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1.
A gene encoding β-1,3-1,4-glucanase was cloned by polymerase chain reaction (PCR) from Bacillus subtilis MA139. Sequencing result showed 97% homology to the corresponding gene from Bacillus licheniformis. The open reading frame (ORF) of the gene contained 690 bp coding for a 226 amino-acid matured protein with the estimated
molecular weight of 24.44 kDa. The β-1,3-1,4-glucanase gene was subcloned into an expression vector of pET28a and expressed
in Escherichia coli BL21 and then purified by metal affinity chromatography using a nickel–nitrilotriacetic acid (Ni–NTA) column. The purified
β-1,3-1,4-glucanase demonstrated 24.05 and 12.52 U ml-1 activities for the substrates of barley β-glucan and lichenan, respectively, and the specific activities were 728.79 and
379.1 U mg-1 for them, respectively. The optimal temperature and pH of the purified enzyme were 40°C and 6.4, respectively. When barley
β-glucan was used as the substrate, K
m was 5.34 mg ml-1, and K
cat showed 7,206.71 S-1, thus the ratio of K
cat and K
m was 1,349.67 ml s-1 mg-1. The activity of β-1,3-1,4-glucanase was affected by a range of metal ions or ethylenediaminetetraacetic acid (EDTA). 相似文献
2.
New Thermostable Amylase from <Emphasis Type="Italic">Bacillus cohnii</Emphasis> US147 with a Broad pH Applicability 总被引:1,自引:0,他引:1
Ghorbel RE Maktouf S Massoud EB Bejar S Chaabouni SE 《Applied biochemistry and biotechnology》2009,157(1):50-60
A new thermophilic bacterial strain identified as Bacillus cohnii US147 was isolated from the southern Tunisian soil. The identification was based on physiological tests and molecular techniques
related to the 16S ribosomal ribonucleic acid. The isolated strain produced amylase, which was purified. This amylase had
an apparent molecular mass of 30 kDa as estimated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Amylase
US147 showed K
m and V
max values of 0.7 mg/ml and 2.2 U/ml, respectively, with starch as the substrate. The enzyme was active in acid and basic pH
and had a maximal activity on starch at pH 9 and 70 °C. The enzyme was stable at pH 9 for 72 h and retained half of its activity
after incubation at 70 °C for 150 min. A partially inhibition (15%, 25%, 23%, 20%, and 22%) was obtained with 1 mM SDS, 1 mM
NaBO3, 1 mM H2O2, 1 mM Zn+2, and 5 mM ethylenediamine tetraacetic acid (EDTA), respectively. The amylase recovered its original activity by the addition
of 10 mM Ca 2+ to the 5 mM EDTA. These properties indicated a possible use of this amylase in starch saccharification, in detergent, and
in other industrial applications. 相似文献
3.
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
4.
Yao Guo Fengxia Lu Haizhen Zhao Yanchong Tang Zhaoxin Lu 《Applied biochemistry and biotechnology》2010,162(2):498-509
A gene of glucose oxidase (GOD) from Aspergillus niger Z-25 was cloned and sequenced. The entire open reading frame (ORF) consisted of 1,818 bp and encoded a putative peptide of
605 amino acids. The gene was fused to the pPICZαA plasmid and overexpressed in Pichia pastoris SMD1168. The recombinant GOD (rGOD) was secreted into the culture using MF-α factor signal peptide under the control of the
AOX1 promoter. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that rGOD exhibited a single band at around
94 kDa. The maximal GOD activity of approximately 40 U/mL was achieved in shake flask by induction under optimal conditions
after 7 days. rGOD was purified by ammonium sulfate precipitate leading to a final specific activity of 153.46 U/mg. The optimum
temperature and pH of the purified enzyme were 40 °C and 6.0, respectively. Over 88% of maximum activity was maintained below
40 °C. And the recombinant enzyme displayed a favorable stability in the pH range from 4.0 to 8.0. The Lineweaver–Burk plotting
revealed that rGOD exhibited a K
m value of 16.95 mM and a K
cat value of 484.26 s−1. 相似文献
5.
Yun Wang Jin-Zhu Song Qian Yang Zhi-Hua Liu Xiao-Mei Huang Yan Chen 《Applied biochemistry and biotechnology》2010,162(3):843-854
A gene encoding chitin deacetylase was cloned by polymerase chain reaction from Aspergillus nidulans. Sequencing result showed 40% homology to the corresponding gene from Colletotrichum lindemuthianum. The complete gene contains an open reading frame of 747 nucleotides encoding a sequence of 249 amino acid residues. The
chitin deacetylase gene was subcloned into a pET28a expression vector and expressed in Escherichia coli BL21 and then purified by metal affinity chromatography using a His-bind column. The purified chitin deacetylase demonstrated
an activity of 0.77 U ml−1 for the glycol chitin substrates, and its specific activity was 4.17 U mg−1 for it. The optimal temperature and pH of the purified enzyme were 50 °C and 8.0, respectively. When glycol chitin was used
as the substrate, K
m was 4.92 mg ml−1, and K
cat showed 6.25 s−1, thus the ratio of K
cat and K
m was 1.27 ml s−1 mg−1. The activity of chitin deacetylase was affected by a range of metal ions and ethylenediaminetetraacetic acid. 相似文献
6.
Chitinase was purified from the culture medium of Bacillus licheniformis SK-1 by colloidal chitin affinity adsorption followed by diethylamino ethanol-cellulose column chromatography. The purified
enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of chitinase 72 (Chi72) were 72 kDa and 4.62 (Chi72) kDa, respectively. The purified chitinase revealed two activity optima
at pH 6 and 8 when colloidal chitin was used as substrate. The enzyme exhibited activity in broad temperature range, from
40 to 70°C, with optimum at 55°C. It was stable for 2 h at temperatures below 60°C and stable over a broad pH range of 4.0–9.0
for 24 h. The apparent K
m and V
max of Chi72 for colloidal chitin were 0.23 mg ml−1 and 7.03 U/mg, respectively. The chitinase activity was high on colloidal chitin, regenerated chitin, partially N-acetylated chitin, and chitosan. N-bromosuccinamide completely inhibited the enzyme activity. This enzyme should be a good candidate for applications in the
recycling of chitin waste. 相似文献
7.
Wild type (WT) DNA sequence, which encoded a mature β-mannanase of Aspergillus sulphureus, composed of 1,152 nucleotides (nt), was amplified from pUCm-T-mann by polymerase chain reaction (PCR). Based on this DNA
fragment, mutants designated as E206G and E314G were constructed by overextension PCR (OE-PCR). Glutamic acids of the 206th and 314th sites in the amino acid sequence of
β-mannanase were separately replaced by glycine in these two mutants. The WT and mutant genes were ligated into prokaryotic
vector pET-28a (+) and transformed into the Escherichia coli BL21 strain, respectively. The recombinant enzyme proteins were expressed by IPTG induction and detected by Western blot.
The recombinant proteins purified with Ni-NTA column were dialyzed to correctly refold. The WT recombinant β-mannanase showed
optimal activity at 50 °C and pH 2.4. The kinetic parameters of K
m and V
max for purified β-mannanase were 1.38 mg/ml and 72.99 U/mg, respectively. However, the mutant proteins did not show any activity.
It was demonstrated that E206 and E314 were the catalytic residues of β-mannanase. 相似文献
8.
Li S Zuo Z Niu D Singh S Permaul K Prior BA Shi G Wang Z 《Applied biochemistry and biotechnology》2011,164(5):581-592
A putative α-amylase gene, designated as RoAmy, was cloned from Rhizopus oryzae. The deduced amino acid sequence showed the highest (42.8%) similarity to the α-amylase from Trichoderma viride. The RoAmy gene was successfully expressed in Pichia pastoris GS115 under the induction of methanol. The molecular weight of the purified RoAmy determined by sodium dodecyl sulfate polyacrylamide
gel electrophoresis was approximately 48 kDa. The optimal pH and temperature were 4–6 and 60 °C, respectively. The enzyme
was stable at pH ranges of 4.5–6.5 and temperatures below 50 °C. Purified RoAmy had a K
m and V
max of 0.27 mg/ml and 0.068 mg/min, respectively, with a specific activity of 1,123 U/mg on soluble starch. Amylase activity
was strongly inhibited by 5 mM Cu2+ and 5 mM Fe2+, whereas 5 mM Ca2+ showed no significant effect. The RoAmy hydrolytic activity was the highest on wheat starch but showed only 55% activity
on amylopectin relative to soluble corn starch, while the pullulanase activity was negligible. The main end products of the
polysaccharides tested were glucose and maltose. Maltose reached a concentration of 74% (w/w) with potato starch as the substrate. The enzyme had an extremely high affinity (K
m = 0.22 mM) to maltotriose. A high ratio of glucose/maltose of 1:4 was obtained when maltotriose was used at an initial concentration
of 40 mM. 相似文献
9.
Wanli Liu Pengjun Shi Qiang Chen Peilong Yang Guozeng Wang Yaru Wang Huiying Luo Bin Yao 《Applied biochemistry and biotechnology》2010,162(1):1-12
A xylanase-encoding gene, xyn11F63, was isolated from Penicillium sp. F63 CGMCC1669 using degenerated polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR techniques.
The full-length chromosomal gene consists of 724 bp, including a 73-bp intron, and encodes a 217 amino acid polypeptide. The
deduced amino acid sequence of xyn11F63 shows the highest identity of 70% to the xylanase from Penicillium sp. strain 40, which belongs to glycosyl hydrolases family 11. The gene was overexpressed in Pichia pastoris, and its activity in the culture medium reached 516 U ml−1. After purification to electrophoretic homogeneity, the enzyme showed maximal activity at pH 4.5 and 40°C, was stable at
acidic buffers of pH 4.5–9.0, and was resistant to proteases (proteinase K, trypsin, subtilisin A, and α-chymotrypsin). The
specific activity, K
m, and V
max for oat spelt xylan substrate was 7,988 U mg−1, 22.2 mg ml−1, and 15,105.7 μmol min−1 mg−1, respectively. These properties make XYN11F63 a potential economical candidate for use in feed and food industrial applications. 相似文献
10.
An exoinulinase has been isolated, purified and characterised from a commercially available broth of Aspergillus ficuum. The enzyme was purified 4.2-fold in a 21% yield with a specific activity of 12,300 U mg−1(protein) after dialysis, ammonium sulphate fractionation and Sephacryl S-200 size exclusion and ion exchange chromatography.
The molecular weight of this enzyme was estimated to be 63 kDa by SDS-PAGE. It exhibited a pH and temperature optima of 5.4
and 50 °C respectively and under such conditions the enzyme remained stable with 96% and 63.8% residual activity after incubation
for 12 h and 72 h respectively. The respective K
m and V
max values were 4.75 mM and 833.3 μmol min−1 ml−1, respectively. Response surface methodological statistical analysis was evaluated for the maximal production of fructose
from the hydrolysis of pure commercial chicory inulin. Incubation of the dialyzed crude exoinulinase (100 U/ml, 48 h, 50 °C,
150% inulin, pH 5.0) produced the highest amount of fructose (106.4 mg/ml) under static batch conditions. The purified exoinulinase
was evaluated for fructose production and the highest amount (98 mg/ml) was produced after 12 h incubation at 50 °C, 150%
inulin pH 5.0. The use of a crude exoinulinase preparation is economically desirable and the industrial production of fructose
from inulin hydrolysis is biotechnologically feasible. 相似文献
11.
A psychrotrophic fungus identified as Trichoderma sp. SC9 produced 36.7 U/ml of xylanase when grown on a medium containing corncob xylan at 20 °C for 6 days. The xylanase
was purified 37-fold with a recovery yield of 8.2%. The purified xylanase appeared as a single protein band on SDS-PAGE with
a molecular mass of approximately 20.5 kDa. The enzyme had an optimal pH of 6.0, and was stable over pH 3.5–9.0. The optimal
temperature of the xylanase was 42.5 °C and it was stable up to 35 °C at pH 6.0 for 30 min. The xylanase was thermolabile
with a half-life of 23.9 min at 45 °C. The apparent K
m
values of the xylanase for birchwood, beechwood, and oat-spelt xylans were found to be 3, 2.1, and 16 mg/ml respectively.
The xylanase hydrolyzed beechwood xylan and birchwood xylan to yield mainly xylobiose as end products. The enzyme-hydrolysed
xylotriose, xylotetraose, and xylopentose to produce xylobiose, but it hardly hydrolysed xylobiose. A xylanase gene (xynA) with an open reading frame of 669 nucleotide base pairs (bp), encoding 222 amino acids, from the strain was cloned and sequenced.
The deduced amino acid sequence of XynA showed 85% homology with Xyn2 from a mesophilic strain of Trichoderma viride. 相似文献
12.
Cloning, Expression, and Identification of a Novel Extracellular Cold-Adapted Alkaline Protease Gene of the Marine Bacterium Strain YS-80-122 总被引:1,自引:0,他引:1
Fang Wang Jianhua Hao Chengye Yang Mi Sun 《Applied biochemistry and biotechnology》2010,162(5):1497-1505
As one of the most important groups of industrial enzymes, cold-adapted protease has been studied widely. An extracellular
cold-adapted alkaline protease metalloproteinase (MP), produced by a marine bacterium strain YS-80-122, has been purified.
The NH2-amino acid sequence of the purified alkaline protease MP was ANGTSSAFTQ, which was identical to that of the serralysin from
Pseudomonas sp. “TAC II 18”. The MP structural gene (lupA gene) was cloned by inverse PCR, and the open reading frame of 1,443 bp encoded a 463 amino acid protein (without signal
peptide). Sequence alignment reveals that the alkaline protease MP belongs to the serralysin-type metalloproteases. The recombinant
protein LupA was expressed in Escherichia coli, and Western blotting confirmed that the LupA was homologous to the cold-adapted alkaline protease MP. 相似文献
13.
14.
Characterization of Thermo-stable Endoinulinase from a New Strain <Emphasis Type="Italic">Bacillus Smithii</Emphasis> T7 总被引:1,自引:0,他引:1
A new thermophilic inulinase-producing strain, which grows optimally at 60 °C, was isolated from soil samples with medium
containing inulin as a sole carbon source. It was identified as a Bacillus smithii by analysis of 16s rDNA. Maximum inulinase yield of 135.2 IU/ml was achieved with medium pH7.0, containing inulin 2.0%, (NH4)H2PO4 0.5%, yeast extract 0.5%, at 50 °C 200 rpm shaker for 72-h incubation. The purified inulinase from the extracellular extract
of B. smithii T7 shows endoinulinolytic activity. The optimum pH for this endoinulinase is 4.5 and stable at pH range of 4.0–8.0. The optimum
temperature for enzyme activity was 70 °C, the half life of the endoinulinase is 9 h and 2.5 h at 70 °C and 80 °C respectively.
Comparatively lower Michaelis–Menten constant (4.17 mM) and higher maximum reaction velocity (833 IU/mg protein) demonstrate
the endoinulinase’s greater affinity for inulin substrate. These findings are significant for its potential industrial application. 相似文献
15.
Kyoung-Mi Lee Ah-Reum Joo Marimuthu Jeya Kyoung-Min Lee Hee-Jung Moon Jung-Kul Lee 《Applied biochemistry and biotechnology》2011,163(1):25-39
A high cellobiohydrolase (CBH)-producing strain was isolated and identified as Penicillium purpurogenum KJS506 according to the morphology and comparison of internal transcribed spacer rDNA gene sequence. When rice straw and
corn steep powder were used as carbon and nitrogen sources, respectively, a maximum CBH activity of 2.6 U mg-protein−1, one of the highest among CBH-producing microorganisms, was obtained. The optimum temperature and pH for CBH production were
30 °C and 4.0, respectively. The increased production of CBH in P. purpurogenum culture at 30 °C was confirmed by two-dimensional electrophoresis followed by MS/MS sequencing of the partial peptide. The
internal amino acid sequences of P. purpurogenum CBH showed a significant homology with hydrolases from glycoside hydrolase family 7. The extracellular CBH was purified to
homogeneity by sequential chromatography of P. purpurogenum culture supernatants on a DEAE-sepharose column, a gel filtration column, and then on a Mono Q column with fast-protein liquid
chromatography. The purified CBH was a monomeric protein with a molecular weight of 60 kDa and showed broad substrate specificity
with maximum activity towards p-nitrophenyl β-d-cellobiopyranoside. P. purpurogenum CBH showed t
1/2 value of 4 h at 60 °C and V
max value of 11.9 μmol min−1 mg-protein−1 for p-nitrophenyl-d-cellobiopyranoside. Although CBHs have been reported, the high specific activity distinguishes P. purpurogenum CBH. 相似文献
16.
17.
Dengeti Shrinivas Gunashekaran Savitha Kumar Raviranjan Gajanan Ramchandra Naik 《Applied biochemistry and biotechnology》2010,162(7):2049-2057
A highly thermostable alkaline xylanase was purified to homogeneity from culture supernatant of Bacillus sp. JB 99 using DEAE-Sepharose and Sephadex G-100 gel filtration with 25.7-fold increase in activity and 43.5% recovery.
The molecular weight of the purified xylanase was found to be 20 kDA by SDS-PAGE and zymogram analysis. The enzyme was optimally
active at 70 °C, pH 8.0 and stable over pH range of 6.0–10.0.The relative activity at 9.0 and 10.0 were 90% and 85% of that
of pH 8.0, respectively. The enzyme showed high thermal stability at 60 °C with 95% of its activity after 5 h. The K
m and V
max of enzyme for oat spelt xylan were 4.8 mg/ml and 218.6 μM min−1 mg−1, respectively. Analysis of N-terminal amino acid sequence revealed that the xylanase belongs to glycosyl hydrolase family
11 from thermoalkalophilic Bacillus sp. with basic pI. Substrate specificity showed a high activity on xylan-containing substrate and cellulase-free nature. The hydrolyzed product
pattern of oat spelt xylan on thin-layer chromatography suggested xylanase as an endoxylanase. Due to these properties, xylanase
from Bacillus sp. JB 99 was found to be highly compatible for paper and pulp industry. 相似文献
18.
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
19.
Vinod Kumar Punesh Sangwan A. K. Verma Sanjeev Agrawal 《Applied biochemistry and biotechnology》2014,173(2):646-659
Phytic acid is the major storage form of organic phosphorus in nature- and plant-based animal feed. It forms insoluble complexes with nutritionally important metals and proteins that are unavailable for monogastric or agastric animals. Phytases initiate the stepwise hydrolysis of phytic acid and release inorganic orthophosphate. In the present investigation, the phytase gene from a phytase producing Bacillus licheniformis strain PB-13 was successfully expressed in Escherichia coli BL21. Recombinant phytase ‘rPhyPB13’ was found to be catalytically active, with an activity of 0.97 U/mL and specific activity of 0.77 U/mg. The rPhyPB13 was purified to 14.10-fold using affinity chromatography. Similar to other β-propeller phytases, purified rPhyPB13 exhibited maximal activity at pH 6.0–6.5 and 60 °C in the presence of 1 mM Ca2+ and was highly active over a wider pH range (pH 4.0–8.0) and high temperature (80 °C). It has shown maximum activity towards Na-phytate as substrate. The observed K m , V max and k cat of purified rPhyPB13 were 1.064 mM, 1.32 μmol/min/mg and 27.46 s?1, respectively. PhyPB13 was resistant to trypsin inactivation, activated in presence of Ca2+ and inhibited in presence of EDTA. Crude rPhyPB13 has good digestion efficiency for commercial feed and soybean meal. These results indicate that PhyPB13 is a β-propeller phytase that has application potential in aquaculture feed. 相似文献
20.
Monia Blibech Raoudha Ellouz Ghorbel Ines Fakhfakh Patricia Ntarima Katheleen Piens Abir Ben Bacha Semia Ellouz Chaabouni 《Applied biochemistry and biotechnology》2010,160(4):1227-1240
The highest β-mannanase activity was produced by Penicillium occitanis Pol6 on flour of carob seed, whereas starch-containing medium gave lower enzymes titles. The low molecular weight enzyme
was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography procedures.
The purified β-mannanase (ManIII) has been identified as a glycoprotein (carbohydrate content 5%) with an apparent molecular
mass of 18 kDa. It was active at 40 °C and pH 4.0. It was stable for 30 min at 70 °C and has a broad pH stability (2.0–12.0).
ManIII showed K
m, V
max, and K
cat values of 17.94 mg/ml, 93.52 U/mg, and 28.13 s−1 with locust bean gum as substrate, respectively. It was inhibited by mannose with a K
I of 0.610−3 mg/ml. ManIII was activated by CuSO4 and CaCl2 (2.5 mM). However, in presence of 2.5 mM Co2+, its activity dropped to 60% of the initial activity. Both N-terminal and internal amino acid sequences of ManIII presented
no homology with mannanases of glycosides hydrolases. During incubation with locust bean gum and Ivory nut mannan, the enzyme
released mainly mannotetraose, mannotriose, and mannobiose. 相似文献