共查询到20条相似文献,搜索用时 15 毫秒
1.
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
2.
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
3.
A β-1,3-glucanase was detected, using laminarin as substrate, in the culture broth of Chaetomium sp. Major activity was associated with a 70 kDa protein band visualized on a polyacrylamide gel. β-1,3-Glucanase was purified
by a one-step, native gel purification procedure. Optimal activity was observed at pH 6.0 and 30 °C (over 30 min). It could
degrade cell walls of plant pathogens including Rhizoctonia solani, Gibberella zeae, Fusarium sp., Colletotrichum gloeosporioides and Phoma sp. The N-terminal amino acid residues of the purified β-1,3-glucanase are PYQLQTP, which do not exhibit homology to other fungal β-1,3-glucanases
suggesting it may be a novel enzyme.
Received 20 July 2005; Revisions requested 2 August 2005 and 27 September 2005; Revisions received 16 September 2005 and 3
November 2005; Accepted 6 November 2005 相似文献
4.
Amylolytic industrial polyploid strains of Saccharomyces cerevisiae (ATCC 4126, ATCC 9763 and ATCC 24858) expressing a glucoamylase gene (GAM1) or an α-amylase gene (AMY) from Debaryomyces occidentalis were developed. The glucoamylase activity of S. cerevisiae ATCC 9763 expressing the GAM1 gene was 3.7-times higher than that of D. occidentalis. On the other hand, α-amylase activity in the corresponding strain expressing the D. occidentalis
AMY gene increased 10-times relative to D. occidentalis. These two recombinant yeast strains expressing the GAM1 gene and AMY gene, respectively were cultured simultaneously to produce both glucoamylase and α-amylase for efficient one-step utilization
of starch. Growth, substrate utilization and enzyme activity of these strains are described. 相似文献
5.
Alkaline stable (pH 7.75–12.5) urease from Sporosarcina ureae was purified over 400-fold by ion exchange and hydrophobic interaction chromatography. The cytoplasmic enzyme was remarkably
active with a specific activity of greater than 9300 μmol urea degraded min-1 mg protein-1 at pH 7.5, where it has optimal activity. Although S. ureae is closely related to Bacillus pasteurii, known to posses a homopolymeric urease containing 1 nickel per subunit [M
r=65000], the S. ureae enzyme is comprised of three subunits [apparent M
r=63100 (α), 14500 (β), and 8500 (γ)] in an estimated ∝βγ stoichiometry and contains 2.1±0.6 nickel ions per ∝βγ unit as measured
by atomic absorption spectrometry. Stationary phase cultures sometimes possessed low levels of urease activity, but the specific
activity of cell extracts of partially purified urease preparations from such cultures could be elevated by heat treatment,
dilution, or dialysis to values comparable to those observed in samples from exponentially grown cells. 相似文献
6.
To evaluate the potential of the production of the ectomycorrhizal fungus Tricholoma matsutake to produce carbohydrases, (1) the distribution of carbohydrase activities among the different strains (18 strains) was investigated
and (2) the abilities of T. matsutake and saprophytic fungi to produce β-glucosidase were compared. The results showed that the carbohydrase productions patterns
of T. matsutake still resemble one another. Moreover, this fungus exhibited markedly higher β-glucosidase than did the saprophytic mushrooms.
Tricholoma matsutake showed weak production of α-amylase and α-glucosidase in a static cultur filtrate. On the other hand, glucoamylase activity
was not observed. Surprisingly, we discovered that β-glucosidase demonstrated strong activity. This finding suggests that
this fungus has saprotrophic abilities. The carbohydrase production systems in T. matsutake were characterized from our experimental results. Also, we point out some weak points in the carbohydrase production systems
of T. matsutake. 相似文献
7.
For the first time, a β-glucosidase gene from the edible straw mushroom, Volvariella volvacea V1-1, has been over-expressed in E. coli. The gene product was purified by chromatography showing a single band on SDS-PAGE. The recombinant enzyme had a molecular
mass of 380 kDa with subunits of 97 kDa. The maximum activity was at pH 6.4 and 50 °C over a 5 min assay. The purified enzyme
was stable from pH 5.6–8.0, had a half life of 1 h at 45 °C. The β-glucosidase had a Km of 0.2 mM for p-nitrophenyl-β-D-glucopyranoside. 相似文献
8.
An N-acetylglucosaminidase produced by Streptomyces cerradoensis was partially purified giving, by SDS-PAGE analysis, two main protein bands with Mr of 58.9 and 56.4 kDa. The Km and Vmax values for the enzyme using p-nitrophenyl-β-N-acetylglucosaminide as substrate were of 0.13 mM and 1.95 U mg−1 protein, respectively. The enzyme was optimally activity at pH 5.5 and at 50 °C when assayed over 10 min. Enzyme activity
was strongly inhibited by Cu2+ and Hg2+ at 10 mM, and was specific to substrates containing acetamide groups such as p-nitrophenyl-β-N-acetylglucosaminide and p-nitrophenyl-β-D-N,N′-diacetylchitobiose. 相似文献
9.
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
10.
The filamentous fungus Stachybotrys sp has been shown to possess a rich β-glucosidase system composed of five β-glucosidases. One of them was already purified
to homogeneity and characterized. In this work, a second β-glucosidase was purified and characterized. The filamentous fungal
A19 strain was fed-batch cultivated on cellulose, and its extracellular cellulases (mainly β-glucosidases) were analyzed.
The purified enzyme is a monomeric protein of 78 kDa molecular weight and exhibits optimal activity at pH 6.0 and at 50°C.
The kinetic parameters, K
m and V
max, on para-nitro-phenyl-β-d-glucopyranosid (p-NPG) as a substrate were, respectively, 1.846 ± 0.11 mM and 211 ± 0.08 μmol min−1 ml−1. One interesting feature of this enzyme is its high stability in a wide range of pH from 4 to 10. Besides its aryl β-glucosidase
activity towards salicin, methylumbellypheryl-β-d-glucoside (MU-Glc), and p-NPG, it showed a true β-glucosidase activity because it splits cellobiose into two glucose monomers. This enzyme has the
capacity to synthesize short oligosaccharides from cellobiose as the substrate concentration reaches 30% with a recovery of
40%. We give evidences for the involvement of a transglucosylation to synthesize cellotetraose by a sequential addition of
glucose to cellotriose. 相似文献
11.
Teng D Fan Y Yang YL Tian ZG Luo J Wang JH 《Applied microbiology and biotechnology》2007,74(5):1074-1083
β-1,3-1,4-glucanase (EC3.2.1.73) as an important industrial enzyme has been widely used in the brewing and animal feed additive
industry. To improve expression efficiency of recombinant β-1,3-1,4-glucanase from Bacillus licheniformis EGW039(CGMCC 0635) in methylotrophic yeast Pichia pastoris GS115, the DNA sequence encoding β-1,3-1,4-glucanase was designed and synthesized based on the codon bias of P. pastoris, the codons encoding 96 amino acids were optimized, in which a total of 102 nucleotides were changed, the G+C ratio was simultaneously
increased from 43.6 to 45.5%. At shaking flask level, β-1,3-1,4-glucanase activity is 67.9 and 52.3 U ml−1 with barley β-glucan and lichenan as substrate, respectively. At laboratory fermentor level, the secreted protein concentration
is approximately 250 mg l−1. The β-1,3-1,4-glucanase activity is 333.7 and 256.7 U ml−1 with barley β-glucan and lichenan as substrate, respectively; however, no activity of this enzyme on cellulose is observed.
Compared to the nonoptimized control, expression level of the optimized β-1,3-1,4-glucanase based on preferred codons in P. pastoris shown a 10-fold higher level. The codon-optimized enzyme was approximately 53.8% of the total secreted protein. The optimal
acidity and temperature of this recombinant enzyme were pH 6.0 and 45°C, respectively. 相似文献
12.
Pectin is one of the major cell wall polysaccharides found in dicotyledonous plants. We have solubilized and partially purified
a β-(1→4)-galactosyltransferase (GalT) involved in the synthesis of the β-(1→4)-galactan side chains of pectin. The enzyme
protein was almost completely solubilized by mixing a crude microsomal preparation of etiolated 6-day-old soybean (Glycine max Merr.) hypocotyls with a detergent, Triton X-100 (0.75%, w/v), in buffer. The solubilized enzyme was partially purified by
ion-exchange chromatography. The crude membrane-bound GalT transferred Gal from UDP-Gal onto 2-aminobenzamide (AB)-derivatized
β-(1→4)-galactoheptaose (Gal7-AB), leading to the formation of Gal8–11-AB by attachment of a series of one to four galactosyl residues; this is similar to what has previously been observed for
2-aminopyridine-derivatized β-(1→4)-galactooligomer acceptors (Konishi et al. in Planta 218:833–842, 2004). The partially purified GalT, by contrast, was able to transfer more than 25 galactosyl residues and elongated the chains
to about Gal35-AB, thus almost reaching the length (43–47 Gal units) of native β-(1→4)-galactan side chains found in pectic polysaccharides
from soybean cotyledons (Nakamura et al. in Biosci Biotechnol Biochem 66:1301–1313, 2002). Enzyme activity increased with increasing chain length of β-(1→4)-galactooligomers and reached maximal activity at heptaose,
whereas galactooligomers higher than heptaose showed lower acceptor efficiency.
Sugars described in this paper belong to the d-series unless otherwise noted. 相似文献
13.
An extracellular β-1,3-glucanase with antifungal properties was secreted by the novel mycoparasite, Periconia byssoides. The glucanase has a molecular mass of 35 kDa estimated by SDS-PAGE. Its optimum activity was at pH 6.0 and 50°C (over 2 h).
The purified β-1,3-glucanase was capable of degrading cell walls, and inhibiting mycelia growth and spore germination of plant
pathogenic fungi including Fulvia fulva, Fusarium sp. and Rhizoctonia solani. The N-terminal amino acid residues of the purified β-1,3-glucanase are LKNGGPSFGA, which do not have any homology with previously described glucanases, suggesting it may be a
novel member of the fungal β-1,3-glucanases.
Chao Lin and Jinkui Yang contributed equally to this work. 相似文献
14.
Neustroev KN Golubev AM Sinnott ML Borriss R Krah M Brumer H Eneyskaya EV Shishlyannikov S Shabalin KA Peshechonov VT Korolev VG Kulminskaya AA 《Glycoconjugate journal》2006,23(7-8):501-511
Comparative studies of the transglycosylation and hydrolytic activities have been performed on the Rhodothermus marinus β-1,3-glucanase (laminarinase) and its M133A, M133C, and M133W mutants. The M133C mutant demonstrated near 20% greater rate
of transglycosylation activity in comparison with the M133A and M133W mutants that was measured by NMR quantitation of nascent
β(1-4) and β(1-6) linkages. To obtain kinetic probes for the wild-type enzyme and Met-133 mutants, p-nitrophenyl β-laminarin oligosaccharides of degree of polymerisation 2–8 were synthesized enzymatically. Catalytic efficiency
values, k
cat/K
m, of the laminarinase catalysed hydrolysis of these oligosaccharides suggested possibility of four negative and at least three
positive binding subsites in the active site. Comparison of action patterns of the wild-type and M133C mutant in the hydrolysis
of the p-nitrophenyl-β-D-oligosac- charides indicated that the increased transglycosylation activity of the M133C mutant did not result
from altered subsite affinities. The stereospecificity of the transglycosylation reaction also was unchanged in all mutants;
the major transglycosylation products in hydrolysis of p-nitrophenyl laminaribioside were β-glucopyranosyl-β-1,3-D-glucopy- ranosyl-β-1,3-D-glucopyranose and β-glucopyranosyl-β-1,
3-D-glucopyranosyl-β-1,3-D-glucpyranosyl-β-1,3-D- glucopyranoxside.
In a memoriam of Dr. Kirill N. Neustroev. All we, his friends and colleagues, mourn for his sudden death. He was a bright
and talented scientist, brilliant manager and good friend. 相似文献
15.
Hua Li Jing Chen Anna Li Duo-Chuan Li 《World journal of microbiology & biotechnology》2007,23(9):1297-1303
A thermostable extracellular β-1,3-glucanase from Chaetomium thermophilum was purified to homogeneity by fractional ammonium sulfate precipitation, Pheny1-Sepharose hydrophobic interaction chromatography,
ion exchange chromatography on DEAE-Sepharose and gel filtration on Sephacryl S-100. SDS-PAGE of the purified enzyme showed
a single protein band of molecular weight 76.3 kDa. The enzyme exhibited optimum catalytic activity at pH 6.0 and 60 °C. It
was thermostable at 50 °C, and retained 90% activity after 60 min at 60 °C. The half-life at 65 °C, 70 °C and 80 °C was 55 min,
21.5 min, and 5 min, respectively. The N-terminal amino acid sequence (8 residues) of the enzyme was HWLGDIPH. The HPLC analysis
showed that the only enzymatic product formed from laminarin by the purified β-1,3-glucanase was glucose, indicating that
the enzyme is an exo-β-1,3-glucanase (EC 3.2.1.58). 相似文献
16.
Ginsenoside Rb1 is the most predominant ginsenoside in Panax species (ginseng) and the hydrolysis of this ginsenoside produces pharmaceutically active compounds. Caulobacter leidyia GP45, one of the isolates having strong β-glucosidase-producing activity, converted ginsenoside Rb1 to the active metabolites by 91%. The structures of the resultant metabolites were identified by NMR. Ginsenoside Rb1 had been consecutively converted to ginsenoside Rd (1), F2 (2) and compound K (3) via the hydrolyses of 20-C β-(1→6)-glucoside, 3-C β-(1→2)-glucoside, and 3-C β-glucose of ginsenoside Rb1. 相似文献
17.
Nascimento AM Brandão MG Oliveira GB Fortes IC Chartone-Souza E 《Antonie van Leeuwenhoek》2007,92(1):95-100
The activity of Eremanthus erythropappus oil (EO) and some of its compounds and their potential synergistic interaction with ampicillin against different strains
of Staphylococcus aureus were investigated. Determination of chemical composition of EO by gas chromatography-mass spectrometry (GC/MS) and bioguided
chemical fractionation led to the identification of β-bisabolene as the main active compound. A synergistic bactericidal activity
of EO or β-bisabolene with ampicillin against Staphylococcus aureus was observed in a time-kill assay. EO and β- bisabolene have the potential to restore the effectiveness of ampicillin against
resistant S. aureus. 相似文献
18.
Youn-Ju Jung Ju-Soon Yoo Yong-Seok Lee In-Hye Park Sun-Hee Kim Sang-Cheol Lee Masaaki Yasuda Soo-Yeol Chung Yong-Lark Choi 《Biotechnology and Bioprocess Engineering》2007,12(3):265-270
In this study, the extracellular enzyme activity ofBacillus sp. A8-8 was detected on LB agar plates containing 0.5% of the following substrates: carboxymethylcellulose (CMC), xylan,
cellulose, and casein, respectively. The β-1,3-1,4 glucanase produced fromBacillus sp. A8-8 was purified by ammonium sulfate and hydrophobic chromatography. The molecular size of the protein was estimated
by SDS-PAGE as approximately 33 kDa. The optimum pH and temperature for the enzyme activity were 6.0 and 60°C, respectiveley.
However, enzyme activity was shown over a broad range of pH values and temperatures. The purified β-1,3-1,4 glucanase retained
over 70% of its original activity after incubation at 80°C for 2 h, and showed over 40% of its original activity within the
pH range of 9 to 12. This suggests that β-1,3-1,4 glucanase fromBacillus sp. A8-8 is thermostable and alkalistable. In addition, β-1,3-1,4 glucanase had higher substrate specificity to lichenan
than to CMC. Finally the activity of the endoglucanase was inhibited by Fe3+, Mg2+, and Mn2+ ions. However Co2+ and Ca2+ ions were increased its activity.
These authors contributed equally to this work. 相似文献
19.
β-Mannanase catalyzes endo-wise hydrolysis of the backbone of mannan and heteromannan, which are abundant in the cell wall
structure of ungerminated leguminous seeds. The mature β-mannanase originated from Bacillus subtilis was expressed in Pichia pastoris, a methylotrophic yeast, using the leader peptide sequence of Saccharomyces cerevisiae
α-factor. The cultivation of β-mannanase expressing Pichia pastoris yields up to 1.8 g/L protein. In the supernatant the activity of the 40 kDa—total mannanase attained a level of 1102.0 IU/mL.
The properties of the β-mannanase were characterized. Optimum pH and temperature for the recombinant enzyme were 5.5 and 50°C respectively. The enzyme
was stable at pH 5.0–10.0 and maintained over 30% original activity after incubating at 70°C for 30 min.
__________
Translated from China Biotechnology, 2005, 26(7): 52–56 [译自: 中国生物工程杂志] 相似文献
20.
Theodoros Goulas Athanasios Goulas George Tzortzis Glenn R. Gibson 《Applied microbiology and biotechnology》2009,82(6):1079-1088
Four different β-galactosidases (previously named BbgI, BbgII, BbgIII and BbgIV) from Bifidobacterium bifidum NCIMB41171 were overexpressed in Escherichia coli, purified to homogeneity and their biochemical properties and substrate preferences comparatively analysed. BbgI was forming
a hexameric protein complex of 875 kDa, whereas BbgII, BbgIII and BbgIV were dimers with native molecular masses of 178, 351
and 248 kDa, respectively. BbgII was the only enzyme that preferred acidic conditions for optimal activity (pH 5.4–5.8), whereas
the other three exhibited optima in more neutral pH ranges (pH 6.4–6.8). Na+ and/or K+ ions were prerequisite for BbgI and BbgIV activity in Bis–Tris-buffered solutions, whereas Mg++ was strongly activating them in phosphate-buffered solutions. BbgII and BbgIII were slightly influenced from the presence
or absence of cations, with Mg++, Mn++ and Ca++ ions exerting the most positive effect. Determination of the specificity constants (k
cat/K
m) clearly indicated that BbgI (6.11 × 104 s−1 M−1), BbgIII (2.36 × 104 s−1 M−1) and especially BbgIV (4.01 × 105 s−1 M−1) are highly specialised in the hydrolysis of lactose, whereas BbgII is more specific for β-d-(1→6) galactobiose (5.59 × 104 s−1 M−1) than lactose (1.48 × 103 s−1 M−1). Activity measurements towards other substrates (e.g. β-d-(1→6) galactobiose, β-d-(1→4) galactobiose, β-d-(1→4) galactosyllactose, N-acetyllactosamine, etc.) indicated that the β-galactosidases were complementary to each other by hydrolysing different substrates and thus contributing in a different
way to the bacterial physiology. 相似文献