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1.
An extracellular α-amylase produced by a cassava-fermenting strain ofMicrococcus luteus was purified 26-fold by gel filtration and ion-exchange chromatography. The molar mass was estimated to be approximately
56 kDa. The optimum temperature of the enzyme was 30°C, optimum pH 6.0 and optimum substrate concentration was 0.6% (W/V). Treatment of the enzyme at 70°C for 10 min resulted in 70% loss of activity. The activation energy was determined to be
34.8 kJ/mol. The activity of the enzyme was enhanced by Mg2+, Ca2+, K+, Na+ and inhibited by EDTA, KCN and citric acid. The enzyme may find some application in local food processing. 相似文献
2.
Jinender Jain Anil Kumar Davender Singh 《Preparative biochemistry & biotechnology》2013,43(8):718-724
AbstractA novel protease-resistant and thermostable phytase from Bacillus subtilis subsp. subtilis JJBS250 was purified 36-fold to homogeneity with a combination of ammonium sulfate precipitation followed by Q-Sepharose and Sephadex G-50 chromatographic techniques. The estimated molecular mass of the purified phytase was 46?kDa by electrophoresis with optimal activity at pH 7.0 and 70?°C. About 19% of original activity was maintained at 80?°C for 10?min. Phytase activity was stimulated in presence of surfactants like Tween-20, Tween-80, and Triton X-100 and metal ions like Ca+2, K+, and Co+2 and it was inhibited by SDS and Mg+2, Al+2, and Fe+2. Purified enzyme showed specificity to different salts of phytic acid and values of Km and Vmax were 0.293?mM and 11.49 nmoles s?1, respectively for sodium phytate. The purified enzyme was resistant to proteases (trypsin and pepsin) that resulted in amelioration of food nutrition with simultaneous release of inorganic phosphate, reducing sugars, and soluble protein. 相似文献
3.
Aspergillus flavus grown in a liquid medium containing pectin as the sole carbon source produced extracellular enzymes which degraded the 1,4-α-d-glycosidic bonds of pectin. The products of degradation were characteristic of substances produced by transeliminase. Synthesis
of this enzyme was repressed by the addition of sucrose, glucose, fructose and maltose. The crude enzyme was partially purified
by a combination of ultrafiltration and ammonium sulfate precipitation. The partially purified enzyme was separated by molecular
exclusion chromatography into three components A, B and C, with molar masses ranging from 13.2 to 64 kDa. Only fraction B
exhibited enzymic activity and further fractionated by ion-exchange chromatography into four components I–IV. Among these
components, only fractions I and II possessed transeliminase activity. Both fractions had an optimum activity at pH 8.5 and
35°C, and were stimulated by Ca2+, Mg2+, Na+ and K+ but inhibited by EDTA and DNP. The apparentK
m for the degradation of pectin by fractions I and II were 6.2 and 8.0 g/L, respectively. 相似文献
4.
An extracellular polygalacturonase was isolated from 5-day culture filtrates of Thermoascus aurantiacus CBMAI-756 and purified by gel filtration and ion-exchange chromatography. The enzyme was maximally active at pH 5.5 and 60–65°C.
The apparent K
m with citrus pectin was 1.46 mg/ml and the V
max was 2433.3 μmol/min/mg. The apparent molecular weight of the enzyme was 30 kDa. The enzyme was 100% stable at 50°C for 1 h
and showed a half-life of 10 min at 60°C. Polygalacturonase was stable at pH 5.0–5.5 and maintained 33% of initial activity
at pH 9.0. Metal ions, such as Zn+2, Mn+2, and Hg+2, inhibited 50, 75 and 100% of enzyme activity. The purified polygalacturonase was shown to be an endo/exo-enzyme, releasing
mono, di and tri-galacturonic acids within 10 min of hydrolysis. 相似文献
5.
An acidic polygalacturonase (PG) secreted by Rhizopus oryzae MTCC-1987 in submerged fermentation condition has been purified to electrophoretic homogeneity using ammonium sulphate fractionation and anion exchange chromatography on diethylaminoethyl cellulose. The purified enzyme gave a single protein band in sodium dodecyl sulphatepolyacrylamide gel electrophoresis analysis with a molecular mass corresponding to 75.5 kDa. The K m and k cat values of the PG were 2.7 mg/mL and 2.23 × 103 s?1, respectively, using citrus polygalacturonic acid as the substrate. The optimum pH of the purified PG was 5.0 and it does not loose activity appreciably if left for 24 hours in the pH range from 5.0 to 12.0. The optimum temperature of purified enzyme was 50°C and the enzyme does not loose activity below 30°C if exposed for two hours. The purified enzyme showed complete inhibition with 1 mM Ag+, Hg2+ and KMnO4, while it was stimulated to some extent by Co2+. The purified PG exhibited retting of Crotalaria juncea fibre in absence of ethylenediaminetetraacetic acid. 相似文献
6.
Kui Wang Gang Li Shi Qin Yu Chen Ting Zhang Yu Huan Liu 《Applied microbiology and biotechnology》2010,88(1):155-165
A novel β-galactosidase gene, zd410, was isolated by screening a soil metagenomic library. Sequence analysis revealed that zd410 encodes a protein of 672 amino acids with a predicted molecular weight of 78.6 kDa. The recombinant ZD410 was expressed and
purified in Pichia pastoris, with a yield of ca. 300 mg from 1 L culture. The purified enzyme displayed optimal activity at 38°C and pH 7.0. Given that
the enzyme had 54% of the maximal activity at 20°C and 11% of the maximal activity at close to 0°C, ZD410 was regarded as
a cold-adapted β-galactosidase. ZD410 displays high enzymatic activity for its synthetic substrate-ONPG (o-nitrophenyl-β-d-galactopyranoside, 243 U/mg) and its natural substrate-lactose (25.4 U/mg), while its activity was slightly stimulated by
addition of Na+, K+, or Ca2+ at low concentrations. ZD410 is a good candidate of β-galactosidases for food industry after further study. 相似文献
7.
Youshuang Zhu Haibo Zhang Mingle Cao Zhenzhen Wei Feng Huang Peiji Gao 《Biotechnology and Bioprocess Engineering》2011,16(5):1027-1035
Production of laccase using a submerged culture of Trametes versicolor sdu-4 was optimized using a central composite design of the Response Surface Methodology. Optimized conditions gave a laccase
yield of 4,213 U/L which was approximately three times of that in basal medium. The laccase was purified to homogeneity using
a three-step process. The overall yield of the purification was 58%, with a purification fold of 11.4 and a specific activity
of 1320.7 U/mg protein. The molecular mass of the laccase was 60 kDa. The optimum pH values of the enzyme were 2.2, 3.7, and
7 for the oxidations of ABTS, DMP, and syringaldazine, respectively. The enzyme had adaptability to a broad pH range and high
temperature and wsa stable at pH 3.0 ∼ 10.0. The half-life of this laccase at 70°C was 2.2 h. Methyl red, 2-bromophenol, and
4-bromophenol were oxidized by the purified laccase in the absence of mediators. Purified laccase was effective in the decolorization
of several dyes and was not inhibited by Cu2+, Mn2+, Zn2+, Na+, K+, Mg2+, Ba2+, and Ca2+ at 5 mM. These excellent characteristics made it a highly attractive candidate for industrial use. 相似文献
8.
9.
Paula Marcela Duque Jaramillo Jürgen Andreaus Geraldo Pereira de Souza Neto Carlos Frederico de Souza Castro Edivaldo Ximenes Ferreira Filho 《Biocatalysis and Biotransformation》2015,33(5-6):310-322
An extracellular pectinase (PEC-I) was isolated from the crude extract of Aspergillus oryzae when grown on passion fruit peel (PFP) as the carbon source and partially purified by ultra filtration, gel filtration and ion-exchange chromatography procedures. Pectinase activity was predominantly found in the retentate. The pectinase from retentate (PEC-Ret) was most active at 50?°C and pH 7.0 and stable at 50?°C with a half-life of approximately 8?h. PEC-I showed higher activity at pH 4.5 and 55?°C, 70?°C and 75?°C and was inhibited by cations (Ag+, Fe2+, Fe3+, Co2+, Ca2+ and Hg2+), EDTA, tannic acid and vanillin. On the other hand, PEC-I was activated by Cu2+, ferulic acid, cinnamic acid and 4-hydroxybenzoic acid. The gel under denaturing conditions of PEC-Ret and PEC-I samples showed a protein band of ~45?kDa coincident with that found by staining for pectinase activity. In the bioscouring of cotton fabric the PEC-Ret pectinase preparation led to a better wettability and removed more pectin from the cotton fibers than the commercial enzyme preparation Viscozyme L, but was less effective than a commercial alkaline pectate lyase preparation and alkaline scouring. The incubation of PEC-Ret with guava juice resulted in a 4.15% decrease in juice viscosity. 相似文献
10.
Fang Gong Tong Zhang Zhenming Chi Jun Sheng Jing Li Xianghong Wang 《Biotechnology and Bioprocess Engineering》2008,13(5):533-539
The extracellular inulinase of the marine yeast Pichia guilliermondii strain 1 was purified to homogeneity resulting in a 7.2-fold increase in specific inulinase activity. The molecular mass
of the purified enzyme was estimated to be 50.0 kDa. The optimal pH and temperature for the purified enzyme were 6.0 and 60°C,
respectively. The enzyme was activated by Mn2+, Ca2+, K+, Li+, Na+, Fe3+, Fe2+, Cu2+, and Co2+, but Mg2+, Hg2+, and Ag+ inhibited activity. The enzyme was strongly inhibited by phenylmethanesulphonyl fluoride (PMSF), iodoacetic acid, EDTA, and
1, 10-phenanthroline. The K
m and V
max values of the purified inulinase for inulin were 21.1 mg/mL and 0.08 mg/min, respectively. A large number of monosaccharides
were detected after the hydrolysis of inulin. The deduced protein sequence from the cloned P. guilliermondii strain 1 inulinase gene contained the consensus motifs R-D-P-K-V-F-W-H and W-M-N-D-P-N-G, which are conserved among the inulinases
from other microorganisms. 相似文献
11.
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
12.
Hideaki Yamada Akihiko Nagao Hiroshi Nishise Yoshiki Tani 《Bioscience, biotechnology, and biochemistry》2013,77(9):2333-2339
NAD+-dependent glycerol dehydrogenase from Cellulomonas sp. NT3060 was purified by a procedure of 10 steps involving crystallization. Dihydroxyacetone was identified as the oxidation product of glycerol with the enzyme. The purified enzyme did not lose activity on heating below 60°C. The enzyme oxidized other alcohols such as 1,2-propanediol, 2,3-butanediol and glycerol-α-monochlorohydrin, beside glycerol. The enzyme activity was inhibited by p-chloromercuribenzoate, Zn2+, Cu2+ and Cd2+. Oxidation of glyberol was activated by Na+ and reduction of dihydroxyacetone was activated by K+ at pH 7.5. 相似文献
13.
A cold-adapted α-amylase (ParAmy) gene from Pseudoalteromonas
arctica GS230 was cloned, sequenced, and expressed as an N-terminus His-tag fusion protein in E. coli. A recombinant protein was produced and purified with DEAE-sepherose ion exchange chromatography and Ni affinity chromatography.
The molecular weight of ParAmy was estimated to be 55 KDa with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). With an optimum
temperature for activity 30 °C, ParAmy showed 34.5% of maximum activity at 0 °C and its activity decreased sharply at above 40 °C. ParAmy was stable in the range of pH 7–8.5 at 30 °C for 1 h. ParAmy was activated by Mn2+, K+ and Na+, and inhibited by Hg2+, Cu2+, and Fe3+. N-Bromosuccinimid showed a significant repressive effect on enzyme activity. The K
m and V
max values of the α-amylase for soluble starch were 7.28 mg/mL and 13.07 mg/mL min, respectively. This research suggests that
Paramy has a good potential to be a cold-stable and alkalitolerant amylase in detergent industry. 相似文献
14.
Purification and Characterization of Extracellular Phytase from a Marine Yeast <Emphasis Type="Italic">Kodamaea ohmeri</Emphasis> BG3 总被引:1,自引:0,他引:1
Li X Chi Z Liu Z Li J Wang X Hirimuthugoda NY 《Marine biotechnology (New York, N.Y.)》2008,10(2):190-197
The extracellular phytase in the supernatant of cell culture of the marine yeast Kodamaea ohmeri BG3 was purified to homogeneity with a 7.2-fold increase in specific phytase activity as compared to that in the supernatant
by ammonium sulfate fractionation, gel filtration chromatography (Sephadex™ G-75), and anion-exchange chromatography (DEAE
Sepharose Fast Flow Anion-Exchange). According to the data from sodium dodecyl sulfate-polyacrylamide gel electrophoresis,
the molecular mass of the purified enzyme was estimated to be 98.2 kDa while the molecular mass of the purified enzyme was
estimated to be 92.9 kDa and the enzyme was shown to be a monomer according to the results of gel filtration chromatography.
The optimal pH and temperature of the purified enzyme were 5.0 and 65°C, respectively. The enzyme was stimulated by Mn2+, Ca2+, K+, Li+, Na+, Ba2+, Mg2+ and Co2+ (at a concentrations of 5.0 mM), but it was inhibited by Cu2+, Hg2+, Fe2+, Fe3+, Ag+, and Zn2+ (at a concentration of 5.0 mM). The enzyme was also inhibited by phenylmethylsulfonyl fluoride (PMSF), iodoacetic acid (at
a concentration of 1.0 mM), and phenylgloxal hydrate (at a concentration of 5.0 mM), and not inhibited by EDTA and 1,10-phenanthroline
(at concentrations of 1.0 mM and 5.0 mM). The K
m, V
max, and K
cat values of the purified enzyme for phytate were 1.45 mM, 0.083 μmol/ml · min, and 0.93 s-1, respectively. 相似文献
15.
Chin-An Hsu Roch-Chui Yu Cheng-Chun Chou 《World journal of microbiology & biotechnology》2006,22(4):355-361
Summary β-galactosidase from Bifidobacterium longum CCRC 15708 was first extracted by ultrasonication then purified by Q Fast-Flow chromatography and gel chromatography on a
Superose 6 HR column. These steps resulted in a purification of 15.7-fold, a yield of 29.3%, and a specific activity of 168.6 U mg−1 protein. The molecular weight was 357 kDa as determined from Native-PAGE. Using o-nitrophenyl-β-d-galactopyranoside (ONPG) as a substrate, the pH and temperature optima of the purified β-galactosidase were 7.0 and 50 °C,
respectively. The enzyme was stable at a temperature up to 40 °C and at pH values of 6.5–7.0. K
m and V
max for this purified enzyme were noted to be 0.85 mM and 70.67 U/mg, respectively. Na+ and K+ stimulated the enzyme up to 10-fold, while Fe3+, Fe2+, Co2+, Cu2+, Ca2+, Zn2+, Mn2+ and Mg2+ inhibited the activity of β-galactosidase. Furthermore, although glucose, galactose, maltose, or raffinose exerted little
or no effect on the β-galactosidase activity, lactose and fructose inhibited the enzyme activity. The effect of lactose on
the enzyme activity for ONPG is probably a case of competitive inhibition.
A relatively high specific activity of β-galactosidase from B. longum CCRC 15708 could be obtained by Q Fast-Flow chromatography and gel chromatography on a Superose 6 HR column. In some aspects,
particularly the activation by monovalent cations, the properties of β-galactosidase of B. longum CCRC 15708 are different from those obtained from other sources.
Data collected in the present study are of value and indispensable when β-galactosidase from B. longum CCRC 15708 is employed in practical application. 相似文献
16.
Xylaria regalis, a wood-grown ascomycete isolated in Taiwan, produces β-glucosidase (EC 3.2.1.21) extracellularly. The β-glucosidase was
purified to homogeneity by ammonium sulfate precipitation, ion-exchange, and gel filtration chromatography. The molecular
mass of the purified enzyme was estimated to be 85 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. With
p-nitrophenyl β-D-glucopyranoside (PNPG) as the substrate at pH 5.0 and 50°C, the K
m
was 1.72 mM and V
max
was 326 μmol/min/mg. Optimal activity with PNPG as the substrate was at pH 5.0 and 50°C. The enzyme was stable at pH 5.0
at temperatures up to 50°C. The purified β-glucosidase was active against PNPG, cellobiose, sophorose, and gentiobiose, but
did not hydrolyze lactose, sucrose, Avicel, and o-nitrophenyl β-D-galactopyranoside. The activity of β-glucosidase was stimulated by Ca2+, Mg2+, Mn2+, Cd2+ and β-mercaptoethanol, and inhibited by Ag+, Hg2+, SDS, and p-chloromercuribenzoate (PCMB).
Received: 30 March 1996 / Accepted: 3 May 1996 相似文献
17.
S. Singh 《Folia microbiologica》1995,40(5):529-533
Urease extracted from an alkaliphilic diazotrophic cyanobacteriumNostoc calcicola was partially purified and some of its properties were studied. Urease purified 39-fold from the crude enzyme extract showed
its optimum activity at pH 7.5 and at 40°C with aK
m value of 120 μmol/L. The enzyme was found to be sensitive to metal cations, particularly Hg2+, Ag+ and Cu2+. 4-Hydroxymercuribenzoate (a mercapto-group inhibitor) and acetohydroxamic acid (a chelating agent of nickel) inhibited,
the enzyme activity completely. These results suggest the involvement of an SH-group and Ni2+ in the activity of urease fromN. calcicola. 相似文献
18.
F. -M. Zhu B. Du H. -S. Gao C. -J. Liu J. Li 《Applied Biochemistry and Microbiology》2010,46(6):626-632
Protoplasts of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 were prepared using cellulose and snail enzyme with 0.6 M NaCl as osmotic stabilizer. Protoplast fusion has been performed
using 35% polyethylene glycol 4,000 with 0.01 mM CaCl2. The fused protoplasts have been regenerated on regeneration medium and fusants were selected for further studies. An intracellular
(β-glucosidase (EC 3.2.1.21) was purified from the protoplast fusant of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 and characterized. The enzyme was purified 138.85-fold by ammonium sulphate precipitation, DE-22 ion exchange and Sephadex
G-150 gel filtration chromatography with a specific activity of 297.14 U/mg of protein. The molecular mass of the purified
enzyme was determined to be about 125 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme
had an optimum pH of 5.4 and temperature of 65°C, respectively. This enzyme showed relatively high stability against pH and
temperature and was stable in the pH range of 3.0–6.6. Na+, K+, Ca2+, Mg2+ and EDTA completely inhibited the enzyme activity at a concentration of 10 mM. The enzyme activity was accelerated by Fe3+. The enzyme activity was strongly inhibited by glucose, the end product of glucoside hydrolysis. The K
m and V
max values against salicin as substrate were 0.035 mM and 1.7215 μmol min−1, respectively. 相似文献
19.
Beris FS De Smet L Karaoglu H Canakci S Van Beeumen J Belduz AO 《Journal of microbiology (Seoul, Korea)》2011,49(4):641-650
The G2ALT gene was cloned and sequenced from the thermophilic bacterium Anoxybacillus gonensis G2. The gene is 666 bp long and encodes a protein 221 amino acids in length. The gene was overexpressed in E. coli and purified to homogeneity and biochemically characterized. The enzyme has a molecular mass of 24.5 kDa and it could be
classified as a member of the family of bacterial aluminium resistance proteins based on homology searches. When this fragment
was expressed in E. coli, it endowed E. coli with Al tolerance to 500 μM. The purified G2ALT protein is active at a broad pH range (pH 4.0–10.0) and temperature range
(25°C–80°C) with optima of 6.0 and the apparent optimal temperature of 73°C respectively. Under optimal conditions, G2ALT
exhibited a low ATPase activity with K
m
− and V
max
− values of 10±0.55 μM and 26.81±0.13 mg Pi released/min/mg enzyme, respectively. The ATPase activity of G2ALT requires Mg2+ and Na+ ions, while Zn2+ and Al3+ stimulate the activity. Cd2+ and Ag+ reduced the activity and Li+, Cu2+, and Co2+ inhibited the activity. Known inhibitors of most ATPases, like such as β-mercaptoethanol and ouabain, also inhibited the
activity of the G2ALT. These biochemical characterizations suggested that G2ALT belongs to the PP-loop ATPase superfamily
and it can be responsible for aluminium tolerance in A. gonensis G2. 相似文献
20.
Summary A metalloprotease from sorghum malt variety KSV8-I was purified by a combination of 4-M sucrose fractionation, ion-exchange
chromatography on Q-Sepharose (Fast flow), gel-filtration chromatography on Sephadex G-100 and hydrophobic interaction chromatography
on phenyl-Sepharose CL-4B. The enzyme was purified 7.9-fold to give a 13.4% yield relative to the total activity in the crude
extract and a final specific activity of 2128.7 U mg−1 protein. SDS-PAGE revealed a single migrating protein band corresponding to a relative molecular mass of 35 kDa. The purified
enzyme had optimal activity at 60 °C and maximal temperature stability between 40 and 60 °C but retained over 77% of its initial
activity after incubation at 70 °C for 30 min. Both pH optimum and maximal stability were at 7.0 but 60% of the activity remained
after 24 h between pH 5.0 and 8.0. Using 0.2 ml of 5 mM solution of each metal ion, the purified protease was slightly (P<0.05) inhibited by Zn2+, appreciably (P<0.01) inhibited by Ca2+ and Co2+ and highly significantly (P<0.001) inhibited by Ag+, Ba2+, Hg2+, Mn2+ and Pb2+. The enzyme was equally highly significantly (P<0.001) inhibited by EDTA and hydrolysed casein to give the following kinetic constants: Km = 21.0 mg ml−1; Vmax = 8.2 μmol ml1 min−1 and Ki = 0.390 mM. 相似文献