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1.
Growing cells of a thermophilic strain ofBacillus brevis, producer of thermostable α-amylase, were immobilized by entrapment in agar gel. Optimum immobilization conditions for effective
α-amylase production in batch fermentations were established (gel concentration 3%, initial biomass concentration in the gel
0.8% (W/V), and preculture age—late exponential phase). The dynamics of α-amylase synthesis by the biocatalysts obtained under the
optimal conditions was compared with that of free cells and the operational stability of the biocatalysts was studied in semicontinuous
cultivation experiments. Maximum α-amylase yields (252% of the control) were achieved after the second cycle of cultivation.
Scanning electron microscopy was used to characterize the bacteria entrapped in agar gel. 相似文献
2.
A simple structured model for biomass and extracellular enzyme production with recombinant Saccharomyces cerevisiae YPB-G 总被引:1,自引:0,他引:1
A simple structured model is proposed for simulating batch cultivation data on growth, substrate utilization, and heterologous
enzyme production of recombinant Saccharomyces cerevisiae YPB-G. The enzyme is a fusion protein displaying α-amylase and glucoamylase activities. Cell growth is modulated mainly by
intracellular substrate and ethanol concentrations. Intracellular substrate concentration is evaluated by means of the extracellular
substrate and biomass concentrations. Extracellular α-amylase and glucoamylase activities are taken to depend on biomass concentration.
The nine parameters of the proposed model are determined using nonlinear estimation techniques, and the model is validated
against experiments not used in parameter determination. The model developed simulates glucose consumption, cell mass, α-amylase
and glucoamylase production in a batch system. Simulation and experimental results are found to be in good agreement. Journal of Industrial Microbiology & Biotechnology (2002) 29, 111–116 doi:10.1038/sj.jim.7000281
Received 07 January 2002/ Accepted in revised form 22 May 2002 相似文献
3.
As found during continuous cultivation ofBacillus licheniformis on a semisynthetic medium (glucose or maltose as C source), the specific rate of α-amylase production is proportional to
growth rate but is repressed by higher substrate concentrations. Besides glucose or maltose, peptone was also used as an alternative
carbon source during cultivation. The specific rate of production of the enzyme on maltose is half that found with glucose. 相似文献
4.
J. L. Meers 《Antonie van Leeuwenhoek》1972,38(1):585-590
The production ofα-amylase (α-1,4 glucan-4-glucan hydrolase; E.C. 3.2.1.1.) by a strain ofBacillus licheniformis has been studied in batch and continuous cultures. The synthesis of this enzyme was shown to be repressed by glucose or other
low-molecular-weight metabolisable sugars. Consequently, amylase production in a medium which contained “liquified” starch
only began after the low-molecular-weight sugars had been dissimilated. Thereafter, the dextrins in the medium were degraded
by amylase produced by the bacteria to yield further quantities of metabolisable sugars. These sugars were continuously dissimilated
by the growing organisms and never accumulated to concentrations where they would repress further amylase synthesis. A clear
analogy could thus be drawn with bacteria growing in a carbon-limited environment in a chemostat. Therefore,α-amylase production byB. licheniformis organisms growing in 3-litre chemostats was studied. No evidence was obtained to infer that an inducer was necessary for
amylase production, and it was concluded that the prime factors influencing amylase production, in this species at least,
were growth rate and catabolite repression. 相似文献
5.
Hiroyuki Uehara Bok Du Choi Enoch Y. Park Mitsuyasu Okabe 《Biotechnology and Bioprocess Engineering》2000,5(1):7-12
The expression of the mouse α-amylase gene in the methylotrophic yeast,P. pastoris was investigated. The mouse α-amylase gene was inserted into the multi-cloning site of a Pichia expression vector, pPIC9,
yielding a new expression vector pME624. The plasmid pME624 was digested withSalI orBglII, and was introduced intoP. pastoris strain GS115 by the PEG1000 method. Fifty-three transformants were obtained by the transplacement of pME624 digested withSalI orBglII into theHIS
4 locus (38 of Mut+ clone) or into theAOX1 locus (45 of Muts clone). Southern blot was carried out in 11 transformants, which showed that the mouse α-amylase gene was integrated into
thePichia chromosome. When the second screening was performed in shaker culture, transformant G2 showed the highest α-amylase activity,
290 units/ml after 3-day culture, among 53 transformants. When this expression level of the mouse α-amylase gene is compared
with that in recombinantSaccharomyces cerevisiae harboring a plasmid encoding the same mouse α-amylase gene, the specific enzyme activity is eight fold higher than that of
the recombinantS. cerevisiae. 相似文献
6.
E Dobreva A Tonkova V Ivanova M Stefanova L Kabaivanova D Spasova 《Journal of industrial microbiology & biotechnology》1998,20(3-4):166-170
Cells of Bacillus licheniformis 44MB82-G immobilized on different polymer membranes were used for production of thermostable α-amylase. The α-amylase yields
of the membrane-immobilized cells were affected by the reactive chemical groups of the carriers and the spacer size. Formaldehyde-activated
polysulphone membranes (PS-FA) were the most suitable for effective immobilization. The highest amylase yield (62% increase
of the control) and operational stability (97% residual activity after 480 h repeated batch cultivation) were obtained with
this system. This was confirmed by scanning electron micrographs. An additional increase of α-amylase production by PS-FA-membrane
immobilized cells was achieved in a fluidized-bed reactor.
Received 20 March 1997/ Accepted in revised form 08 January 1998 相似文献
7.
An extracellular raw-starch-digesting α-amylase was isolated from Geobacillus thermodenitrificans HRO10. The culture conditions for the production of α-amylase by G. thermodenitrificans HRO10 was optimized in 1.2–l bioreactor using full 24 and 32 factorial designs. From the optimal reaction conditions, a model (Y = − 594.206 − 0.178T2 − 8.448pH2 + 6.020TpH − 0.005T2pH2) was predicted, which was then used for α-amylase production. In the bioreactor studies, the enzyme yield under optimized
conditions (pH 7.1, 49°C) was 30.20 U/ml, a 51% improvement over the results (19.97 U/ml) obtained when the traditional one-factor-at-a-time
method was employed. This α-amylase does not require extraneous calcium ions for activity, which may be a commercially important
observation. 相似文献
8.
Summary The concentration and productivity of -amylase increased remarkably, 15- and 11-fold respectively, in a continuous culture of Bacillus caldolyticus DSM 405 compared with batch culture, provided starch was used as the sugar source in a casitone medium. In the casitone medium with or without glucose hardly any improvement of enzyme production was observed in continuous culture. The addition of a small amount of starch to the glucose-casitone medium had a marked effect in stimulating amylase formation in continuous culture but no effect in batch culture.It was suggested that the higher production of -amylase in the continuous culture using starch as the inducer was partly related to the predominance of some conditional non-sporulating variants with a higher amylase forming activity and to derepression of the enzyme at a low glucose concentration. 相似文献
9.
Zhi-Hua Feng Yuan-Shan Wang Yu-Guo Zheng 《Biotechnology and Bioprocess Engineering》2011,16(5):894-900
Alpha-amylase inhibitors are widely used by the pharmaceutical and agricultural industries, such as the treatment of diabetes
and obesity and insect controller. Here, we developed a colorimetric method to screen for α-amylase inhibitor producing strains or mutants with higher α-amylase inhibitor productivity. This method relies on absorbance changes at 402 nm that are due to the inhibition of α-amylase catalyzed hydrolysis of 2-Chloro-4-nitrophenyl-4-O-β-D-galactopyranosyl-maltoside by α-amylase inhibitors. The assay can be performed on a microtiter plate, making it simple and convenient. Using this method,
α-amylase inhibitor producing strains and mutants with higher α-amylase inhibitor productivity can be rapidly screened. One strain, ZJB-08196, with the highest α-amylase inhibition was isolated and identified as Actinoplanes utahensis, and one mutant with higher acarbose production was obtained by screening 3,000 variants using this method. 相似文献
10.
Growth, differential rate of α-amylase synthesis and production characteristics ofBacillus subtilis DP 1 (isolate from starch materials) in comparison with 10Bacillus strains were examined in batch fermentation. The effect of the carbon and nitrogen source was evaluated with regard to cell
growth and enzyme production. The pH optimum of enzyme activity was 6.5 and temperature optimum 60°C. 相似文献
11.
G. Satheesh kumar M. Subhosh Chandra K. V. Mallaiah P. Sreenivasulu Yong-Lark Choi 《Biotechnology and Bioprocess Engineering》2010,15(3):435-440
In this study, the production of extracellular thermostable α-amylase by newly isolated thermophilic Alicyclobacillus acidocaldarius was detected on LB agar plates containing 1.0% soluble potato starch and incubated at 60°C. This extracellular α-amylase
was purified to homogeneity by ammonium sulphate precipitation followed by Sephadex and ion-exchange chromatography. The α-amylase
was purified to 8.138 fold homogeneity with a final recovery of 58% and a specific activity of 3,239 U/mg proteins. The purified
α-amylase appeared as a single protein band on SDS-PAGE with a molecular mass of 94.5 kDa. Non-denaturing PAGE analysis showed
one major band associated with enzyme activity, indicating the absence of isoenzymes. A TLC analysis showed maltose as major
end product of the enzyme. The optimum assay temperature and pH for enzyme activity were 60°C and 6.0 respectively; however,
the enzyme activity was stable over a wide range of pH and temperatures. The α-amylase retained its activity in the presence
of the denaturing agents — SDS, Triton X-100, Tween-20, Tween-80, and was significantly inhibited by EDTA and urea. Calcium
ions increased the enzyme activity, while Hg2+, Zn2+, and Co2+ had inhibitory effects. The K
m and V
max values were found to be 2.9 mg/mL and 7936 U/mL respectively. 相似文献
12.
Summary Potato peel was found to be a superior substrate for solid state fermentation, compared to wheat bran, for the production
of α-amylase by two thermophilic isolates of Bacillus licheniformis and Bacillus subtilis. Under optimal conditions, B. licheniformis produced 270 units/ml and 175 units/ml of α-amylase on potato peel and wheat bran, respectively, while the corresponding
values for B. subtilis were 600 units/ml and 265 units/ml. The enzyme from B.␣licheniformis was optimally active at 90 °C and pH 9.0, while that from B. subtilis at 60 °C and pH 7.0. The nature of the experimental data permitted excellent polynomial fits, on the basis of which, two
master equations, corresponding to the isolated strains, were derived for estimation of enzyme activity for any set of values
of temperature, particle size, moisture, and incubation time within the indicated ranges. 相似文献
13.
α-Amylase (EC 3.2.1.1) expression was found in calli of French bean (Phaseolus vulgaris L. cv Goldstar). We examined enzyme activity in the calli to investigate influence of gibberellin and sugars on enzyme expression.
After subculture of the calli, α-amylase activity decreased, and then increased at a stationary phase of callus growth. Exogenous
application of gibberellin and an inhibitor of gibberellin synthesis, uniconazole, did not have any significant effects on
the enzyme expression. Sugar starvation increased the activity, while addition of metabolizable sugars, such as sucrose, glucose
and maltose, to the medium repressed expression. Addition of 6% mannitol, a non-metabolizable sugar, to the medium induced
higher α-amylase expression as compared to addition of 3% mannitol. This result suggests that osmotic stress enhances α-amylase
activity in the calli. Furthermore, high concentrations of agar in the medium increased α-amylase activity in the calli. It
is probable that high concentrations of agar prevented incorporation of nutrient into the calli and induced the α-amylase
activity in the calli. 相似文献
14.
Purification of extracellular α-amylase from Bacillus subtilis KIBGE HAS was carried out by ultrafiltration, ammonium sulfate precipitation and gel filtration chromatography. The enzyme
was purified to homogeneity with 96.3-fold purification with specific activity of 13011 U/mg. The molecular weight of purified
α-amylase was found to be 56,000 Da by SDS-PAGE. Characteristics of extracellular α-amylase showed that the enzyme had a Km
and V
max value of 2.68 mg/ml and 1773 U/ml, respectively. The optimum activity was observed at pH 7.5 in 0.1 M phosphate buffer at
50°C. The amino acid composition of the enzyme showed that the enzyme is rich in neutral/non polar amino acids and less in
acidic/polar and basic amino acids. The N-terminal protein sequence of 10 residues was found to be as Ser-Ser-Asn-Lys-Leu-Thr-Thr-Ser-Trp-Gly
(S-S-N-K-L-T-T-S-W-G). Furthermore, the protein was not N-terminally blocked. The sequence of α-amylase from B. subtilis KIBGE HAS was a novel sequence and showed no homology to other reported α-amylases from Bacillus strain. 相似文献
15.
Analysis of the kinetics of α-amylase production in a batch and a fed-batch culture ofBacillus subtilis made it possible to derive a konetic model of the process describing mutual interactions between growth and production. The
specific growth rate is limited by the concentration of both corn-steep liquor and starch. Higher concentrations of reducing
sugars in the medium also inhibit growth. The overall production of α-amylase is a result of an equilibrium between the rate
of enzyme production and its degradation due to the effect of environment. The actual specific production rate is directly
proportional to the specific production rate is directly proportional to the specific growth rate (characterizing the physiological
state of the culture) and is inhibited by higher concentrations of corn-steep liquor in the medium. 相似文献
16.
Jari Olavi Vehmaanperä Matti Pellervo Korhola 《Applied microbiology and biotechnology》1986,23(6):456-461
Summary Theα-amylase gene ofBacillus amyloliquefaciens has previously been cloned into pUB110 to give the recombinant plasmid, pKTH10 (Palva 1982. Gene 19:81–87). Strains transformed
by this plasmid are promising candidates for industrialα-amylase production. The stability of pKTH10 was determined in variousB. subtilis strains possessing specific alleles which affect the level ofα-amylase secretion.B. subtilis strains carrying pKTH10 were cultivated in starch-containing medium for up to 50 generations without antibiotic selection
and then screened for the presence of pKTH10. The plasmid proved stable enough (< 1.0% cured after 50 generations) for industrial
batchwise enzyme production in two strains, but in asacU9 strain (thesacU9 mutation increases concominantly the production ofα-amylase levansucrase and proteases) 99.9% of cells had lost pKTH10 after 50 generations, although the parental plasmid (pUB110)
was stable in this strain (0.09% cured after 50 generations). The instability of pKTH10 in thesacU9 strain seems somehow to be related to high expression of the clonedα-amylase gene: when grown in a medium restrictingα-amylase production, only 0.53% ofsacU9 cells had lost pKTH10 after 50 generations. 相似文献
17.
The effect of biomass concentration on the formation of Aspergillus oryzaeα-amylase during submerged cultivation with A. oryzae and recombinant A. nidulans strains has been investigated. It was found that the specific rate of α-amylase formation in chemostats decreased significantly
with increasing biomass concentration in the range of approx. 2–12 g dry weight kg−1. When using a recombinant A. nidulans strain in which the gene responsible for carbon catabolite repression of the A. oryzaeα-amylase gene (creA) was deleted, no significant decrease in the specific rate of α-amylase formation was observed. On the basis of the experimental
results, it is suggested that the low value of the specific α-amylase productivity observed at high biomass concentration
is caused by slow mixing of the concentrated feed solution in the viscous fermentation medium.
Received: 13 January 2000 / Received revision: 30 June 2000 / Accepted: 1 July 2000 相似文献
18.
Kamakshi Gupta P. K. Mishra Pradeep Srivastava 《Biotechnology and Bioprocess Engineering》2009,14(2):207-212
Lovastatin, a hypocholesterolemic agent, is a secondary metabolite produced by filamentous microorganism Aspergillus terreus in submerged batch cultivation. Lovastatin production by pellets and immobilized siran cells was investigated in an airlift
reactor. The process was carried out by submerged cultivation in continuous mode with the objective of increasing productivity
using pellet and siran supported growth of A terreus. The continuous mode of fermentation improves the rate of lovastatin production. The effect of dilution rate and aeration
rate were studied in continuous culture. The optimum dilution rate for pellet was 0.02 h−1 and for siran carrier was 0.025 h−1. Lovastatin productivity using immobilized siran carrier (0.0255 g/L/h) was found to be greater than pellets (0.022 g/L/h).
The productivity by both modes of fermentation was found higher than that of batch process which suggests that continuous
cultivation is a promising strategy for lovastatin production. 相似文献
19.
Neeta D. Kalve Purushottam R. Lomate Vandana K. Hivrale 《Arthropod-Plant Interactions》2012,6(2):213-220
Insects feeding on stored grains cause considerable damage to harvested cereals and legumes every year. The use of α-amylase
inhibitors to interfere with the pest’s digestion process has become an interesting alternative biocontrolling agent. In this
study, we have studied the interactions of α-amylase inhibitors from Albizia lebbeck seeds with the amylases of coleopteran and lepidopteran insect pests. We isolated and purified the α-amylase inhibitor using
acetone precipitation and gel filtration chromatography. Two prominent activity bands of α-amylase inhibitors were detected
in electrophoretic analysis using 8% starch PAGE. We found that the α-amylase inhibitor, isolated as a monomer, had a molecular
weight of 14.4 kDa. The α-amylase inhibitor was purified 36.15-fold with gel filtration chromatography. Its specific activity
was determined at 14.4 U/mg/min. Feeding analysis of Tribolium confusum larvae on a diet containing purified α-amylase inhibitor from Albizia lebbeck revealed that survival of the larvae was severely affected, with the highest mortality rate occurring on the fifth day of
feeding. We found that the isolated α-amylase inhibitor inhibits T. confusum and Helicoverpa armigera α-amylases in electrophoretic analysis as well as in solution assays. The isolated α-amylase inhibitor was found to be resistant
to commercial protease as well as T. confusum and H. armigera digestive proteinases. The isolated α-amylase inhibitor was degraded by heating above 60°C. Our results suggest that A. lebbeck α-amylase inhibitor could be a useful future biocontrolling agent. 相似文献
20.
Pyrococcus woesei (DSM 3773) α-amylase gene was cloned into pET21d(+) and pYTB2 plasmids, and the pET21d(+)α-amyl and pYTB2α-amyl vectors obtained
were used for expression of thermostable α-amylase or fusion of α-amylase and intein in Escherichia coli BL21(DE3) or BL21(DE3)pLysS cells, respectively. As compared with other expression systems, the synthesis of α-amylase in
fusion with intein in E. coli BL21(DE3)pLysS strain led to a lower level of inclusion bodies formation—they exhibit only 35% of total cell activity—and
high productivity of the soluble enzyme form (195,000 U/L of the growth medium). The thermostable α-amylase can be purified
free of most of the bacterial protein and released from fusion with intein by heat treatment at about 75°C in the presence
of thiol compounds. The recombinant enzyme has maximal activity at pH 5.6 and 95°C. The half-life of this preparation in 0.05
M acetate buffer (pH 5.6) at 90°C and 110°C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following
incubation for 2 h at 120°C. Maltose was the main end product of starch hydrolysis catalyzed by this α-amylase. However, small
amounts of glucose and some residual unconverted oligosaccharides were also detected. Furthermore, this enzyme shows remarkable
activity toward glycogen (49.9% of the value determined for starch hydrolysis) but not toward pullulan. 相似文献