共查询到20条相似文献,搜索用时 15 毫秒
1.
Khomaini Hasan Wangsa Tirta Ismaya Idar Kardi Yandi Andiyana Susanti Kusumawidjaya Safri Ishmayana Toto Subroto Soetijoso Soemitro 《Biologia》2008,63(6):1044-1050
α-Amylase from Saccharomycopsis fibuligera R-64 was successfully purified by butyl Toyopearl hydrophobic interaction chromatography, followed by Sephadex G-25 size
exclusion and DEAE Toyopearl anion exchange chromatography. The enzyme has a molecular mass of 54 kDa, as judged by SDS PAGE
analysis. Upon tryptic digestion, two major fragments with relative molecular masses of 39 kDa and 10 kDa, which resemble
the A/B and C-terminal domains in the homologous Taka-amylase, were obtained and were successfully separated with the Sephadex
G-50 size exclusion column. The 39-kDa fragment demonstrated a similar amylolytic activity to that of the undigested enzyme.
However, it was found that the K
m value of the 39-kDa fragment was about two-times higher than that of the undigested enzyme. Moreover, thermostability studies
showed a lower half-life time for the 39-kDa fragment. These findings suggest that the 39-kDa fragment is the catalytic domain,
while the 10-kDa fragment is the C-terminal one, which plays a role in thermostability and starch binding. Although the undigested
enzyme is able to act on raw starches at room temperature, with maize starches as the best substrate, neither the undigested
enzyme nor the fragments adsorb the tested raw starches. These results propose Saccharomycopsis fibuligera α-amylase as a raw starch-digesting but not adsorbing amylase, with a similar domain organization to that of Taka-amylase A. 相似文献
2.
cDNAs of barley α-amylase andA. niger glucoamylase were cloned in oneE. coli-yeast shuttle plasmid resulting in the construction of expression secretion vector pMAG15. pMAG15 was transformed intoS. cerevisiae GRF18 by protoplast transformation. The barley α-amylase andA. niger glucoamylase were efficiently expressed under the control of promoter and terminator of yeast PGK gene and their own signal
sequence. Over 99% of the enzyme activity expressed was secreted to the medium. The recombinant yeast strain, S.cerevisiae GRF18 (pMAG15), hydrolyzes 99% of the starch in YPS medium containing 15% starch in 47 h. The glucose produced can be used
for the production of ethanol.
Project supported by the Guangdong Natural Science Foundation. 相似文献
3.
Michelin M Ruller R Ward RJ Moraes LA Jorge JA Terenzi HF Polizeli Mde L 《Journal of industrial microbiology & biotechnology》2008,35(1):17-25
An extracellular glucoamylase produced by Paecilomyces variotii was purified using DEAE-cellulose ion exchange chromatography and Sephadex G-100 gel filtration. The purified protein migrated
as a single band in 7% PAGE and 8% SDS-PAGE. The estimated molecular mass was 86.5 kDa (SDS-PAGE). Optima of temperature and
pH were 55 °C and 5.0, respectively. In the absence of substrate the purified glucoamylase was stable for 1 h at 50 and 55 °C,
with a t
50 of 45 min at 60 °C. The substrate contributed to protect the enzyme against thermal denaturation. The enzyme was mainly activated
by manganese metal ions. The glucoamylase produced by P. variotii preferentially hydrolyzed amylopectin, glycogen and starch, and to a lesser extent malto-oligossacarides and amylose. Sucrose,
p-nitrophenyl α-d-maltoside, methyl-α-d-glucopyranoside, pullulan, α- and β-cyclodextrin, and trehalose were not hydrolyzed. After 24 h, the products of starch hydrolysis,
analyzed by thin layer chromatography, showed only glucose. The circular dichroism spectrum showed a protein rich in α-helix.
The sequence of amino acids of the purified enzyme VVTDSFR appears similar to glucoamylases purified from Talaromyces emersonii and with the precursor of the glucoamylase from Aspergillus oryzae. These results suggested the character of the enzyme studied as a glucoamylase (1,4-α-d-glucan glucohydrolase). 相似文献
4.
vanKuyk PA Benen JA Wösten HA Visser J de Vries RP 《Applied microbiology and biotechnology》2012,93(1):285-293
AmyR is commonly considered a regulator of starch degradation whose activity is induced by the presence of maltose, the disaccharide
building block of starch. In this study, we demonstrate that the role of AmyR extends beyond starch degradation. Enzyme activity
assays, genes expression analysis and growth profiling on d-glucose- and d-galactose-containing oligo- and polysaccharides showed that AmyR regulates the expression of some of the Aspergillus niger genes encoding α- and β-glucosidases, α- and β- galactosidases, as well as genes encoding α-amlyases and glucoamylases. In
addition, we provide evidence that d-glucose or a metabolic product thereof may be the inducer of the AmyR system in A. niger and not maltose, as is commonly assumed. 相似文献
5.
Ochratoxin A is a mycotoxin produced by several Aspergillus and some Penicillium species which may be present in food and feed products. It can be enzymatically hydrolyzed into ochratoxin α and l-β-phenylalanine, thereby decreasing its toxicity. The ochratoxin A degradation capacity of Aspergillus niger is well known and here we report the isolation and purification of a novel enzyme from A. niger that hydrolyzes this mycotoxin. A wheat germ medium supplemented with ochratoxin A was used to produce the enzyme, which
was purified from culture filtrate by acetone precipitation and anion exchange chromatography. An overall purification of
2.5-fold with a recovery of 68% and a final specific activity of 36 U/mg was obtained. The enzyme is a metalloenzyme as it
was inhibited at 10 mM EDTA, whereas PMSF had no effect. The ochratoxin A hydrolytic enzyme presented a V
max of 0.44 μM/min and a K
m of 0.5 mM when the reaction was carried out at pH 7.5 and 37°C. 相似文献
6.
Bo Young Jeon Soo Jin Kim Dae Hee Kim Byung Kwan Na Doo Hyun Park Hung Thuan Tran Ruihong Zhang Dae Hee Ahn 《Biotechnology and Bioprocess Engineering》2007,12(5):566-573
Aspergillus niger hyphae were found to grow with unliquefied potato starch under aerobic conditions, but did not grow under anaerobic conditions.
The raw culture ofA. niger catalyzed saccharification of potato starch to glucose, producing approximately 12 g glucose/L/day/ The extracellular enzyme
activity was decreased in proportion to incubation time, and approximately 64% of initial activity was maintained after 3
days. At 50°C,A. niger hyphae growth stopped, while the extracellular enzyme activity peaked. On the basis of theA. niger growth property and enzyme activity, we designed a serial bioreactor system composed of four different reactors. Fungal hyphae
were cultivated in reactor I at 30°C, uniquefied starch was saccharified to glycose by a fungal hyphae culture in reactors
II and III at 50°C, and glucose was fermented to ethanol bySaccharomyces cerevisiae in reactor IV. The total glucose produced by fungal hyphae in reactor I and saccharification in reactor II was about 42 g/L/day.
Ethanol production in reactor IV was approximately 22 g/L/day, which corresponds to about 79% of the theoretical maximum produced
from 55 g starch/L/day. 相似文献
7.
Feng B Hu W Ma BP Wang YZ Huang HZ Wang SQ Qian XH 《Applied microbiology and biotechnology》2007,76(6):1329-1338
It has been previously reported that a glucoamylase from Curvularia lunata is able to hydrolyze the terminal 1,2-linked rhamnosyl residues of sugar chains at C-3 position of steroidal saponins. In
this work, the enzyme was isolated and identified after isolation and purification by column chromatography including gel
filtration and ion-exchange chromatography. Analysis of protein fragments by MALDI-TOF/TOF™ proteomics Analyzer indicated
the enzyme to be 1,4-alpha-D-glucan glucohydrolase EC 3.2.1.3, GA and had considerable homology with the glucoamylase from Aspergillus oryzae. We first found that the glucoamylase was produced from C. lunata and was able to hydrolyze the terminal rhamnosyl of steroidal saponins. The enzyme had the general character of glucoamylase,
which hydrolyze starch. It had a molecular mass of 66 kDa and was optimally active at 50°C, pH 4, and specific activity of
12.34 U mg of total protein−1 under the conditions, using diosgenin-3-O-α-L-rhamnopyranosyl(1→4)-[α-L-rhamnopyranosyl (1→2)]-β-D-glucopyranoside (compound II) as the substrate. Furthermore, four kinds of commercial glucoamylases from Aspergillus niger were investigated in this work, and they had the similar activity in hydrolyzing terminal rhamnosyl residues of steroidal
saponin.
This project was supported by the National Natural Science Foundation of China (NSFC; 30572333). 相似文献
8.
Efficient production of recombinant barley α-amylase has been achieved in Aspergillus niger. The cDNA encoding α-amylase isozyme 1 (AMY1) and its signal peptide was placed under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter and the A. nidulans trpC gene terminator. Secretion yields up to 60 mg/l were obtained in media optimised for α-amylase activity and low protease
activity. The recombinant AMY1 (reAMY1) was purified to homogeneity and found to be identical to native barley AMY1 with respect
to size, pI, and immunoreactivity. N-terminal sequence analysis of the recombinant protein indicated that the endogenous plant signal
peptide is correctly processed in A. niger. Electrospray ionisation/mass spectrometry gave a molecular mass for the dominant form of 44 960 Da, in accordance with the
loss of the LQRS C-terminal residues; glycosylation apparently did not occur. The activities of recombinant and native barley
α-amylases are very similar towards insoluble and soluble starch as well as 2-chloro-4-nitrophenol β-d-maltoheptaoside and amylose (degree of polymerisation = 17). Barley α-amylase is the first plant protein efficiently secreted
and correctly processed by A. niger using its own signal sequence.
Received: 22 August 1997 / Received revision: 21 November 1997 / Accepted: 29 November 1997 相似文献
9.
An amylase was purified from the culture filtrate ofTermitomyces clypeatus by ammonium sulphate precipitation, DEAE-Sephadex chromatography and gel filtration on Bio-Gel P-200 column. The electrophoretically
homogeneous preparation also exhibited hydrolytic activity (in a decreasing order) on amylose, xylan, amylopectin, glycogen,
arabinogalactan and arabinoxylan. The enzyme had characteristically endo-hydrolytic activity on all the substrates tested
and no xylose, glucose, arabinose or glucuronic acid could be detected even after prolonged enzymatic digestion of the polysaccharides.
Interestingly the enzyme had similar pH optima (5.5), temperature optima (55°C), pH stability (pH 3–10) and thermal denaturation
kinetics when acted on both starch and xylan (larch wood) .K
m values were found to be 2.63 mg/ml for amylase and 6.25 mg/ml for xylanase activity. Hill’s plot also indicated that the
enzyme contained a single active site for both activities. Hg2+ was found to be most potent inhibitor. Ca2+, a common activator for amylase activity, appeared to be an inhibitor for this enzyme. Thus it appeared that the enzyme had
multisubstrate specificity acting as α-amylase on starch and also acting as xylanase on side chain oligosaccharides of xylan
containing α-linked sugars. 相似文献
10.
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
11.
Francisco M. Reyes-Sosa Fernando P. Molina-Heredia Miguel A. De la Rosa 《Applied microbiology and biotechnology》2010,86(1):131-141
Little information is yet available on the α-amylases of cyanobacteria. Here, the presence of an α-amylase in the cyanobacterium
Nostoc sp. PCC 7119 is first demonstrated. A gene (amy1) encoding a cytoplasmic α-amylase (Amy1) protein has been identified, cloned, and overexpressed in Escherichia coli cells. The recombinant protein is a 56.7-kDa monomer, which has been purified to electrophoretic homogeneity by affinity
chromatography. The substrate specificity and end product analyses confirm that it is a calcium-dependent α-amylase enzyme,
which exhibits its maximum activity at 31°C and at pH between 6.5 and 7.5. The Amy1 protein breaks down mainly starch, is
also able to cleave glycogen and dextrin, and exhibits no activity against xylan or pullulan. So the enzyme cannot efficiently
attack the maltodextrins with degrees of polymerization below that of maltooctaose. Maltotriose, maltose, and maltotetraose
are the major products of the enzymatic reaction with starch as substrate. The enzyme shows a very high turnover number against
soluble potato starch (3,420 ± 270 s−1), as compared with other α-amylases reported in the literature. The high catalytic efficiency and relatively low optimum
temperature of the Nostoc Amy1 protein make this previously unexplored group of cyanobacterial enzymes of great interest for further physiological
studies and industrial applications. 相似文献
12.
Glucoamylase II (EC 3.2.1.3) fromAspergillus niger has 31 % α-helix, 36 %Β- structure and rest aperiodic structure at pH 4.8 as analysed by the method of Provencher and Glockner (1981,Biochemistry, 20,33). In the near ultra-violet circular dichroism spectrum the enzyme exhibits peaks at 304, 289, 282 and 257 nm and troughs
at 285, 277 and 265 nm respectively. The enzyme activity and structure showed greater stability at pH 4.8 than at pH 7.0,
were highly sensitive to alkaline pH but less sensitive to acid pH values. The enzyme retained most of its catalytic activity
and structure even on partial removal of carbohydrate moieties by periodate treatment but was less stable at higher temperatures
and storage at 30‡C. Reduction of the periodate treated enzyme did not reverse the loss of stability. Binding of the synthetic
substrate,p-nitrophenyl-α-D-glucoside, perturbed the environment around aromatic amino acids and caused a decrease in the
ordered structure. 相似文献
13.
Haiyan Sun Xiangyang Ge Weiguo Zhang 《World journal of microbiology & biotechnology》2007,23(5):603-613
Penicillium sp. X−1, isolated from decayed raw corn, produced high level of raw-starch-digesting glucoamylase (RSDG) under solid state
fermentation (SSF). Maximum enzyme yield of 306.2 U g−1 dry mouldy bran (DMB) was obtained after 36 h of culture upon optimized production. The enzyme could hydrolyse both small
and large granule starches but did not adsorb on raw starch. The enzyme exhibited maximum activity at 65°C and pH 6.5, which
provided an opportunity of synergism with α-amylase. It significantly hydrolysed 15% (w/v) raw corn starch slurry in synergism
with the commercial α-amylase and a degree of hydrolysis of 92.4% was obtained after 2 h of incubation. 相似文献
14.
InbredDrosophila melanogaster stocks were surveyed for α-glucosidases with nondenaturing gel electrophoresis using a fluorogenic substrate to stain the
gels. The glucosidase most active under these conditions is polymorphic. We established that the polymorphism is genetic in
origin and that the glucosidase was not likely to be a previously characterized enzyme. The gene encoding the enzyme was mapped
cytogenetically to 33 A1-2- 33A8-B1, confirming that this is an enzyme not yet reported inD. melanogaster. The enzyme was partially purified by elution from nondenaturing gels, which enabled us to establish that it has optimal
activity at pH 6 and interacts most strongly with α-1–4 glucosides. A developmental and tissue survey suggested that this
enzyme could have a purely digestive role or be involved in carbohydrate metabolism inside the organism. We propose that this
enzyme is involved in either starch digestion or glycogen metabolism. 相似文献
15.
Fusarium venenatum A3/5 was transformed using the Aspergillus niger expression plasmid, pIGF, in which the coding sequence for the F. solani f. sp. pisi cutinase gene had been inserted in frame, with a KEX2 cleavage site, with the truncated A. niger glucoamylase gene under control of the A. niger glucoamylase promoter. The transformant produced up to 21 U cutinase l−1 in minimal medium containing glucose or starch as the primary carbon source. Glucoamylase (165 U l−1 or 8 mg l−1) was also produced. Both the transformant and the parent strain produced cutinase in medium containing cutin. 相似文献
16.
Gulelat D. Haki Alfredo J. Anceno Sudip K. Rakshit 《World journal of microbiology & biotechnology》2008,24(11):2517-2524
Bacillus sp. GRE1 isolated from an Ethiopian hyperthermal spring produced raw-starch digesting, Ca2+-independent thermostable α-amylase. Enzyme production in shake flask experiments using optimum nutrient supplements and environmental
conditions was 2,360 U l−1. Gel filtration chromatography yielded a purification factor of 33.6-fold and a recovery of 46.5%. The apparent molecular
weight of the enzyme was 55 kDa as determined by SDS-PAGE. Presence or absence of Ca2+ produced similar temperature optima of 65–70°C. The optimum pH was in the range of 5.5–6.0. The enzyme maintained 50% of
its original activity after 45 min of incubation at 80°C and was stable at a pH range of 5.0–9.0. The V
max and K
m values for soluble starch were 42 mg reducing sugar min−1 and 4.98 mg starch ml−1, respectively. Strong inhibitors of enzyme activity included Cu2+, Zn2+ and Fe2+. The enzyme coding gene and the deduced protein translation revealed a characteristic but markedly atypical homology to Bacillus species α-amylase sequences. The enzyme hydrolyzed wheat, corn and tapioca starch granules efficiently below their gelatinization
temperatures. Rather than the higher oligosaccharides normally produced by Bacillus α-amylases operating at high temperatures, maltose was the major hydrolysis product with the present enzyme. 相似文献
17.
Kumar GR Sharma A Kumari M Jagannadham MV Debnath M 《European biophysics journal : EBJ》2011,40(8):923-935
Equilibrium unfolding of A. niger RNase with chemical denaturants, for example GuHCl and urea, and thermal unfolding have been studied as a function of pH
using fluorescence, far-UV, near-UV, and absorbance spectroscopy. Because of their ability to affect electrostatic interactions,
pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins. ANS
binding studies have been conducted to enable understanding of the folding mechanism of the protein in the presence of the
denaturants. Spectroscopic studies by absorbance, fluorescence, and circular dichroism and use of K2D software revealed that
the enzyme has α + β type secondary structure with approximately 29% α-helix, 24% β-sheet, and 47% random coil. Under neutral
conditions the enzyme is stable in urea whereas GuHCl-induced equilibrium unfolding was cooperative. A. niger RNase has little ANS binding even under neutral conditions. Multiple intermediates were populated during the pH-induced unfolding
of A. niger RNase. Urea and temperature-induced unfolding of A. niger RNase into the molten globule-like state is non-cooperative, in contrast to the cooperativity seen with the native protein,
suggesting the presence of two parts/domains, in the molecular structure of A. niger RNase, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of the A state (molten globule state) of A. niger RNase is unique, because a low concentration of denaturant not only induces structural change but also facilitates transition
from one molten globule like state (AMG1) into another (IMG2). 相似文献
18.
Lee MH Yang SJ Kim JW Lee HS Kim JW Park KH 《Extremophiles : life under extreme conditions》2007,11(3):537-541
A gene that encodes the enzyme Pyrococcus furiosus cyclodextrin glucanotransferase (PFCGT) was cloned in Escherichia
coli. PFCGT was highly expressed in recombinant E. coli after compensation for codon usage bias using the pRARE plasmid. Purified PFCGT was extremely thermostable with an optimal
temperature and pH of 95°C and 5.0, respectively, retaining 97% of its activity at 100°C. Incubation at 60°C for 20 min during
the purification process led to a 1.5-fold increase in enzymatic activity. A time course assay of the PFCGT reaction with
starch indicated that cyclic α-1,4-glucans with DPs greater than 20 were produced at the beginning of the incubation followed
by an increase in β-CD. The major final product of PFCGT cyclization was β-CD, and thus the enzyme is a β-CGTase. 相似文献
19.
Petrus J. van Zyl V. Moodley S. H. Rose R. L. Roth W. H. van Zyl 《Journal of industrial microbiology & biotechnology》2009,36(4):611-617
The β-mannanase gene (man1) from Aspergillus aculeatus MRC11624 (Izuka) was patented for application in the coffee industry. For production of the enzyme, the gene was originally
cloned and expressed in Saccharomyces cerevisiae. However the level of production was found to be economically unfeasible. Here we report a 13-fold increase in enzyme production
through the successful expression of β-mannanase of Aspergillus aculeatus MRC11624 in Aspergillus niger under control of the A. niger glyceraldehyde-3-phosphate dehydrogenase promoter (gpd
P) and the A. awamori glucoamylase terminator (glaAT). The effect of medium composition on mannanase production was evaluated, and it was found that the glucose concentration
and the organic nitrogen source had an effect on both the volumetric enzyme activity and the specific enzyme activity. The
highest mannanase activity levels of 16,596 nkat ml−1 and 574 nkat mg−1 dcw were obtained for A. niger D15[man1] when cultivated in a process-viable medium containing corn steep liquor as the organic nitrogen source and high glucose
concentrations. 相似文献
20.
Yanan Cao Yaru Wang Kun Meng Yingguo Bai Pengjun Shi Huiying Luo Peilong Yang Zhigang Zhou Zhifang Zhang Bin Yao 《Applied microbiology and biotechnology》2009,83(5):875-884
A novel α-galactosidase gene (aga-F75) from Gibberella sp. F75 was cloned and expressed in Escherichia coli. The gene codes for a protein of 744 amino acids with a 24-residue putative signal peptide and a calculated molecular mass
of 82.94 kDa. The native structure of the recombinant Aga-F75 was estimated to be a trimer or tetramer. The deduced amino
acid sequence showed highest identity (69%) with an α-galactosidase from Hypocrea jecorina (Trichoderma reesei), a member of the glycoside hydrolase family 36. Purified recombinant Aga-F75 was optimally active at 60°C and pH 4.0 and
was stable at pH 3.0–12.0. The enzyme exhibited broad substrate specificity and substantial resistance to neutral and alkaline
proteases. The enzyme K
m values using pNPG, melibiose, stachyose, and raffinose as substrates were 1.06, 1.75, 54.26, and 8.23 mM, respectively. Compared
with the commercial α-galactosidase (Aga-A) from Aspergillus niger var. AETL and a protease-resistant α-galactosidase (Aga-F78) from Rhizopus sp. F78, Aga-F75 released 1.4- and 4.9-fold more galactose from soybean meal alone, respectively, and 292.5- and 8.6-fold
more galactose from soybean meal in the presence of trypsin, respectively. The pH and thermal stability and hydrolytic activity
of Aga-F75 make it potentially useful in the food and feed industries. 相似文献