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1.
Ana Flávia Azevedo Carvalho Maurício Boscolo Roberto da Silva Henrique Ferreira Eleni Gomes 《Journal of microbiology (Seoul, Korea)》2010,48(4):452-459
An α-glucosidase enzyme produced by the fungus Thermoascus aurantiacus CBMAI 756 was purified by ultra filtration, ammonium sulphate precipitation, and chromatography using Q Sepharose, Sephacryl
S-200, and Superose 12 columns. The apparent molecular mass of the enzyme was 83 kDa as determined in gel electrophoresis.
Maximum activity was observed at pH 4.5 at 70°C. Enzyme showed stability stable in the pH range of 3.0–9.0 and lost 40% of
its initial activity at the temperatures of 40, 50, and 60°C. In the presence of ions Na+, Ba2+, Co2+, Ni2+, Mg2+, Mn2+, Al3+, Zn2+, Ca2+ this enzyme maintained 90–105% of its maximum activity and was inhibited by Cr3+, Ag+, and Hg2+. The enzyme showed a transglycosylation property, by the release of oligosaccharides after 3 h of incubation with maltose,
and specificity for short maltooligosaccharides and α-PNPG. The Km measured for the α-glucosidase was 0.07 μM, with a Vmax of 318.0 μmol/min/mg. 相似文献
2.
V. Cabiaux A. Phalipon R. Wattiez P. Falmagne J. M. Ruysschaert M. Kaczorek 《Molecular microbiology》1988,2(3):339-346
The toxB gene of Corynebacterium diphtheriae bacteriophage β encoding the B fragment of diphtheria toxin was cloned into an inducible expression vector. When expressed In Escherichia coli, fragment B was not proteolysed and was indistinguishable, by immunological criteria, from wild-type C. diphthsriae derived fragment B. Soluble fragment B was partially purified from the cytoplasm by saline precipitation steps and was shown to compete with the wild-type diphtheria toxin for binding to receptors of sensitive eukaryotic cells. A complete diphtheria toxin was reconstituted by formation of the disulphide bridge between purified fragment A and recombinant fragment B, which migrates at the expected Mr on Western blots and which was able to block protein synthesis by ADP-ribosylation of elongation factor–2, thereby indicating that the recombinant fragment B had retained its biological activity. 相似文献
3.
The -glucosidase (-d-glucoside glucohydrolase, EC 3.2.1.20) of Pseudomonas fluorescens W was partially purified by (NH4)2SO4 fractionation, Sephadex G-200 and DEAE-cellulose column chromatography. The enzyme showed great specificity for maltose hydrolysis, with very little action against polymeric forms. Sucrose, isomaltose, -methylglucoside, and maltobionic acid were not hydrolyzed. Turanose was a strong competitive inhibitor, and glucose a weaker one. Tris (2-amino-2-hydroxymethylpropan-1:3-diol) inhibited enzyme activity significantly only at alkaline pH. Mercuric, cupric, and silver cations strongly inhibited, and EDTA (ethylenediaminetetraacetate) weakly inhibited the enzyme. The isolated enzyme was rather unstable even at 4° C, and was destroyed by freezing and lyophilization. Inositol and albumin had a slightly protective effect. Sulfhydryl-binding reagents strongly inhibited the enzyme.Abbreviations PNPG
paranitrophenyl--d-glucoside
- PCMB
parachloromercuribenzoate
- DEAE
diethylaminoethyl cellulose
- NEM
N-ethylmaleimide
- EDTA
ethylenediaminetetraacetate 相似文献
4.
Glutamate synthase (EC 1 4 1 14) was purified to homogeneity from a cell\|free extract of Streptomyces lincolnensis by precipitation with streptomycin sulfate and ammonium sulfate, and column chromatography on DEAE\| cellulose, Sepharose 6B, DEAE\|sephadex A\|50, hydroxyapatite and Sephadex G\|150. The enzyme activity is stabilized by addition of α ketoglutarate, PMSF,EDTA, β mercaptoethanol and glycerol. The native enzyme has a molecular weight of 138 000 and is composed of two nonidentical subunits with molecular weights of 81 000 and 57 000. Spectroscopic examination of the enzyme gave absorption maximum at 280 and none at 380 and 440 nm, indicating the absence of iron and flavin. The enzyme shows optimum activity at pH 7.2 and 30℃. Km values for α ketoglutarate, L\|glutamine and NADH were 417, 435, and 52.1 μmol/L, respectively. When NADPH was substituted for NADH as reductant, there was approximately 13% of the control activity. The activity of this glutamate synthase is inhibited by its products (i.e. glutamate and NAD), several metal ions, amino acids and tricarboxylic acid cycle intermediates. 相似文献
5.
The starch-degrading yeastCandida tsukubaensis CBS 6389 secreted amylase at high activity when grown in a medium containing soluble starch. The extracellular α-amylase
activity was very low. The major amylase component was purified by DEAE-Sephadex A-50 chromatography and Ultrogel AcA 44 gel
filtration and characterized as a glucoamylase. The enzyme proved to be a glycoprotein with a molecular weight of 56000. The
glucoamylase had a temperature optimum at 55°C and displayed highest activity in a pH range of 2.4–4.8. Acarbose strongly
inhibited the purified glucoamylase. Debranching activity was present as demonstrated by the release of glucose from pullulan. 相似文献
6.
Larissa A. Balabanova Irina Yu. Bakunina Olga I. Nedashkovskaya Ilona D. Makarenkova Tatiana S. Zaporozhets Natalia N. Besednova Tatiana N. Zvyagintseva Valery A. Rasskazov 《Marine biotechnology (New York, N.Y.)》2010,12(1):111-120
An α-galactosidase capable of converting B red blood cells into the universal blood type cells at the neutral pH was produced
by a novel obligate marine bacterium strain KMM 701 (VKM B-2135 D). The organism is heterotrophic, aerobic, and halophilic
and requires Na+ ions and temperature up to 34°C for its growth. The strain has a unique combination of polysaccharide-degrading enzymes.
Its single intracellular α-galactosidase exceeded other glycoside hydrolases in the level of expression up to 20-fold. The
α-galactosidase was purified to determine the N-terminal amino acid sequences and new activities. It was found to inhibit
Corynebacterium diphtheria adhesion to host buccal epithelium cell surfaces with high effectiveness. The nucleotide sequence of the homodimeric α-galactosidase
indicates that its subunit is composed of 710 amino acid residues with a calculated Mr of 80,055. This α-galactosidase shares structural property with 36 family glycoside hydrolases. The properties of the enzyme
are likely to be highly beneficial for medicinal purposes. 相似文献
7.
Ravi-Kumar K Venkatesh KS Umesh-Kumar S 《Applied microbiology and biotechnology》2007,74(5):1011-1015
The 53-kDa amylase secreted by Aspergillus niger due to proteolytic processing of the precursor starch-hydrolyzing enzyme was resistant to acarbose, a potent α-glucosidase
inhibitor. The enzyme production was induced when A. niger was grown in starch medium containing the inhibitor. Antibodies against the precursor enzyme cross-reacted with the 54-kDa
Taka-amylase protein of A. oryzae. It resembled Taka-amylase in most of its properties and also hydrolyzed starch to maltose of α-anomeric configuration. However,
it did not degrade maltotriose formed during the reaction and was not inhibited by zinc ions. 相似文献
8.
Seonghun Kim Doo-Byoung Oh Ohsuk Kwon Hyun Ah Kang 《Applied microbiology and biotechnology》2010,88(4):893-903
Sialidases can be used to transfer sialic acids from sialoglycans to asialoglycoconjugates via the trans-glycosylation reaction
mechanism. Some pathogenic bacteria decorate their surfaces with sialic acids which were often scavenged from host sialoglycoconjugates
using their surface-localized enzymes. In this study, we constructed an in vitro trans-sialylation system by reconstructing
the exogenous sialoglycoconjugate synthesis system of pathogens on the surfaces of yeast cells. The nanH gene encoding an extracellular sialidase of Corynebacterium diphtheriae was cloned into the yeast surface display vector pYD1 based on the Aga1p–Aga2p platform to immobilize the enzyme on the surface
of the yeast Saccharomyces cerevisiae. The surface-displayed recombinant NanH protein was expressed as a fully active sialidase and also transferred sialic acids
from pNP-α-sialoside, a sialic acid donor substrate, to human-type asialo-N-glycans. Moreover, this system was capable of attaching sialic acids to the glycans of asialofetuin via α(2,3)- or α(2,6)-linkage.
The cell surface-expressed C. diphtheriae sialidase showed its potential as a useful whole cell biocatalyst for the transfer of sialic acid as well as the hydrolysis
of N-glycans containing α(2,3)- and α(2,6)-linked sialic acids for glycoprotein remodeling. 相似文献
9.
Summary An -glucosidase was purified from Aspergillus carbonarious CCRC 30414 over 20 fold with 37 % recovery. Its molecular mass was estimated to be 328 kDa by gel filtration with an optimum pH from 4.2 to 5.0, and pI=5.0. The optimum temperature is at 60°C over 40 min. The enzyme was partially inhibited by 5 mM Ag+, Hg2+, Ba2+, Pb2+, and Aso4
+. 相似文献
10.
Toxigenic Corynebacterium diphtheriae was grown from skin lesions of 44 indigent patients seen at the emergency or out-patient departments of this hospital, 43 of them within the last 16 months of the study period. In all cases staphylococci or hemolytic streptococci were also present in the wounds. An increase in the incidence of clinical diphtheria occurred in the few months preceding and overlapping the period of recognition of the cutaneous infections. The gravis strains, which accounted for the majority of the infections, were sensitive to erythromycin and to penicillin, but were relatively resistant to cloxacillin. 相似文献
11.
《Bioscience, biotechnology, and biochemistry》2013,77(6):1142-1143
The transglycosylation reaction of partially purified β-glucosidase from sesame seeds with cellobiose is described. Sesame β –glucosidase was partially purified by ammonium sulfate fractionation and gel filtration. The molecular weight of the enzyme was 200,000 by gel filtration. Sesame β-glucosidase showed strong transfer activity to synthesize the trisaccharide from cellobiose. The optimum pH and temperature of the transglycosylation reaction were pH 4.0 and 60°C. 相似文献
12.
Arzu Coleri Cihan Birgul Ozcan Cumhur Cokmus 《World journal of microbiology & biotechnology》2009,25(12):2205-2217
We have partially purified and characterized two new thermostable exo-α-1,4-glucosidases (E.C.3.2.1.20) isolated from Geobacillus sp. A333 and thermophilic bacterium A343 strains. A333 α-glucosidase showed optimum activity at 60°C, pH 6.8 and had a value
of 1.38 K
m for the pNPG substrate, whereas these results were found to be 65°C, 7.0 and 0.85, respectively for A343 enzyme. Specificity for 20
different substrates and thin layer chromatography studies demonstrated that the A333 enzyme had high transglycosylation activity,
and A343 had wide substrate specificity. The substrate specificity of A333 α-glucosidase was determined as maltose, dextrin,
turanose, maltotriose, maltopentaose, meltotetraose, maltohexaose and phenyl-α-d-glycopyranoside. On the other hand, the A343 α-glucosidase mostly hydrolyzed dextrin, turanose, maltose, phenyl-α-d-glucopyranoside, maltotriose, maltotetraose, maltopentaose, isomaltose, saccharose and kojibiose by acting α-1,2, α-1,3,
α-1,4 and α-1,6 bonds of these substrates. The relative activites of A333 and A343 enzymes were determined to be 83 and 92%
when incubated at 60°C for 5 h whereas, the pH of 50% inactivation at 60°C for 15 h were determined to be pH 4.5/10.0 and
pH 5.0/10.0, respectively. In addition, the results not only showed that both of the α-glucosidases were stable in a wide
range of pH and temperatures, but were also found to be resistant to most of the denaturing agents, inhibitors and metal ions
tested. With this study, thermostable exo-α-1,4-glucosidases produced by two new thermophilic strains were characterized as
having biotechnological potential in transglycosylation reactions and starch hydrolysis processes. 相似文献
13.
Abstract A 8-glucosidase was isolated from Candida guilliermondii, a yeast capable of growth on cellobiose. The enzyme was partially purified by treatment with polyethylcneimine and ammonium sulfate precipitation. Further purification was achieved by affinity chromatography using a Sepharose 4B matrix to which oxidized salicin was coupled through adipic dihydrazide. The final product was a 12.5-fold purification of the crude extract with a recovery of 27% of the initial enzyme activity. Polyacryl-amide disc electrophoresis of the purified enzyme gave a single band. A Km of 1.25 × 10?4M was obtained using p_-nitrophenyl-β-D_-glucopyranoside as the substrate. The optimum pH for enzyme activity was 6.8. Maximum activity was observed at a temperature of 37°C. Enzyme activity was completely inhibited by Hg++, Pb++, and Zn++ ions. The molecular weight of the enzyme is 48, 000 as estimated by sucrose density gradient centri-fugation. 相似文献
14.
Aureobasidium pullulans produced an intracellular-glucosidase. The enzyme was purified 124-fold by solubilization with Triton X-100, Q-Sepharose treatment, hydroxylapatite, octyl-Sepharose column chromatography, and gel filtration on Sephacryl S-200, and had a specific activity of 316.82 U/mg protein. The enzyme displayed an optimum pH for its action at 4.0 and was fully stable at pH 3.0–6.0 at 50°C. The-glucosidase was completely stable up to 60°C and had an optimum activity at 60°C. The partially purified enzyme preparation hydrolyzed maltose, isomaltose, sucrose, and trehalose at relative rates of 100, 60, 47, and 50, respectively, and had little or no activity on polysaccharides. TheK
m
value for maltose hydrolysis at pH 4.0 and 50°C was 1.85mm. The enzyme was not adsorbed onto raw corn starch and showed little raw starch degradation. The-glucosidase did not require any metal ion for activity. This represents the first characterization of intracellular-glucosidase fromA. pullulans.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned. 相似文献
15.
The high-molar mass from of β-glucosidase fromAspergillus niger strain NIAB280 was purified to homogeneity with a 46-fold increase in purification by a combination of ammonium sulfate precipitation,
hydrophobic interaction, ion-exchange and gel-filtration chromatography. The native and subunit molar mass was 330 and 110
kDa, respectively. The pH and temperature optima were 4.6–5.3 and 70°C, respectively. TheK
m andk
cat for 4-nitrophenyl β-d-glucopyranoside at 40°C and pH 5 were 1.11 mmol/L and 4000/min, respectively. The enzyme was activated by low and inhibited
by high concentrations of NaCl. Ammonium sulfate inhibited the enzyme. Thermolysin periodically inhibited and activated the
enzyme during the course of reaction and after 150 min of proteinase treatment only 10% activity was lost with concomitant
degradation of the enzyme into ten low-molar-mass active bands. When subjected to 0–9 mol/L transverse urea-gradient-PAGE
for 105 min at 12°C, the nonpurified β-glucosidase showed two major bands which denatured at 4 and 8 mol/L urea, respectively,
with half-lives of 73 min. 相似文献
16.
S M Kotwal M M Gote M I Khan J M Khire 《Journal of industrial microbiology & biotechnology》1999,23(1):661-667
The thermophilic fungus Humicola sp constitutively produces intracellular α-galactosidase (1.33 U mg−1 protein) within 48 h at 45°C in shaken flasks, when grown in a medium containing 7% wheat bran extract as a carbon source
and 0.5% yeast extract as a nitrogen source. The enzyme has been purified to homogeneity by ultrafiltration, ethanol precipitation,
DEAE cellulose and Sephacryl S-300 chromatography with a 124-fold increase in specific activity and 29.5% recovery. The molecular
weight of the enzyme is 371.5 kDa by gel filtration on Sephacryl S-300 and 87.1 kDa by SDS-polyacrylamide gel electrophoresis.
The enzyme has an optimum temperature of 65°C and an optimum pH of 5.0. Humicola α-galactosidase is a glycoprotein with 8.3% carbohydrate content and is acidic in nature with a pI of 4.0. The K
m
S for p-nitrophenyl-α-D-galactopyranoside, O-nitrophenyl-α-D-galactopyranoside, raffinose and stachyose are 0.279, 0.40, 1.45 and 1.42 mM respectively. The enzyme activity was strongly
inhibited by Ag+ and Hg2+. D-Galactose inhibited α-galactosidase competitively and the inhibition constant (K
i) for galactose was 11 mM.
Received 28 January 1999/ Accepted in revised form 07 April 1999 相似文献
17.
Vladimir Bezjak 《Antonie van Leeuwenhoek》1954,20(1):269-272
Summary The purpose of this study was to find out whether there existed any difference betweenC. diphtheriae typemitis always present in the nasal cavity of ozaena patients (the so-calledC. belfanti) andC. diphtheriae of themitis type found in diphtheria patients or carriers.
Studying in details all the morphological, cultural and biochemical properties, a difference was found to exist in the reduction
of nitrates. This test was investigated in 55 strains ofC. belfanti and 45 strains of themitis type ofC. diphtheriae. All the strains ofC. belfanti yielded negative results in the reduction of nitrates, while all strains ofC. diphtheriae typemitis reduced nitrates within 24 hours.
The value of this observation was shorty discussed. 相似文献
18.
Bravo-Torres JC Calvo-Méndez C Flores-Carreón A López-Romero E 《Antonie van Leeuwenhoek》2003,84(3):169-178
A soluble α-glucosidase presumably involved in the general carbohydrate metabolism was purified from E. histolytica trophozoites by a three-step procedure consisting of ion exchange, size exclusion and adsorption chromatographies in columns
of Mono Q, Sepharose CL-6B and hydroxyapatite, respectively. After the last step, the enzyme was enriched about 673-fold over
the starting material with a yield of 18%. SDS-PAGE revealed the presence in the purified preparations of two polypeptides
of comparable intensity exhibiting molecular weights of 43 and 68 kDa. These results and the molecular weight of 243 kDa determined
by gel filtration, suggest that the native enzyme is a heterotetramer consisting of two copies of each subunit. Some properties
were investigated to determine the role of this activity in glycoprotein processing. Analysis of linkage specificity using
a number of substrates indicated a preferential hydrolysis of isomaltose (α1,6) with much less activity on nigerose (α1,3)
and maltose (α1,4). Trehalose (α1,1), kojibiose (α1,2) and cellobiose (β1,4) were not cleaved at all. As expected, isomaltose
competed away hydrolysis of 4-methylumbelliferyl-α-D-glucoside with a higher efficiency than nigerose and maltose. Hydrolysis
of the fluorogenic substrate was competitively inhibited by glucose and 6-deoxy-D-glucose with comparable Ki values of 0.23 and 0.22 mM, respectively. Sensitivity of the enzyme to the α-glucosidase inhibitors 1-deoxynojirimycin, castanospermine
and australine largely depended on the substrate utilized to determine activity. 1-Deoxynojirimycin and castanospermine inhibited
isomaltose hydrolysis in a competitive manner with Ki values of 1.2 and 1.5 μM, respectively. The properties of the purified enzyme are consistent with a general glycosidase probably
involved in glycogen metabolism.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
19.
Ascorbic acid (AA) increased the phosphatase activity (pH 6.8) in 10,000 g supernatants from Anacystis nidulans. The enzyme activated by AA was deactivated by dehydroascorbic acid (DHAA). The modulation by AA/DHAA of phosphatase activity in Anacystis appears to be specific; a number of other redox compounds, known to modulate other enzymes, had no effect on the Anacystis phosphatase. A purified phosphatase preparation from Anacystis was also deactivated by DHAA. In contrast, the purified enzyme was not activated by AA, suggesting that a factor mediating the effect of AA was lost during purification. Another factor was found to protect the purified phosphatase against deactivation by DHAA. The enzyme was characterized as a phosphatase with a broad substrate specificity, an apparent molecular weight of 19,000, and a pH optimum of 6.0–7.0. Dialysis of the enzyme preparation against EDTA abolished the phosphatase activity which could be restored by Zn2+ ions and partially restored by Co2+ ions. Crude extracts also contained a latent enzyme, the phosphatase activity of which could be detected in the presence of Co2+ ions only. Zn2+ ions did not activate this enzymatically inactive protein. The Co2+-dependent phosphatase had an apparent mol. wt. of 40,000, a broad substrate specificity, and an alkaline pH-optimum. Infection of Anacystis cultures by cyanophage AS-1 resulted in a decrease in phosphatase activity. The enzyme present in 10,000 g supernatants from infected cells could not be modulated by the AA/DHAA system.Abbreviations AA
ascorbic acid
- DEAE
diethylamino ethyl
- DHAA
dehydroascorbic acid
- EDTA
ethylene-diaminetetra-acetate
- G6PDH
glucose-6-phosphate dehydrogenase
- GSH
reduced glutathione
- GSSG
oxidized glutathione
- HMP
hexose monophosphate
- P
i
inorganic phosphorus
- pNPP
p-nitrophenylphosphate
- pNP
p-nitrophenol
- Tris
Tris(hydroxymethyl)-aminomethane 相似文献
20.
Yasuhiko Nakamura Yuko Ikeda Naoko Ebihara Keiko Tabuchi 《Bioscience, biotechnology, and biochemistry》2013,77(3):779-780
Candida pelliculosa var. acetaetherius was found to produce a β-glucosidase intracellularly. The enzyme was purified 200-fold by fractionation with ammonium sulfate and chromatography on Sephadex G-100 and DEAE Sepharose CL-6B. After polyacrylamide gel electrophoresis of the final fraction, one protein band corresponding to β-glucosidase was detected. The molecular weights determined by SDS-PAGE and by Sephacryl S-300 chromatography were 90,000 and 360,000, respectively, suggesting that the enzyme was a tetramer. The enzyme was a glycoprotein and its isoelectric point was at pH 4.9. It’s optimum pH and temperature were 6.5 and 50°C, respectively. The enzyme activity was inhibited by Zn2 +, Hg2 +, Cu2 +, Co2 +, p-chloromercuribenzoate, and glucose. Inhibition by glucose was competitive with a Ki value of 6.5 mm. Specificity studies for substrates indicated that the enzyme was specific for the p-configuration of sugars. Km values measured at 50°C were 0.5 mm for p-nitrophenyl-β-glucoside and 37 mm for cellobiose. 相似文献