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1.
Taibi Z Saoudi B Boudelaa M Trigui H Belghith H Gargouri A Ladjama A 《Applied biochemistry and biotechnology》2012,166(3):663-679
An extracellular thermostable xylanase from a newly isolated thermophilic Actinomadura sp. strain Cpt20 was purified and characterized. Based on matrix-assisted laser desorption–ionization time-of-flight mass
spectrometry analysis, the purified enzyme is a monomer with a molecular mass of 20,110.13 Da. The 19 residue N-terminal sequence
of the enzyme showed 84% homology with those of actinomycete endoxylanases. The optimum pH and temperature values for xylanase
activity were pH 10 and 80 °C, respectively. This xylanase was stable within a pH range of 5–10 and up to a temperature of
90 °C. It showed high thermostability at 60 °C for 5 days and half-life times at 90 °C and 100 °C were 2 and 1 h, respectively.
The xylanase was specific for xylans, showing higher specific activity on soluble oat-spelt xylan followed by beechwood xylan.
This enzyme obeyed the Michaelis–Menten kinetics, with the K
m and k
cat values being 1.55 mg soluble oat-spelt xylan/ml and 388 min−1, respectively. While the xylanase from Actinomadura sp. Cpt20 was activated by Mn2+, Ca2+, and Cu2+, it was, strongly inhibited by Hg2+, Zn2+, and Ba2+. These properties make this enzyme a potential candidate for future use in biotechnological applications particularly in
the pulp and paper industry. 相似文献
2.
Xiaoyu Fu Pengfu Liu Ling Lin Yuzhi Hong Xiaoluo Huang Xin Meng Ziduo Liu 《Applied biochemistry and biotechnology》2010,160(6):1627-1636
By constructing a genomic library, an endoglucanase gene (cel9P) was cloned from Paenibacillus sp. BME-14 which was isolated from the sea. It had an open-reading frame of 1,629 bp, encoding a peptide of 542-amino acid
residue with a calculated molecular mass of 60 kDa. The enzyme showed the highest amino acid identity of 52% with other known
endoglucanases and had a C-terminal catalytic domain belonging to the glycosyl hydrolases family 9. The optimum pH and temperature
for enzymatic activity was pH 6.5 and 35 °C. The metal ions of Ca2+, Mg2+, and Mn2+ had a positive effect on the activity while Hg2+, Cu2+, and EDTA had a negative effect. Notably, Cel9P had 65% of the maximal activity at 5 °C. Based on the special characteristic
of Cel9P, it had a potential significance for study of cold-active mechanism and industry applications. 相似文献
3.
A phosphite dehydrogenase gene (ptdhK) consisting of 1,011-bp nucleotides which encoding a peptide of 336 amino acid residues was cloned from Pseudomonas sp. K. gene ptdhK was expressed in Escherichia coli BL21 (DE3) and the corresponding recombinant enzyme was purified by metal affinity chromatography. The recombinant protein
is a homodimer with a monomeric molecular mass of 37.2 kDa. The specific activity of PTDH-K was 3.49 U mg−1 at 25 °C. The recombinant PTDH-K exhibited maximum activity at pH 3.0 and at 40 °C and displayed high stability within a
wide range of pHs (5.0 to 10.5). PTDH-K had a high affinity to its natural substrates, with K
m values for sodium phosphite and NAD of 0.475 ± 0.073 and 0.022 ± 0.007 mM, respectively. The activity of PTDH-K was enhanced
by Na+, NH4+, Mg2+, Fe2+, Fe3+, Co2+, and EDTA, and PTDH-K exhibited different tolerance to various organic solvents. 相似文献
4.
Jaya Ram Simkhada Seung Sik Cho Seong Ju Park Poonam Mander Yun Hee Choi Hyo Jeong Lee Jin Cheol Yoo 《Applied biochemistry and biotechnology》2010,162(5):1457-1470
Organic solvent- and detergent-resistant proteases are important from an industrial viewpoint. However, they have been less
frequently reported and only few of them are from actinomycetes. A metalloprotease from Streptomyces olivochromogenes (SOMP) was purified by ion exchange with Poros HQ and gel filtration with Sepharose CL-6B. Apparent molecular mass of the
enzyme was estimated to be 51 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and gelatin zymography. The
activity was optimum at pH 7.5 and 50 °C and stable between pH 7.0 and 10.0. SOMP was stable below 45 °C and Ca2+ increased its thermostability. Ca2+ enhanced while Co2+, Cu2+, Zn2+, Mn2+, and Fe2+ inhibited the activity. Ethylenediaminetetraacetic acid and ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, but not phenylmethylsulfonyl fluoride, aprotinin, and pefabloc SC, significantly suppressed the activity,
suggesting that it might be a metalloprotease. Importantly, it is highly resistant against various detergents, organic solvents,
and oxidizing agents, and the activity is enhanced by H2O2. The enzyme could be a novel protease based on its origin and peculiar biochemical properties. It may be useful in biotechnological
applications especially for organic solvent-based enzymatic synthesis. 相似文献
5.
Wang H Gong Y Xie W Xiao W Wang J Zheng Y Hu J Liu Z 《Applied biochemistry and biotechnology》2011,164(8):1323-1338
A novel glycoside hydrolases family 57 gene (gh-57) was found from a metagenomic fosmid library constructed from a black smoker chimney sample 4143-1 from the Mothra hydrothermal
vent at the Juan de Fuca Ridge. Sequence and homology analysis using BLAST revealed that it had high similarity to gh-57 family. Conserved domain research revealed that the novel gh-57 contained a Glyco-hydro-57 domain and five conserved regions, including two putative catalytic residues Glu154 and Asp263. The three-dimensional features of the protein and its homologue from Pyrococcus horikoshii OT3 known as α-amylase were generated by homology modeling. The gh-57 gene was cloned, expressed, and purified in Escherichia coli using pQE system. Enzyme activity revealed that the recombinant protein could hydrolyze soluble starch and demonstrated amylase
activity. It showed an optimal pH of 7.5, an optimal temperature of 90°C, and its thermostability at 90°C could remain over
50% enzyme activity for 4 h. The enzyme activity could be increased by DTT and Mg2+ while an inhibitory effect was observed with EDTA, ATP, and Ca2+. These results showed that the gh-57 gene was a novel thermostable amylase from oceanic microorganisms. 相似文献
6.
Qi Wu Chen Li Chenglei Li Hui Chen Liu Shuliang 《Applied biochemistry and biotechnology》2010,160(1):129-139
The collagenase, produced extracellular by Bacillus pumilus Col-J, was purified by ammonium sulfate precipitation followed by two gel filtrations, involving Sephadex G-100 column and
Sepharose Fast Flow column. Purified collagenase has a 31.53-fold increase in specific activity of 87.33 U/mg and 7.00% recovery.
The collagenase has a relative molecular weight of 58.64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
The optimal temperature for the enzyme reaction was 45 °C. More than 50% of the original activity still remained after 5 min
of incubation at 70 °C or 10 min at 60 °C. The maximal enzyme activity of collagenase was obtained at pH 7.5, and it was stable
over a pH range of 6.5–8.0. The collagenase activity was strongly inhibited by Mn2+, Pb2+, ethylenediamine tetraacetic acid, ethylene glycol tetraacetic acid, and β-mercaptoethanol. However, Ca2+ and Mg2+ greatly increased its activity. The collagenase from B. pumilus Col-J showed highly specific activity towards the native collagen from calf skin. The K
m and V
max of the enzyme for collagen were 0.79 mg/mL and 129.5 U, respectively. 相似文献
7.
Biochemical Characterization of Raw-starch-digesting Alpha Amylase Purified from <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> 总被引:1,自引:0,他引:1
Gangadharan D Nampoothiri KM Sivaramakrishnan S Pandey A 《Applied biochemistry and biotechnology》2009,158(3):653-662
Alpha amylase (E.C. 3.2.1.1) of Bacillus amyloliquefaciens produced by submerged fermentation was purified to near homogeneity by ion exchange chromatography. Through the process 38.6-fold
increase in purity with a specific activity of 72 U/mg proteins was obtained. The apparent molecular weight of the purified
enzyme was found to be 58 kDa by SDS-PAGE. The enzyme was relatively stable between pH 5.0–8.0 and temperature between 50
and 60°C. The enzyme did not show any obligate requirement of metal ions but Ca2+ and Cu2+ enhanced the enzyme activity marginally and the thermostability was enhanced in the presence of Ca2+ ions. The purified enzyme exhibited maximal substrate specificity for amylose and efficiency in digesting various raw starches.
The K
m and V
max of the enzyme was determined using both amylose and soluble starch as substrate. The analysis of the hydrolyzed products
of soluble starch by thin layer chromatography showed the yield of maltosaccharides after 6 h of hydrolysis. 相似文献
8.
Lokendra Kumar Balvinder Singh Dilip Kumar Adhikari Joydeep Mukherjee Debashish Ghosh 《Applied biochemistry and biotechnology》2012,166(7):1723-1735
Purification and characterization of halotolerant, thermostable alkaline l-glutaminase from a Bacillus sp. LKG-01 (MTCC 10401), isolated from Gangotri region of Uttarakhand Himalaya, is being reported in this paper. Enzyme has
been purified 49-fold from cell-free extract with 25% recovery (specific activity 584.2 U/mg protein) by (NH4)2SO4 precipitation followed by anion exchange chromatography and gel filtration. Enzyme has a molecular weight of 66 kDa. l-Glutaminase is most active at pH 11.0 and stable in the pH range 8.0–11.0. Temperature optimum is 70 °C and is completely
stable after 3 h pre-incubation at 50 °C. Enzyme reflects more enhanced activity with 1–20% (w/v) NaCl, which is further reduced to 80% when NaCl concentration was increased up to 25%. l-Glutaminase is almost active with K+, Zn2+, and Ni2+ ions and K
m and V
max values of 240 μM and 277.77 ± 1.1 U/mg proteins, respectively. Higher specific activity, purification fold, better halo-tolerance,
and thermostability would make this enzyme more attractive for food fermentation with respect to other soil microbe derived
l-glutaminase reported so far. 相似文献
9.
Sasithorn Uttatree Pakorn Winayanuwattikun Jittima Charoenpanich 《Applied biochemistry and biotechnology》2010,162(5):1362-1376
The benzene tolerant Acinetobacter baylyi isolated from marine sludge in Angsila, Thailand could constitutively secrete lipolytic enzymes. The enzyme was successfully
purified 21.89-fold to homogeneity by ammonium sulfate precipitation and gel-permeable column chromatography with a relative
molecular mass as 30 kDa. The enzyme expressed maximum activity at 60°C and pH 8.0 with p-nitrophenyl palmitate as a substrate and found to be stable in pH and temperature ranging from 6.0-9.0 to 60-80°C, respectively.
A study on solvent stability revealed that the enzyme was highly resisted to many organic solvents especially benzene and
isoamyl alcohol, but 40% inhibited by decane, hexane, acetonitrile, and short-chain alcohols. Lipase activity was completely
inhibited in the presence of Fe2+, Mn2+, EDTA, SDS, and Triton X-100 while it was suffered detrimentally by Tween 80. The activity was enhanced by phenylmethylsulfonyl
fluoride (PMSF), Na+, and Mg2+ and no significant effect was found in the presence of Ca2+ and Li+. Half of an activity was retained by Ba2+, Ag+, Hg+, Ni2+, Zn2+, and DTT. The enzyme could hydrolyze a wide range of p-nitrophenyl esters, but preferentially medium length acyl chains (C8-C12). Among natural oils and fats, the enzyme 11-folds favorably catalyzed the hydrolysis of rice bran oil, corn oil, sesame
oil, and coconut oil in comparison to palm oil. Moreover, the transesterification activity of palm oil to fatty acid methyl
esters (FAMEs) revealed 31.64 ± 1.58% after 48 h. The characteristics of novel A. baylyi lipase, as high temperature stability, organic solvent tolerance, and transesterification capacity from palm oil to FAMEs,
indicate that it could be a vigorous biocatalyzer in the prospective fields as bioenergy industry or even in organic synthesis
and pharmaceutical industry. 相似文献
10.
Wei Zhao Aisheng Xiong Xiaoyan Fu Feng Gao Yongsheng Tian Rihe Peng 《Applied biochemistry and biotechnology》2010,162(8):2157-2165
To obtain a high level expression of phytase with favorable characteristics, a codon-optimized phytase gene from Citrobacter freundii was synthesized and transferred into Pichia pastoris. Small-scale expression experiments and activity assays were used to screen positive colonies. After purified by Ni2+–NTA agarose affinity column, the characterizations of the recombinant phytase were determined. The recombinant phytase (r-phyC)
had two distinct pH optima at 2.5 and 4.5 and an optimal temperature at 50 °C. It retained more than 80% activity after being
incubated under various buffer (pH 1.5–8.0) at 37 °C for 1 h. The specific activity, Km, and Vmax values of r-phyC for sodium phytate were 2,072 ± 18 U mg−1, 0.52 ± 0.04 mM, and 2,380 ± 84 U mg−1 min−1, respectively. The enzyme activity was significantly improved by 1 mM of K+, Ca2+, and Mg2+. These characteristics contribute to its potential application in feed industry. 相似文献
11.
Kouakou TH Kouadio YJ Kouamé P Waffo-Téguo P Décendit A Mérillon JM 《Applied biochemistry and biotechnology》2009,158(2):285-301
Polyphenol oxidases (PPOs) were isolated from cell suspensions of two cultivars of cotton (Gossypium hirsutum L.), and their biochemical characteristics were studied. PPO from Coker 312, an embryogenic cultivar, showed a highest affinity
to catechol 20 mM, and PPO from R405-2000, a nonembryogenic cultivar, showed a highest affinity to 4-methylcatechol 20 mM.
The optimal pH for PPO activity was 7.0 and 6.0 for Coker 312 and R405-2000, respectively. The enzyme had an optimal temperature
of 25 °C and was relatively stable at 20–30 °C. Reducing sodium metabisulfite, ascorbic acid, dithiothreitol, SnCl2, and FeCl3 markedly inhibited PPO activity, whereas its activity was highly enhanced by Mg2+, Ca2+, and Mn2+ and was moderately inhibited by Ba2+, Cu2+, and Zn2+. The analysis revealed a single band on the sodium dodecyl sulfate polyacrylamide gel electrophoresis which corresponded
to a molecular weight of 55 kDa for Coker 312 and 42 kDa for R405-2000. 相似文献
12.
A heparinase-producing fungus was isolated, and the strain was taxonomically characterized as Aspergillus flavus by morphophysiological and 26S rRNA gene homology studies. The culture produced intracellular heparinase enzyme, which was
purified 40.5-fold by DEAE-Sephadex A-50, CM-Sephadex C-50, and Sephadex G-100 column chromatography. Specific activity of
the purified enzyme was found to be 44.6 IU/μg protein and the molecular weight of native as well as reduced heparinase was
24 kDa, showing a monomeric unit structure. Peptide mass spectrum showed poor homogeneity with the database in the peptide
bank. The enzyme activity was maximum at 30 °C in the presence of 300 mM NaCl at pH 7.0. In the presence of Co2+, Mn2+ ions, and reducing agents (β-mercaptoethanol, dithiothreitol), enzyme activity was enhanced and inhibited by iodoacetic acid.
These observations suggested that free sulfohydryl groups of cysteine residues were necessary for catalytic activity of the
enzyme. The enzyme was also inhibited by histidine modifier, DEPC, which suggests that along with cysteine, histidine may
be present at its active site. The enzyme showed a high affinity for heparin as a substrate with K
m and V
max as 2.2 × 10−5 M and 30.8 mM min−1, respectively. The affinity of the enzyme for different glycosaminoglycans studied varied, with high substrate specificity
toward heparin and heparin-derived polysaccharides. Depolymerization of heparin and fractionation of the oligosaccharides
yielded heparin disaccharides as main product. 相似文献
13.
Aminopeptidases catalyze the cleavage of specific amino acids from the amino terminus of protein or peptide substrates. A
proline-specific aminopeptidase was purified to homogeneity from the culture-free extract of Streptomyces lavendulae ATCC 14162 in sequential steps comprising ammonium sulfate precipitation, ultra-filtration, and column chromatography on
Q-sepharose and Sephadex G-100. The purified protein showed approximately 60 kDa in SDS-PAGE and was optimally active at pH
6.5 and 40 °C. Kinetic studies showed a K
m and V
max of 0.23 mM and 0.087 μmol/min, respectively, using Pro-p-NA, the substrate with maximum specificity. Enzyme activity was inhibited by PMSF and ions like Zn2+, Co2+, and Ni2+. However, unlike other aminopeptidases, the activity was enhanced in the presence of DTT, 1,10-phenanthroline, EDTA, amastatin,
and bestatin. Ions like Ca2+, Mg2+, and Mn2+ also enhanced the activity. 相似文献
14.
An extracellular thermostable α-galactosidase producing Aspergillus terreus
GR strain was isolated from soil sample using guar gum as sole source of carbon. It was purified to apparent homogeneity by
acetone precipitation, gel filtration followed by DEAE-Sephacel chromatographic step. The purified enzyme showed a single
band after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the purified enzyme
after SDS-PAGE was 108 kDa. The enzyme showed optimum pH and temperature of 5.0 and 65 °C, respectively, for artificial substrate
pNPαGal. α-Galactosidase from A. terreus
GR is found to be thermostable, as it was not inactivated after heating at 65 °C for 40 min. The K
m for pNPαGal, oNPαGal, raffinose, and stachyose are 0.1, 0.28, 0.42, and 0.33 mM, respectively. Inhibitors such as 1,10-phenanthroline,
phenylmethylsulfonyl fluoride, ethylenediaminetetraacetic acid, mercaptoethanol, and urea have no effect, whereas N-bromosuccinamide inhibited enzyme activity by 100%. Among metal ions tested, Mg2+, Ni2+, Ca2+, Co2+, and Mn2+ had no effect on enzyme activity, but Ag+, Hg2+, and Cu2+ have inhibited complete activity. 相似文献
15.
Kouakou TH Dué EA Kouadio NE Niamké S Kouadio YJ Mérillon JM 《Applied biochemistry and biotechnology》2009,157(3):575-592
Two peroxidases, cPOD-I and rPOD-II, have been isolated and purified from cotton cell suspension and their biochemical characteristics
studied. rPOD-II from R405-2000, a non-embryogenic cultivar, has higher activity than cPOD-I derived from Coker 312, which
developed an embryogenic structure. The cPOD-I and rPOD-II had molecular mass of 39.1 and 64 kDa respectively, as determined
by SDS-PAGE. Both enzymes showed high efficiency of interaction with the guaiacol at 25 mM. The optimal pH for cPOD-I and
rPOD-II activity was 5.0 and 6.0, respectively. The enzyme had an optimum temperature of 25 °C and was relatively stable at
20–30 °C. The isoenzymes were highly inhibited by ascorbic acid, dithiothreitol, sodium metabisulfite, and β-mercaptoethanol.
Their activities were highly enhanced by Al3+, Fe3+, Ca2+, and Ni2+, but they were moderately inhibited by Mn2+ and K+. The enzyme lost 50% to 62% of its activity in the presence of Zn2+ and Hg2+. 相似文献
16.
Aminzadeh S Naderi-Manesh H Khajeh K Naderi-Manesh M 《Applied biochemistry and biotechnology》2006,135(3):193-208
For the first time, a polygalacturonase from the culture broth of Tetracoccosporium sp. was isolated and incubated at 30°C in an orbital shaker at 160 rpm for 48h. The enzyme was purified by ammonium sulfate
precipitation and two-step ion-exchange chromatography and had an apparent molecular mass of 36 kDa, as shown by sodium dodecyl
sulfate (SDS)-polyacrylamide gel electrophoresis. Its optimum activity was at pH 4.3 and 40°C, and the K
m
and V
max values of this enzyme (for polygalacturonic acid) were 3.23 mg/mL and 0.15 μmol/min, respectively. Ag+, Co2+, EDTA, Tween-20, Tween-80, and Triton X-100 stimulated polygalacturonase activity whereas Al3+, Ba2+, Ca2+, Fe2+, Fe3+, Ni2+, Mg2+, Mn2+, and SDS inhibited it. In addition, iodoacetamide and iodoacetic acid did not inhibit enzyme activity at a concentration
of 1 mM, indicating that cysteine residues are not part of the catalytic site of polygalacturonase. We studied the kinetic properties
and thermal inactivation of polygalacturonase. This enzyme exhibited a t
1/2 of 63 min at 60°C and its specific activity, turnover number, and catalytic efficiency were 6.17 U/mg, 113.64 min−1, and 35.18 mL/(min·mg), respectively. The activation energy (ΔE
#) for heat inactivation was 5.341 kJ/mol, and the thermodynamic activation parameters ΔG
#, ΔH
#, and ΔS
# were also calculated, revealing a potential application for the industry. 相似文献
17.
18.
Cyclodextrin glucanotransferase, produced by Bacillus megaterium, was characterized, and the biochemical properties of the purified enzyme were determined. The substrate specificity of the
enzyme was tested with different α-1,4-glucans. Cyclodextrin glucanotransferase displayed maximum activity in the case of
soluble starch, with a K
m value of 3.4 g/L. The optimal pH and temperature values for the cyclization reaction were 7.2 and 60 °C, respectively. The
enzyme was stable at pH 6.0–10.5 and 30 °C. The enzyme activity was activated by Sr2+, Mg2+, Co2+, Mn2+, and Cu2+, and it was inhibited by Zn2+and Ag+. The molecular mass of cyclodextrin glucanotransferase was established to be 73,400 Da by sodium dodecyl sulfate–polyacrylamide
gel electrophoresis, 68,200 Da by gel chromatography, and 75,000 Da by mass spectrometry. The monomer form of the enzyme was
confirmed by the analysis of the N-terminal amino acid sequence. Cyclodextrin glucanotransferase formed all three types of
cyclodextrins, but the predominant product was β-cyclodextrin. 相似文献
19.
Eltayib Hassan Ahmed Tripti Raghavendra Datta Madamwar 《Applied biochemistry and biotechnology》2010,160(7):2102-2113
A mesophilic bacterial culture producing a novel thermostable alkaline lipase was isolated from oil rich soil sample and identified
as Bacillus subtilis EH 37. The lipase was partially purified by ammonium sulfate precipitation and hydrophobic interaction chromatography with
17.8-fold purification and 41.9 U/ml specific activity. The partially purified enzyme exhibited maximum activity at pH 8.0
and at 60 °C. It retained 100% of activity at 50 °C and 60 °C for 60 min. The presence of Ca+2, Mg+2, and Zn2+ exhibited stimulatory effect on lipase activity, whereas Fe+3 and Co+2 reduced its activity. The enzyme retained more than 80% of its initial activity upon exposure to organic solvents, exhibited
107% and 115% activity in the presence of 15% isopropyl alcohol and 30% n-hexane, respectively. The EH 37 lipase also proved
to be an efficient catalyst in synthesis of ethyl caprylate in organic solvent, thus providing a concept of application of
B. subtilis lipase in non-aqueous catalysis. 相似文献
20.
Lignin peroxidase was purified (72-fold) from Acinetobacter calcoaceticus NCIM 2890. The purified lignin peroxidase (55–65 kDa) showed dimeric nature. The maximum enzyme activity was observed at
pH 1.0, between a broad temperature range of 50 and 70°C, at H2O2 concentration (40 mM) and the substrate concentration (n-propanol, 100 mM). Purified lignin peroxidase was able to oxidize a variety of substrates including Mn2+, tryptophan, mimosine, l-Dopa, hydroquinone, xylidine, n-propanol, veratryl alcohol, and ten textile dyes of various groups indicating as a versatile peroxidase. Most of the dyes
decolorized up to 90%. Tryptophan stabilizes the lignin peroxidase activity during decolorization of dyes. 相似文献