely inhibited to maximize sucrose utilization for IMO synthesis (Su and Robyt 1993;
Rabelo et al., 2009).
In this thesis, the effects of reaction factors (concentrations of sucrose, maltose, and
dextransucrase) on the IMO product profile were studied for the first time by
optimization design. A high sucrose concentration was favorable for IMO yield. Enzyme
dosage showed a small effect on the yield and profile of IMOs over a prolonged reaction
time for maximal sucrose conversion. Maltose input, however, had varying effects on
the production of IMO components. It promoted the production of lower DP while
inhibiting that of DP6. This indicates that maltose has a higher affinity to dextransucrase
than do the higher-DP IMOs. Thus, only when maltose concentration was low, did the
intermediate IMO products have a better chance to act as acceptors. The total IMO yield
62
peaked when the sucrose and maltose inputs were at their highest. This was also
observed in a previous study with Lc. mesenteroides NRRL B-512F dextransucrase, in
which the IMO size distribution and maltose consumption were unfortunately not
reported (Rabelo et al., 2009). However, this thesis showed that for a good yield of
higher-DP IMOs, medium maltose (~0.5 M) and high sucrose (1 M) concentrations were
optimal and also resulted in a better maltose utilization.
In vitro fermentation by human fecal bacteria showed that linear maltose-based IMOs
with DP5−7 had a relatively high selectivity toward beneficial bacteria compared to
those with a lower DP (Sanz et al., 2006). The higher IMOs also persist longer in the
colon and can reach the most distal regions, where most chronic intestinal disorders
originate (Sanz et al., 2006). In addition, dextransucrases can simultaneously synthesize
minor α-(1→2)-branched IMOs, which may have enhanced resistance to gastrointestinal
digestion and selectivity toward probiotic bacteria (Sanz et al., 2005). Thus, the
dextransucrase-catalyzed maltose acceptor reaction represents a promising synthetic
pathway for prebiotic IMOs. Dextransucrase from Lc. mesenteroides B-512F was
successfully immobilized for the repetitive production of IMOs (Tanriseven and Doğan
2002). Several purification methods have been developed to remove unwanted
monosaccharides (e.g., glucose and fructose) and disaccharides (e.g., maltose and
sucrose) from IMO products, including adsorption separation and selective fermentation
by yeasts or bacteria (Crittenden and Playne 2002; Yoon et al., 2003; Pan and Lee 2005;
Goffin et al., 2011). Beneficial IMOs can also be produced in situ by bacterial cells
during food fermentation (Katina et al., 2009; Cho et al., 2014).
6.3.2 Synthesis of other GLOSs (III)
Leuconostoc dextransucrases have been used to synthesize GLOSs from a variety of
acceptors, such as lactose, cellobiose, isomaltulose, lactulose, and gentiobiose. These
products have been demonstrated in vitro as potential prebiotics (Ruiz-Matute et al.,
2011; Coelho et al., 2014; Díez-Municio et al., 2014; Barea-Alvarez et al., 2014; GarcíaCayuela et al., 2014; Kothari and Goyal 2015). Although these acceptors are less
effective than maltose, a reasonable yield of acceptor products can be achieved by
increasing the substrate concentrations and the ratio of acceptor to sucrose, which diverts
more sucrose for use in the acceptor reaction at the cost of dextran and leucrose
production (Díez-Municio et al., 2012b). In the Lc. mesenteroides B-512F
dextransucrase-catalyzed synthesis of 2Glc-α-Glcp-lactose (Lac3) using concentrated
sucrose and lactose both at 0.88 M, the trisaccharide yield was 130 g/L corresponding
to a consumption of approximately 36% of the initial lactose (Díez-Municio et al.,
2012b). In this thesis, the lactose product yield was limited by the low lactose input
63
concentration of 0.15 M. The highest Lac3 yield was achieved (with a 27% lactose
consumption) when the sucrose and dextransucrase concentrations were at their highest,
which resulted in the concomitant formation of a large amount of dextran. Under these
conditions for lactose acceptor reaction, a novel side product, isomelezitose (NR3), was
identified and is a potential nutraceutical (Görl et al., 2012).
Dextransucrase-synthesized 2Glc-α-Glcp-lactose has found use as a precursor for the
synthesis of kojibiose, a bioactive disaccharide with low availability due to difficulties
in its isolation or synthesis. After the initial lactose acceptor reaction, the sugars present
in the mixture (fructose, glucose, galactose, and sucrose) were removed by yeast
Saccharomyces cerevisiae treatment. The remaining lactose and 2Glc-α-Glcp-lactose
were then hydrolyzed by Kluyveromyces lactis β-galactosidase without removing the
yeast cells. Kojibiose was obtained with good yield: 38% in weight with respect to the
initial amount of lactose (Díez-Municio et al., 2014). Dextransucrase acceptor reactions
may provide affordable synthesis processes for a variety of rare di- and oligosaccharides,
thus expanding their applications.
64
7. CONCLUSION
In this thesis, two W. confusa dextransucrases (mostly WcE392-rDSR and partly
WcCab3-DSR) were studied for their catalytic properties and dextran- and GLOSsynthesizing functionalities. Focus was given to the structural characterization of their
GLOS products because the information on dextransucrase-synthesized GLOSs was
previously only available for a commercial Lc. mesenteroides dextransucrase.
The effects of pH and temperature on the WcE392-rDSR activity were determined. The
enzyme lost activity quickly above 40 °C and was stabilized by glycerol. It showed
Michaelis-Menten kinetics with a KM of 14.7 mM and a Vmax of 8.2 µmol/(mg∙min) as
determined by the sucrose radioisotope assay. A similar KM value and a 2.5-fold higher
Vmax were obtained by the reducing-value Nelson-Somogyi assay. Dextrans from the
Weissella dextransucrases consisted of 97% α-(1→6) linkages and 3% α-(1→3) linkages,
which resembled that from the commercial Lc. mesenteroides B-512F dextransucrase.
The dextrans obtained showed high molar masses in the range of 107‒108 g/mol in
HPSEC analysis.
The selectivity of WcE392-rDSR towards several acceptors and the acceptor product
patterns were similar to those of Lc. mesenteroides B-512F dextransucrase. The most
efficient acceptor maltose was tested with Weissella dextransucrases and the Lc.
mesenteroides dextransucrase, all exhibiting a similar product profile in HPAEC-PAD
analysis. The GLOS products were isolated according to DP for structural
characterization. Interestingly, in addition to a series of principal products, there seemed
to be another series of minor products. The principal products were identified as linear
maltose-based IMOs, which corresponded to the predominant α-(1→6) linkages in
dextran products from typical dextransucrases. The minor products were confirmed to
form another homologous series, where a single glucosyl residue was α-(1→2) linked to
the reducing residue of linear IMOs. Such branched IMOs have not been reported for a
glucansucrase. The branch was proposed to be added to each member of the linear IMOs.
Unlike maltose, WcE392-rDSR produced dominantly branched trisaccharides with
analogous lactose and cellobiose and with a glucosyl residue α-(1→2) linked to the
acceptor’s reducing end. Moreover, the side product isomelezitose was proven for the
first time to be formed by a dextransucrase. This nonreducing trisaccharide was
apparently synthesized from the fructose acceptor product isomaltulose. An MSn
analysis of the 13C6glc-labeled GLOS products was proven useful in, e.g., distinguishing
fructose-containing side products from products derived from exogenous acceptors.
65
To apply WcE392-rDSR for the synthesis of higher-DP linear IMOs for better prebiotic
properties, the effects of sucrose, maltose, and dextransucrase concentrations on the
IMO product profile were studied here for the first time. According to response surface
modelling, the yields of individual IMOs of DP3−6 were all maximized with the highest
sucrose input (1 M) but varying maltose inputs (0.15−1 M). The optimal maltose
concentration for DP3−4, DP5 and DP6 was at its highest, medium and lowest levels,
respectively. The dextransucrase dosage (1–10 U/g sucrose) had a smaller effect on these
responses.
Overall, this study examined the catalytic properties of Weissella dextransucrases and
the structures of their dextran and GLOS products. Several GLOSs from Weissella
dextransucrases were identified, which enhanced the understanding of the catalytic
mechanism of these enzymes and more generally of typical dextransucrases producing
predominantly α-(1→6) linkages, such as the commercial one from Lc. mesenteroides
B-512F. The study also demonstrated the usability of Weissella dextransucrases in
synthesizing dextran and oligosaccharides for food use.
66
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