Indian Journal of Biotechnology
Vol. 5 (Suppl), July 2006, pp. 337-345
Production of a high maltose-forming, hyperthermostable and Ca2+independent amylopullulanase by an extreme thermophile Geobacillus
thermoleovorans in submerged fermentation
S M Noorwez, M Ezhilvannan and T Satyanarayana*
Department of Microbiology, University of Delhi South Campus, New Delhi 110 021, India
Production of a hyperthermostable, high maltose-forming and Ca2+-independent amylopullulanase by an extreme
thermophile Geobacillus thermoleovorans was studied in submerged fermentation. Parametric optimization led to secretion of
amylopullulanase (2850 U L-1 of α-amylase, 840 U L-1 of pullulanase) in a starch-yeast extract medium (2% starch, 0.1%
ammonium sulphate, 0.3% yeast extract, 0.1% K2HPO4, 0.1% NaCl, 0.01% MgSO4.7H2O and 0.1% Maltose) using 2%
inoculum (12 h) at 70°C and 200 rpm in 20 h. The addition of trace elements, detergents, surfactants and additives did not
stimulate growth or enzyme production. The hydrolysis of pullulan and starch yielded maltotriose, and maltose, maltotriose and
maltotetraose as the major end products, respectively. The cultivation of the organism in a laboratory fermenter showed (i)
abolition of the lag phase, (ii) a 2-fold increase in enzyme production (5880 U L-1 of α-amylase, 1900 U L-1 of pullulanase) and
(iii) a reduction in fermentation time to 7-8 h.
Keywords: Geobacillus thermoleovorans, amylopullulanase, submerged fermentation, starch hydrolysis
IPC Code: Int. Cl.8 C12N9/44; C12R1/01
Introduction
The steep increase in the demand for enzymes as
industrial catalysts has led to rapid developments in
the enzyme industry. The starch processing industry is
unique among industrial enzyme sectors, where
application of thermostable enzymes is essential for
the industry. The potential exploitation of sugars from
natural sources is useful for (i) glucose/fructose syrup
production, (ii) synthesis of non-fermentable
carbohydrates, and (iii) anti-cariogenic and antistaling agents in baking1. Thermostable amylolytic
enzymes of microbial origin are used extensively in
food, chemical, detergent and textile industries, to
convert starch into low molecular weight products. Of
special interest are thermophilic microorganisms
endowed with production of thermostable enzymes
with remarkable properties2.
A
new
class
of
amylolytic
enzymes,
amylopullulanases (Type-II pullulanases) has been
reported in thermophilic bacteria and archaea with
dual specificity for α-1,4- and α-1,6- glycosidic
linkages in starch3,4. These enzymes have great
potential for direct use in (i) starch liquefaction (with
or without α-amylase), (ii) saccharification processes,
________________
*Author for correspondence:
Tel.: 91-11-24112008; Fax: 91-11-2411 5270
E-mail: [email protected]
and (iii) in baking. The need to look for highly
thermostable and thermoactive pullulanases from
thermophiles has been emphasized5. Pullulanases may
prove superior to the commercial thermostable αamylase from B. licheniformis used for industrial
starch liquefaction. Pullulanases may also be useful
for one-step starch conversion into maltodextrins, and
hence, suggested as an alternative to replace αamylases
in
starch
liquefaction.
Certain
amylopullulanases produce maltose, maltotriose, and
maltotetraose (DP2 to DP4) as the major end products
of starch hydrolysis, and, therefore, suggested as
catalysts in one-step liquefaction-saccharification
process for the production of high DP2-DP4 syrups6.
The baking industry is another large consumer of
starch and starch-modifying enzymes. All undesirable
changes (staling), viz. increase of crumb firmness,
loss of crispness of the crust, decrease in moisture
content of the crumb and loss of bread flavour usually
occur upon storage. Staling limits the shelf-life of
baked products, and hence, causes economic losses to
baking industries. Amylolytic enzymes active on
starch have been suggested as anti-staling agents. For
this purpose, α-amylases were added during dough
preparation to generate fermentable compounds and
to improve the retention of softness of baked goods.
However, slight overdose of α-amylase is not
338
INDIAN J BIOTECHNOL, JULY 2006
attractive due to the production of sticky bread.
Alternatively, pullulanases are claimed to (i) alleviate
sticky defect in bread, (ii) increase shelf-life, and (iii)
loaf volume of baked goods7- 9.
In order to develop novel processes, microbes
endowed with thermostable enzymes are desired. For
this, thermophilic microbes with the ability to produce
thermostable enzymes having high operation stability
and a longer shelf-life are warranted10. Very few
investigations have emphasized on strain selection,
their growth and enzyme yield optimization, even
though the level of thermophilic enzyme production is
relatively low11. Several thermophiles are known to
produce Type-II pullulanase (amylopullulanase)12.
Hence, this investigation has been attempted to throw
light on the optimization of medium components and
cultural parameters in submerged fermentation for
amylopullulanase
secretion
by
Geobacillus
thermoleovorans.
Materials and Methods
Source of the Bacterial Strain
The bacterial strain NP33 was isolated from a hot
water spring of the Waimangu Volcanic Valley (New
Zealand), and maintained as described earlier13, 14. The
isolate producing relatively high Ca2+-independent
amylopullulanase was identified as Geobacillus
thermoleovorans by the analysis of partial 16S rDNA
gene sequence [GenBank (Bethesda, Maryland, USA)
accession no. AY427833]. The bacterial strain has
been deposited at Microbial Type Culture and
Collection & Gene Bank (MTCC), Institute of
Microbial Technology, Chandigarh (India) with
accession number MTCC 4219.
Production of Amylopullulanase
The enzyme was produced by cultivating the
bacterium in 250 mL Erlenmeyer flasks containing 50
mL of the basal medium (g L-1: soluble starch, 10.0;
yeast extract, 3.0; ammonium sulphate, 3.0; K2HPO4,
1.0; MgSO4.7H2O, 0.2; NaCl, 1.0 and pH 7.0), in an
incubator shaker (New Brunswick Scientific Co. Inc.,
NJ, USA) at 70ºC for 20 h at 200 rev min-1. A 2 %
cell suspension of G. thermoleovorans prepared from
10 h-old seed culture was used as inoculum. The
culture fluid was centrifuged at 8000 xg for 20 min
and 4ºC (Sorvall RC 5C Plus; Kendro Labs,
Newtown, USA). The cell-free supernatant was used
as the source of extracellular amylopullulanase.
Enzyme Assays
Both α-amylase and pullulanase activities were
determined by measuring the amount of reducing
sugars liberated from 1% soluble starch (Sigma) or
1% pullulan (Sigma) at 80°C according to Bernfeld15
using dinitrosalicylic acid (DNSA) reagent16. One
international unit (IU) of α-amylase or pullulanase is
defined as the amount of enzyme that liberates 1 μmol
of reducing sugar as glucose min-1 mL-1 under the
assay conditions.
Parametric Optimization
Each parameter was examined after taking into
account the previously optimized condition i.e., ‘one
variable at a time’ approach. All the experiments were
performed in triplicate and the average values are
presented.
Amylopullulanase production was studied at
regular intervals during 24 h. To study the effect of
pH, the bacterial strain was cultivated in the basal
medium buffered at different pH [0.1 M citrate buffer
(pH 3.0-5.0), 0.1 M phosphate buffer (pH 6.0-8.0) and
0.1 M glycine-NaOH buffer (pH 9.0-10.0)]. The
effects of temperature and agitation were assessed by
cultivating the bacterial strain at different
temperatures (45-80°C) in shake flasks and agitating
in the ranges between 50 and 250 rev min-1. Inoculum
was prepared from the cultures grown for varying
time intervals (6-16 h), and was used at different
levels (1-6 %) to study the effect of inoculum age and
size on amylopullulanase secretion.
The bacterial strain was also grown in the basal
medium containing different carbon sources (1%)
(glucose, maltose, galactose, sucrose, fructose, xylose,
lactose, raffinose, glycerol, sorbitol, xylitol,
maltodextrins, PEG, PVA, xylan and arabinogalactan)
instead of starch to study their effect on the secretion
of the enzyme. Three different starch sources (Merck
soluble starch, corn starch and tapioca starch, 1%
w/v) were incorporated in the production medium to
select the best. Effects of various concentrations of
soluble starch (0.5-4.0% w/v), and maltose (0.1-1.5%
w/v) supplementation in 2% starch containing
medium were also tested.
Ammonium sulphate present in the basal medium
was substituted with equimolar proportions of
different nitrogen sources (ammonium sulphate,
tryptone, peptone, casein, ammonium nitrate, urea,
potassium nitrate, ammonium hydrogen phosphate,
ammonium chloride and asparagines) to assess the
effect of different organic and inorganic nitrogen
sources on enzyme production.
The effect of different levels of yeast extract (0.11.5% w/v), ammonium sulphate (0.1-1% w/v),
NOORVEZ et al: AMYLOPULLULANASE PRODUCTION BY G. THERMOLEOVORANS
339
MgSO4.7H20 (0.01-0.10% w/v), K2HPO4 (0.05-0.15%
w/v), NaCl (0.1-1.0% w/v) and various cations
(0.01% w/v), additives (0.1% w/v) and detergents
(0.1% w/v) were also studied on enzyme production.
Batch Fermentation in a Lab Fermenter
The bacterial strain was cultivated in 22 L Biostat
C (B Braun Biotech International, Mersungen,
Germany) fermenter containing 10 L of the
amylopullulanase production medium (g L-1: soluble
starch, 20.0; yeast extract, 3.0; ammonium sulphate,
1.0; K2HPO4, 1.0; MgSO4.7H2O, 0.1; maltose, 1.0;
and NaCl, 1.0). The fermenter was operated at 65ºC,
200 rev min-1 and 1 vvm of aeration. The medium was
inoculated with 2% of 12 h-old seed culture. The pH
of the medium was maintained at 7.0 using sterile 1 M
NaOH/HCl. The samples were withdrawn at regular
intervals for determining amylopullulanase titres.
Identification of End-Products of Starch and Pullulan
hydrolysis
The hydrolysis products of 1% starch and 1%
pullulan by the action of amylopullulanase from G.
thermoleovorans at 80°C were analyzed by TLC
(Silica gel) plate 60 F (E Merck AG, Darmstadt,
Germany) and HPLC using an Aminex-HPX-42A
oligosaccharide column (300 by 78 mm; Bio-Rad,
Hercules, California, USA)14,17.
Results and Discussion
The extremely thermophilic bacterium G.
thermoleovorans
secreted
high
titres
of
-1
amylopullulanase (210 U L α-amylase and 48 U L-1
pullulanase) in the basal medium within 20 h in shake
flasks when the organism was cultivated at 70°C, 200
rpm, pH 7.0 with 2% of 12 h old inoculum (Fig. 1).
The action of extracellular amylolytic enzyme of G.
thermoleovorans on starch and pullulan liberated
maltose, maltotriose and other oligosaccharides, and
only maltotriose, respectively. Among different
carbon sources tested, a nearly similar level of
amylopullulanase was produced in starch, maltose,
maltodextrin and glucose. Among the different
starches, soluble starch (2%) supported a high enzyme
production. A low maltose supplementation (0.1%) to
2% starch containing medium further enhanced
enzyme titre (Fig. 2).
A high enzyme production was recorded in organic
nitrogen sources, tryptone and casein, and among
inorganic nitrogen sources, ammonium sulphate and
ammonium nitrate. The enzyme production was high
Fig. 1—Effect of (A) fermentation time, (B) temperature, (C) pH,
and (D) agitation on amylopullulanase production.
at a low concentration of ammonium sulphate (0.1%),
and thereafter, it declined sharply (Fig. 3).
340
INDIAN J BIOTECHNOL, JULY 2006
Fig. 2—Effect of (A) different carbon sources, (B) starch
concentration and (C) maltose supplementation in 2% starch
containing medium on amylopullulanase production.
The amylopullulanase production was enhanced
with an increase in the level of yeast extract upto 0.3 %
and thereafter, it declined (Fig. 3). A low magnesium
concentration (0.01 %) supported a good enzyme titre
(α-amylase 3000 U, pullulanase 840 U L-1) and at
higher levels, the enzyme secretion declined. The
enzyme titre was high at 0.1% K2HPO4 (α-amylase
2200 U, pullulanase 950 U L-1) with a decline on either
side of this concentration.
Surfactants such as Tween-80 and SDS did not
have any observable effect on enzyme production,
Fig. 3—Effect of different concentrations of (A) nitrogen sources,
(B) ammonium sulphate and (C) yeast extract on amylopullulanase
production.
while Triton X-100 lowered the production to about
50%. The presence of glycine also did not affect the
enzyme secretion. Among the cations, cobalt
enhanced enzyme production, but Cu2+ and Mn2+
strongly inhibited the enzyme secretion. However,
iron and calcium did not exert any noticeable effect
on the enzyme production (Table 1).
When G. thermoleovorans was cultivated in a 22 L
laboratory fermenter, the enzyme production levels
[
NOORVEZ et al: AMYLOPULLULANASE PRODUCTION BY G. THERMOLEOVORANS
markedly increased from 830 to 1900 and 2850 to
5880 U L-1 of pullulanase and α-amylase, respectively
(Fig. 4). A peak in enzyme secretion was attained in
7-8 h in fermenter, in contrast to 20 h in the shake
flasks, without any significant increase in biomass.
The production of amylopullulanase was sustainable
in flasks and enhanced in the fermenter (Tables 2 &
3).
Novel starch- and pullulan-degrading enzymes are
classified on the basis of the end-products of
hydrolysis. Maltotriose was the exclusive product of
pullulan hydrolysis by pullulanase action of
amylopullulanase, while its α-amylase activity led to
release of maltose, maltotriose and maltotetraose from
Table 1—Effect of surfactants, detergents, additives and divalent
ions on enzyme production
Enzyme Production (U L-1)*
Source
α-amylase
pullulanase
2924
3100
2920
2810
1560
2563
3252
2863
2657
2842
820
920
918
790
410
533
1098
798
556
810
Control
SDS
Glycine
Tween-80
Triton X-100
Cu++
Co++
Fe++
Mn++
Ca++
341
starch18,19. Thin layer chromatography and HPLC
have been extensively used for the identification of
enzyme reaction products20. As the enzyme of G.
thermoleovorans hydrolyzed pullulan to maltotriose
and starch to maltose, maltotriose and maltotetraose,
it was classified as an amylopullulanase or Type II
pullulanase. This is the first report on the secretion of
amylopullulanase by G. thermoleovorans.
The extracellular enzymes produced by various
members of Bacillus and the related genera play a
vital role in the biotechnology industry. Thermophiles
in general are known to produce low amounts of
biomass and also enzyme titres. Optimization of
various cultural parameters was carried out to increase
the yield of biomass, enzymes and other microbial
products10,11. The production of enzyme in various
media by G. thermoleovorans led to the selection of a
rather simple medium that supported relatively high
enzyme titres. The production of the enzyme was
initiated with the growth of the organism and reached
a peak after 18-20 h in the late logarithmic phase of
growth, followed by a decline in the late stationary
phase. This could be due to lysis of the cells,
depletion of nutrients after vigorous growth or due to
the accumulation of toxic metabolites21. Growth
associated production of amylopullulanase was also
reported in thermophilic species of Bacillus21 and
Clostridium22.
*Mean of three values, S.D within 10%
Table 2—The amylopullulanase production in the optimized
starch-yeast extract medium in flasks and fermenter
Medium
volume
(mL)
50
100
200
400
10000
Volume of Fermentation Enzyme titre (U L -1)*
flask
time
α-amylase Pullulanase
(mL)
(h)
250
20
2 850
830
500
20
2 856
840
1000
20
2 742
794
2000
20
2 665
713
22000
7
5 880
1900
*Mean of three values, S.D within 10%
Fig. 4—Amylopullulanase production profile in the optimized
amylopullulanase production medium in a laboratory fermenter
Table 3—Summary of enzyme production in shake flasks and laboratory fermenter
Enzyme titre (U L-1)*
Levels of optimization
Without optimization in shake flasks
After optimization in shake flasks
After optimization in Lab fermenter
* Mean of three values, S.D with in 10%
Fold increase
α-amylase
pullulanase
α-amylase
pullulanase
210
2 850
5 880
48
840
1900
1
14
28
1
17.5
39.5
342
INDIAN J BIOTECHNOL, JULY 2006
Temperature and pH are the two most important
physical parameters that affect growth as well as
secretion of extracellular enzymes. Temperature and
pH optima for growth and enzyme production by G.
thermoleovorans were 70°C and 7.0, respectively.
Microorganisms are known to produce enzymes
optimally at pH and temperature optimal for their
growth4,19,21.
Factors affecting proper mixing of nutrients and
oxygen usually determine the optimal production of
extracellular enzymes. Agitation rates in the range of
100 to 250 rpm are generally reported for the
production of amylopullulanase from diverse
bacteria19,22. The optimal agitation rate for G.
thermoleovorans was within this range of agitation
rates. The preference for this range of agitation may
be due to proper balance of mixing of nutrients and
their physical availability to the organism. At higher
agitation rates, the uptake of nutrients from the outer
environment may be hindered, as too little time is
available for contact with substrates. This may lead to
reduced uptake of substrates and other nutrients
resulting in poor growth and production of enzymes23.
The age and level of the inoculum are important
parameters as they have a direct bearing on the
growth pattern of the organisms. With increase in the
inoculum level, there was a sharp fall in enzyme
production after 2% (v/v). The production levels
enhanced with increasing the age of the inoculum, and
the optimal titres were attained with overnight grown
culture in B. thermoamyloliquefaciens KP107124. In
Bacillus sp. DSM 405, 1% inoculum of 2 h old
culture supported a high enzyme production. Lower
levels may be expected to take longer time to attain
adequate cell numbers, while higher levels may lead
to competition for nutrients19.
Carbon sources provided for the growth and
production of enzymes have a profound effect on the
production behaviour of the organism. G.
thermoleovorans grew very well on xylitol, sucrose,
lactose and raffinose but the enzyme production was
very low. The growth and enzyme titres were high in
the presence of polysaccharides. Starch has been
found to be the most preferred substrate for the
production of amylopullulanases. Starch (1% w/v) has
been very widely used3,20,25, although 2% starch was
optimal for the production of amylopullulanase by B.
subtilis4 as in G. thermoleovorans. Production of the
enzyme was greater in soluble starch than in tapioca
and corn starches, suggesting structural complexity of
the raw starch grains.
The choice of nitrogen source has also a significant
influence on the secretion of amylopullulanases.
Tryptone, an organic form of nitrogen, has been
extensively
used
for
the
production
of
amylopullulanase by thermophiles, especially the
hyperthermophiles23,25. Ammonium chloride and
ammonium nitrate have also been used for the
cultivation and enzyme production by Bacillus sp.
31833 and Bacillus sp. DSM 40519, respectively.
Ammonium sulphate was used as a nitrogen source
for production of the enzyme by B. circulans F-226,
Bacillus sp. KSM-137827 and Bacillus sp. strain TS2320. The literature survey has suggested that the
thermophilic bacilli appeared to have a preference for
ammonium salts as source of nitrogen for the
production of amylopullulanase. Our findings
are consistent with this trend, and ammonium
sulphate supported a high enzyme production in
G. thermoleovorans.
Magnesium ions play a critical role in diverse
biological functions and their requirement may be
expected for the growth and consequently enzyme
production. At higher concentrations of Mg2+, the
enzyme production was reduced, which may be due to
blockage of protein release into the external medium
as observed in the secretion of α-amylase from B.
brevis No. 4728.
Inorganic phosphate ions being constituents of
cellular biomolecules such as cAMP, nucleic acids
and coenzymes, play a regulatory role in the synthesis
of primary and secondary metabolites in
microorganisms29. There was a linear increase in the
enzyme production with the increase in phosphate
concentration but a sharp decline at higher
concentrations.
Most of the organisms producing amylopullulanase
require yeast extract or other such complex
compounds and trace elements for growth and
enzyme production3,23,30,31. Mesophilic B. subtilis4 and
Bacillus sp. KSM-137827 have also been shown to
require micronutrients for enzyme production. There
was no requirement of additional trace elements for
enzyme production by T. thermosaccharolyticum25,
Bacillus sp. DSM 40519 and Bacillus sp. strain TS2320. G. thermoleovorans appeared to have an
obligate requirement for certain factor(s) available
from yeast extract (vitamins) for growth, although
additional trace elements were not necessary. The
[
NOORVEZ et al: AMYLOPULLULANASE PRODUCTION BY G. THERMOLEOVORANS
addition of copper and manganese to the production
medium decreased the enzyme secretion, while cobalt
enhanced it. There was, however, no increase in the
biomass in presence of cobalt. Iron and calcium did
not show any observable effect on the enzyme
production. Presence of Triton X-100 in the
production medium led to suppression of growth and
enzyme secretion, while Tween-80 and SDS had no
observable effect. Higher levels of detergents are
known to disrupt the cell membrane of the bacterium
leading to its lysis and consequently suppress
growth32.
Catabolite repression is one of the primary factors
regulating the synthesis of exoenzymes33. Glucose
and other easily matabolizable sugars did not repress
the synthesis of enzyme in G. thermoleovorans.
Amylopullulanases have been produced in the
presence of glucose20, while some reports have shown
repression of enzyme synthesis in the presence of
glucose34.
There is a great variation in the regulation of the
exoenzyme synthesis as it may be inducible, partially
or completely constitutive depending on the strain and
the enzyme in question33. The majority of
exoenzymes secreted by bacilli appeared to be at least
partially inducible. Catabolic enzymes are normally
induced by the enzyme substrate, but exoenzymes are
an exception in that they are presumably excreted
because the substrate is unable to enter the cell. The
substrate, therefore, cannot be directly involved in the
induction process. It is now generally accepted, that a
low basal level of constitutive exoenzyme degrades its
exogenous substrate and the resultant low molecular
weight products enter the cell and induce further
enzyme synthesis. These examples are relevant as the
production
of
amylopullulanase
by
G.
thermoleovorans demonstrated both the patterns. In
addition to the induction of enzyme secretion by the
presence of substrate, enzyme titres were further
enhanced in the presence of maltose and
maltodextrins. Maltotetraose has been shown to be an
effective inducer of α-amylase synthesis in B.
stearothermophilus35
and
B.
licheniformis36.
Commercial malto-oligosaccharide mixture was used
as an inducer for amylopullulanase production in
Pyrococcus furiosus and Thermococcus litoralis23.
The ratio of the medium volume to the volume of
the flask determines the mixing properties of the
nutrients as well as oxygen in conjunction with the
agitation rate. With increasing medium volume at
343
fixed agitation rate, the mixing behaviour is expected
to change, especially in the case of aerobic
thermophiles where the amount of dissolved oxygen
is already very low in comparison to ordinary
temperatures. Shake flasks suffer from certain
shortcomings, which affect production. Cotton plugs
were found to limit oxygen transfer rates depending
upon the quality of plugs. While swishing motion of
flasks on a shaker does not allow proper oxygen
transfer when medium volume is high. The
production of metabolites in fermenters provides
several advantages over shake flasks owing to better
control over process parameters such as the control of
pH and aeration besides better mixing of nutrients,
heat and oxygen transfer37. When G. thermoleovorans
was cultivated in the fermenter, there was neither
improvement in enzyme production nor in growth as
compared to shake flasks. The fermentation profile
was similar in both, but the noticeable effect was
reduction in production time from 18-20 h in flasks to
7-8 h in fermenter. This appeared to be due to
improved process parameters such as mixing of
nutrients as well as oxygen in the fermenter as
compared to shake flasks37. The pH of the culture
broth dropped from 7.0 to 4.7 accompanied by the
growth of G. thermoleovorans as reported also in
Fervidobacterium
pennavorans
Ven538
and
22
Clostridium sp. strain EM1 . The addition of 0.1%
maltose as inducer and maintenance of pH at 7.0 led
to a 2-fold increase in the enzyme titre. Since these
are smaller molecules in comparison to starch, they
could be easily taken up by the cells resulting in
enhanced growth and secretion of enzyme. This
enhanced level of excreted enzymes caused
hydrolysis of starch35.
The optimization of media components and cultural
parameters has led to a marked enhancement
in the production of amylopullulanase by
G. thermoleovorans. In view of the potential
applications of amylopullulanase in saccharification
of starch into high maltose syrups in a single step and
its potential application in baking as an antistale
factor, further work on the production of
amylopullulanase by G. thermoleovorans MTCC
4219 is needed to increase the enzyme production by
induced mutagenesis.
Acknowledgement
The authors wish to thank Mr U Gangi Reddy and
Mr M Dileep Kumar, and Mr S Krishnan of The
INDIAN J BIOTECHNOL, JULY 2006
344
Energy and Resources Institute, New Delhi for their
help in the end-product analysis by HPLC, and for
16S rRNA sequence analysis, respectively. We are
grateful to the Department of Science & Technology,
Government of India for the financial support through
grant No.SP/SO/A89-98 during the course of this
investigation.
References
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Bauer M W, Driskill L E & Kelly R M, Glycosyl hydrolases
from hyperthermophilic microorganisms, Curr Opin
Biotechnol, 9 (1998) 141-145.
Sonnleitner B, Biotechnology of thermophilic bacteria–
Growth, products, and application, Adv Biochem Eng
Biotechnol, 28 (1983) 69-138.
Saha B C, Shen G-J, Srivastava K C, LeCureux L W & Zeikus
J G, New thermostable α-amylase-like pullulanase from
thermophilic Bacillus sp. 3183, Enzyme Microb Technol,
11(1989) 760-764.
Takasaki Y, Pullulanase-amylase complex enzyme from
Bacillus subtilis, Agric Biol Chem, 51 (1987) 9-16.
Saha B C & Zeikus J G, Novel highly thermostable
pullulanase from thermophiles, Trends Biotechnol, 7 (1989)
234-238.
Vieille C & Zeikus G J, Hyperthermophilic enzymes: Sources,
uses and molecular mechanisms for thermostability, Microbiol
Mol Biol Rev, 65 (2001) 1-43.
De Stefanis V A & Turner E W, Modified enzyme system to
inhibit bread firming method for preparing same and use of
same in bread and other bakery products, US Pat 4299848, 10
November, 1981; http://www.uspto.gov/patft/index.html.
Sahlstrom S & Brathen E, Effects of enzyme preparations for
baking, mixing time and resting time on bread quality and
bread staling, Food Chem, 58 (1997) 175-180.
Van der Maarel M J E C, Van der Veeen B, Uitdehaag J C M,
Leemhuis H & Dijkhvizen L, Properties and applications of
starch-converting enzymes of the α-amylase family, J
Biotechnol, 94 (2000) 137-155.
Niehaus F, Bertoldo C, Gahler M & Antranikian G,
Extremophiles as a source of novel enzymes for industrial
applications, Appl Microbiol Biotechnol, 51 (1999) 711-729.
Antranikian G, Physiology and enzymology of thermophilic
anaerobic bacteria degrading starch, FEMS Microbiol Rev, 75
(1990) 201-218.
Bertoldo C & Antranikian G, Starch-hydrolyzing enzymes
from thermophilic archaea and bacteria, Curr Opin Chem Biol,
6 (2002) 151-160.
Satyanarayana T, Noorwez S M, Kumar S, Rao J L U M,
Ezhilvannan M et al, Development of an ideal starch
saccharification process using amylolytic enzymes from
thermophiles, Biochem Soc Trans, 32 (2004) 276-278.
Malhotra R, Noorwez S M & Satyanarayana T, Production and
partial characterization of thermostable and calciumindependent α-amylase of an extreme thermophile, Bacillus
thermoleovorans NP54, Lett Appl Microbiol, 31 (2000) 378384.
Bernfeld P, Amylases, α and β, Methods Enzymol, 1 (1955)
149-158.
Miller G L, Use of dinitrosalicylic reagent for determination of
reducing sugars, Anal Chem, 31 (1959) 426-428.
Kumar S & Satyanarayana T, Purification and kinetics of a
raw
starch-hydrolyzing,
thermostable,
and
neutral
glucoamylase of the thermophilic mold Thermomucor indicaeseudaticae, Biotechnol Prog, 19 (2003) 936-944.
18 Abdullah M & French D, Substrate specificity of pullulanase,
Arch Biochem Biophys, 137 (1970) 483-493.
19 Brunswick J M, Kelly C T & Fogarty W M, The
amylopullulanase of Bacillus sp. DSM 405, Appl Microbiol
Biotechnol, 51 (1999) 170-175.
20 Lin L-L, Tsau M-R & Chu W-S, General characteristics of
thermostable amylopullulanases and amylases from the
alkalophilic Bacillus sp. TS-23, Appl Microbiol Biotechnol, 42
(1994) 51-56.
21 Shen G-J, Srivastava K C, Saha B C & Zeikus J G,
Physiological and enzymatic characterization of a novel
pullulan-degrading thermophilic Bacillus strain 3183, Appl
Environ Microbiol, 33 (1990) 340-344.
22 Klingeberg M, Hippe H & Antranikian G, Production of novel
pullulanases at high concentrations by two newly isolated
thermophilic Clostridia, FEMS Microbiol Lett, 69 (1990) 145152.
23 Brown S H & Kelly R M, Characterization of amylolytic
enzymes, having both α-1,4 and α-1,6 hydrolytic activity,
from the thermophilic archaea, Pyrococcus furiosus and
Thermococcus littoralis, Appl Environ Microbiol, 59 (1993)
2614-2621.
24 Suzuki Y, Nagayama T, Nakano H & Oishi K, Purification and
characterization of a maltotriogenic α-amylase I and a
maltogenic α-amylase II capable of cleaving α-1,6-bonds in
amylopectin, Starch/Stärke, 39 (1987) 211-214.
25 Ganghofner D, Kellermann J, Staudenbauer W L &
Bronnenmeier K, Purification and properties of an
amylopullulanase, a glucoamylase and an α-glucosidase in the
amylolytic enzyme system of Thermoanaerobacterium
thermosaccharolyticum, Biosci Biotechnol Biochem, 62 (1998)
302-308.
26 Sata H, Umeda M, Kim C-H, Taniguchi H & Maruyama Y,
Amylase-pullulanase enzyme produced by Bacillus circulans
F-2, Biochim Biophys Acta, 991 (1989) 338-394.
27 Ara K, Saeki K, Igarashi K, Takaiwa M, Uemura T et al,
Purification
and
characterization
of
an
alkaline
amylopullulanase with both α-1,4 and α-1,6 hydrolytic activity
from alkalophilic Bacillus sp. KSM-1378, Biochim Biophys
Acta, 1243 (1995) 315-324.
28 Shaku M, Koike S & Udaka S, Cultural conditions for protein
production by Bacillus brevis No. 47, Agric Biol Chem, 44
(1980) 99-103.
29 Demain A C R, Influence of environment on the control of
enzyme synthesis, J Appl Chem Biotechnol, 22 (1972) 245259.
30 Mathupala S P & Zeikus J G, Improved purification and
biochemical characterization of extracellular amylopullulanase
from Thermoanaerobacter ethanolicus 39E, Appl Microbiol
Biotechnol, 39 (1993) 487-493.
31 Rudiger A, Jorgensen P L & Antranikian G, Isolation and
characterization of a heat-stable pullulanase from the
hyperthermophilic archaeon Pyrococcus woesei after cloning
and expression of its gene in Escherichia coli, Appl Environ
Microbiol, 61 (1995) 567-575.
32 Srivastava R A R & Baruah J N, Culture conditions for
17
NOORVEZ et al: AMYLOPULLULANASE PRODUCTION BY G. THERMOLEOVORANS
production of thermostable amylase by Bacillus
stearothermophilus, Appl Environ Microbiol, 52 (1986) 179184.
33 Priest F G, Extracellular enzyme synthesis in the Genus
Bacillus, Bacteriol Rev, 41 (1977) 711-753.
34 Hyun H H & Zeikus J G, Regulation and genetic
enhancement of glucoamylase and pullulanase production in
Clostridium thermohydrosulfuricum, J Bacteriol, 164 (1985)
1146-1152.
35 Welker N E & Campbell L L, Induction of α-amylase of
Bacillus stearothermophilus by maltodextrins, J Bacteriol,
36
37
38
345
86 (1963) 687-691.
Saito N & Yamamoto K, Regulating factors affecting αamylase production in Bacillus licheniformis, J Bacteriol,
121 (1975) 850-856.
Humphrey A, Shake flask to fermerter: What have we
learnt?, Biotechnol Prog, 14 (1998) 3-7.
Koch R, Canganella F, Hippe H, Jhanke K D & Antranikian
G, Purification and properties of a thermostable pullulanase
from a newly isolated anaerobic bacterium Fervidobacterium
pennavorans Ven5, Appl Environ Microbiol, 63 (1997)
1088-1094.