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1.
2.
Pratibha Dheeran Sachin Kumar Yogesh K. Jaiswal Dilip K. Adhikari 《Applied microbiology and biotechnology》2010,86(6):1857-1866
A newly isolated Geobacillus sp. IIPTN (MTCC 5319) from the hot spring of Uttarakhand's Himalayan region produced a hyperthermostable α-amylase. The microorganism
was characterized by biochemical tests and 16S rRNA gene sequencing. The optimal temperature and pH were 60°C and 6.5, respectively,
for growth and enzyme production. Although it was able to grow in temperature ranges from 50 to 80°C and pH 5.5–8.5. Maximum
enzyme production was in exponential phase with activity 135 U ml−1 at 60°C. Assayed with cassava as substrate, the enzyme displayed optimal activity 192 U ml−1 at pH 5.0 and 80°C. The enzyme was purified to homogeneity with purification fold 82 and specific activity 1,200 U mg−1 protein. The molecular mass of the purified enzyme was 97 KDa. The values of K
m
and V
max were 36 mg ml−1 and 222 μmol mg−1 protein min−1, respectively. The amylase was stable over a broad range of temperature from 40°C to 120°C and pH ranges from 5 to 10. The
enzyme was stimulated with Mn2+, whereas it was inhibited by Hg2+, Cu2+, Zn2+, Mg2+, and EDTA, suggesting that it is a metalloenzyme. Besides hyperthermostability, the novelty of this enzyme is resistance
against protease. 相似文献
3.
C. Sivapathasekaran Palashpriya Das Soumen Mukherjee J. Saravanakumar Mahitosh Mandal Ramkrishna Sen 《International journal of peptide research and therapeutics》2010,16(4):215-222
A marine Bacillus circulans DMS-2 was able to grow and produce biosurfactant on glucose mineral salts medium (GMSM) with a reduction in the surface tension
up to 27 mN m−1. The microorganism produced 1.64 ± 0.1 g l−1 of crude biosurfactant. The lipopeptide nature of the produced biosurfactant was confirmed by primulin and ninhydrin assays
using High Performance Thin Layer Chromatography (HPTLC). Preparative thin layer chromatography (TLC) was performed to purify
the lipopeptides from the crude biosurfactant. The critical micelle concentrations (CMC) of the crude and purified products
were found to be 90 and 40 mg l−1 respectively. Fourier transform infrared spectrophotometer (FTIR) and matrix assisted laser desorption/ionization time of
flight (MALDI-ToF) mass spectral analysis revealed the identity of the produced lipopeptides as surfactin (m/z 1,023 Da) and fengycin (m/z 1,495 Da) isoforms. The purified marine lipopeptides displayed a significant antiproliferative activity against the human
colon cancer cell lines HCT-15 (IC50 80 μg ml−1) and HT-29 (IC50 120 μg ml−1). 相似文献
4.
Two carotenoid 1,2-hydratase (CrtC) genes from the photosynthetic bacteria Rubrivivax gelatinosus and Thiocapsa roseopersicina were cloned and expressed in Escherichia coli in an active form and purified by affinity chromatography. The biochemical properties of the recombinant enzymes and their
substrate specificities were studied. The purified CrtCs catalyze cofactor independently the conversion of lycopene to 1-HO-
and 1,1′-(HO)2-lycopene. The optimal pH and temperature for hydratase activity was 8.0 and 30°C, respectively. The apparent K
m and V
max values obtained for the hydration of lycopene were 24 μM and 0.31 nmol h−1 mg−1 for RgCrtC and 9.5 μM and 0.15 nmol h−1 mg−1 for TrCrtC, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed two protein bands of 44 and
38 kDa for TrCrtC, which indicate protein processing. Both hydratases are also able to convert the unnatural substrate geranylgeraniol
(C20 substrate), which functionally resembles the natural substrate lycopene. 相似文献
5.
A nitrilase gene from Alcaligenes sp. ECU0401 was cloned and overexpressed in Escherichia coli BL21 (DE3) in a soluble form. The encoded protein with a His6-tag was purified to nearly homogeneity as revealed by SDS-PAGE with a molecular weight of approximately 38.5 kDa, and the
holoenzyme was estimated to be composed of 10 subunits of identical size by size exclusion chromatography. The V
max and K
m parameters were determined to be 27.9 μmol min−1 mg−1 protein and 21.8 mM, respectively, with mandelonitrile as the substrate. The purified enzyme was highly thermostable with
a half life of 155 h at 30 °C and 94 h at 40 °C. Racemic mandelonitrile (50 mM) could be enantioselectively hydrolyzed to
(R)-(−)-mandelic acid by the purified nitrilase with an enantiomeric excess of 97%. The extreme stability, high activity and
enantioselectivity of this nitrilase provide a solid base for its practical application in the production of (R)-(−)-mandelic acid. 相似文献
6.
The tolerances of 20 Beauveria bassiana isolates derived from host insects worldwide to UV-B irradiation were assessed quantitatively in multi-dose bioassays. Conidial
suspensions of the isolates smeared on glass slides were exposed to the gradient UV-B doses of 0.1–1.6 J cm−2 (D), which generated from 0.75 to 10.17 min irradiation of weighted 312-nm wavelength at 2.0–2.61 mW cm−2. Irradiated conidia were then incubated for 24 h at 25°C under saturated humidity. The ratio of germination at each dose
over that in the blank control was defined as survival index (I
s). For all isolates, the I
s − D observations fit well with the survival model I
s = 1/[1 + exp(a + bD)] (0.94 ≤ r
2 ≤ 0.99) generated widely spanned lethal doses of 0.154–0.928, 0.240–1.139, and 0.383–1.493 J cm−2 for their losses of 50%, 75%, and 95% viabilities, respectively. These were far below the solar UV-B dose of 2.439 J cm−2 measured in a sunny day during the summer. The large variation of UV-B tolerance among the isolates indicates a necessity
to select UV-tolerant candidates for formulations applied to insect control during summer. The highly efficient bioassay method
was developed to measure accurately the UV-B tolerances of fungal biocontrol agents as lethal doses. 相似文献
7.
Joo GJ 《Biotechnology letters》2005,27(19):1483-1486
An extracellular chitinase from Streptomyces halstedii AJ-7, a broad spectrum antifungal biocontrol agent, was characterized and purified. The apparent molecular weight of the
purified protein was 55 kDa, Km value and Vmax of the protein for colloidal chitin were 3.2 mg ml−1 and 118 μmol h−1, respectively. The growth and chitinase activity of S. halstedii AJ−7 were enhanced by adding of 0.1% killed mycelium of Fusarium oxysporium in a medium containing 0.2% colloidal chitin. 相似文献
8.
A novel alkylsulfatase gene, sdsAP, was cloned from a newly isolated bacterium Pseudomonas sp. S9. It encoded a protein of 675 amino acids with a calculated molecular mass of 74.9 kDa. The protein contained a typical
N-terminal signal peptide of 41 amino acid residues, followed by a metallo-β-lactamase like domain at the N-terminus and a
SCP-2-like domain at the C-terminus. This domain organization mode suggested that it belonged to the type III sulfatase. The
mature alkylsulfatase was overexpressed in Escherichia coli. The optimal temperature and pH of the recombinant SdsAP were 70°C and 9.0, respectively. Notably, at optimal conditions,
the purified recombinant SdsAP had a high specific activity of 23.25 μmol min−1 mg−1, a K
m (app) of 264.3 μmol, and a V
max (app) of 33.8 μmol min−1 mg−1 for SDS. Additionally, it still retained more than 90% activity after incubation at 65°C for 1 h, which was much different
from other alkylsulfatases reported. The recombinant enzyme hydrolyzed the primary alkyl sulfate such as sodium octyl sulfate
and sodium dodecyl sulfate (SDS). It was a Zn2+-containing and Ca2+ activated alkylsulfatase. This is the first report to explore the various characteristics of the heterologous recombinant
alkylsulfatase in details. These favorable properties could make SdsAP attractive to be useful in the degradation of SDS-containing
waste. 相似文献
9.
10.
A phytase with high activity at neutral pH and typical water temperatures (∼25°C) could effectively hydrolyze phytate in aquaculture.
In this study, a phytase-producing strain, Pedobacter nyackensis MJ11 CGMCC 2503, was isolated from glacier soil, and the relevant gene, PhyP, was cloned using degenerate PCR and thermal asymmetric interlaced PCR. To our knowledge, this is the first report of detection
of phytase activity and cloning of phytase gene from Pedobacter. PhyP belongs to beta-propeller phytase family and shares very low identity (∼28.5%) with Bacillus subtilis phytase. The purified recombinant enzyme (r-PhyP) from Escherichia coli displayed high specific activity for sodium phytate of 24.4 U mg−1. The optimum pH was 7.0, and the optimum temperature was 45°C. The K
m, V
max, and k
cat values were 1.28 mM, 71.9 μmol min−1 mg−1, and 45.1 s−1, respectively. Compared with Bacillus phytases, r-PhyP had higher relative activity at 25°C (r-PhyP (>50%), B. subtilis phytase (<8%)) and hydrolyzed phytate from soybean with greater efficacy at neutral pH. These characteristics suggest that
r-PhyP might be a good candidate for an aquatic feed additive in the aquaculture industry. 相似文献
11.
Molecular characterization of phenylalanine ammonia lyase gene from <Emphasis Type="Italic">Cistanche deserticola</Emphasis> 总被引:1,自引:0,他引:1
Hu GS Jia JM Hur YJ Chung YS Lee JH Yun DJ Chung WS Yi GH Kim TH Kim DH 《Molecular biology reports》2011,38(6):3741-3750
We cloned the gene, CdPAL1, from Cistanche deserticola callus using RACE PCR with degenerate primers that were designed based on a multiple sequence alignment of known PAL genes
from other plant species. The gene shows high homology to other known PAL genes registered in GenBank. The recombinant protein
exhibited Michaelis–Menten kinetics with a K
m of 0.1013 mM, V
max of 4.858 μmol min−1, K
cat of 3.36 S−1, and K
cat/K
m is 33,168 M−1 S−1. The enzyme had an optimal pH of 8.5 and an activation energy of 38.92 kJ mol−1 when l-Phenylalanine was used as a substrate; l-tyrosine cannot be used as substrate for this protein. The optimal temperature was 55°C, and the thermal stability results
showed that, after a treatment at 70°C for 20 min, the protein retained 87% activity, while a treatment at 75°C for 20 min
resulted in a loss of over 85% of the enzyme activity. Treatment with heavy metal ions (Hg2+, Pb2+, and Zn2+) showed remarkable inhibitory effects. Among the intermediates from the lignin (cinnamyl alcohol, cinnamyl aldehyde, coniferyl
aldehyde, coniferyl alcohol), phenylpropanoid (cinnamic acid, coumaric acid, caffeic acid, and chlorogenic acid) and phenylethanoid
(tyrosol and salidroside) biosynthetic pathways, only cinnamic acid showed strong inhibitory effects against CdPAL1 activity with a K
i of 8 μM. Competitive inhibitor AIP exhibited potent inhibition with K
i = 0.056 μM. 相似文献
12.
Fusarium moniliforme var. subglutinans was selected from among 100 strains of fungi for producing ginsenoside F1 from ginsenoside Rg1. The enzyme responsible was purified as a single 85 kDa band with a specific activity of 136 U mg−1. It hydrolysed glucose-linked ginsenosides Rb1, Rd and Rg1 but not for other monosaccharide-linked ginsenosides, Rb2, Rc, R1, and Re. Under the optimum conditions of pH 6.0, 50°C, 30 U l−1 of enzyme, and 5 mg Rg1 ml−1, 4 mg F1 ml−1 was produced after 4 h, with a molar yield of 100% and a productivity of 1 g l−1 h−1. This represents the highest productivity and conversion yield of F1 yet reported. 相似文献
13.
Purification, immobilization and characterization of linoleic acid isomerase on modified palygorskite 总被引:2,自引:0,他引:2
Linoleic acid isomerase from Lactobacillus delbrueckii subsp. bulgaricus 1.1480 was purified by DEAE ion-exchange chromatography and gel filtration chromatography. An overall 5.1% yield and purification
of 93-fold were obtained. The molecular weight of the purified protein was ~41 kDa which was analyzed by SDS-PAGE. The purified
enzyme was immobilized on palygorskite modified with 3-aminopropyltriethoxysilane. The immobilized enzyme showed an activity
of 82 U/g. The optimal temperature and pH for the activity of the free enzyme were 30 °C and pH 6.5, respectively; whereas
those for the immobilized enzyme were 35 °C and pH 7.0, respectively. The immobilized enzyme was more stable than the free
enzyme at 30–60 °C, and the operational stability result showed that more than 85% of its initial activity was retained after
incubation for 3 h. The K
m and V
max values of the immobilized enzyme were found to be 0.0619 mmol l−1 and 0.147 mmol h−1 mg−1, respectively. The immobilized enzyme had high operational stability and retained high enzymatic activity after seven cycles
of reuse at 37 °C. 相似文献
14.
The cry8C-type gene designated cry8Ca2, which was cloned and sequenced from a Bacillus thuringiensis isolate HBF-1 in China, consisted of an open reading frame of 3483 bp encoding a protein of 1160 amino-acid residues. Sequence
analysis showed that the Cry8Ca2 protoxin of 130.5 kDa had 99.9% sequence homology with the previously reported Cry8Ca1 protein,
with one mismatch between the two amino-acid sequences. When the Cry8Ca2 toxin was expressed in a crystal-negative strain
of B. thuringiensis (HD-73−), elliptical crystals were produced. Cell extracts from this recombinant strain showed insecticidal activity against Anomala corpulenta larva. Mutant cry8Ca2 genes, produced by polymerase chain reaction amplification with Taq DNA polymerase, were used to develop recombinant B. thuringiensis strains. Mutants producing higher levels of insecticidal activity were identified by bioassay. Thirty-five mutants forming
crystals were characterized, and two of them showed significantly increased insecticidal activity against A. corpulenta larva. The 50% lethality concentrations (LC50) of the two mutants were 0.2334 × 108 and 0.2591 × 108 colony-forming units g−1, considerably lower than the LC50 of the wild-type strain HBF-1 (0.9583 × 108 CFU g−1) and that of B. thuringiensis serovar japonensis strain Buibui (1.0752 × 108 CFU g−1). 相似文献
15.
An artificial fusion protein of Arthrobacter oxydans dextranase and Klebsiella pneumoniae α-amylase was constructed and expressed in Escherichia coli. Most of the expressed protein existed as an insoluble fraction, which was solubilized with urea. The purified fusion enzyme
electrophoretically migrated as a single protein band; M = 137 kDa, and exhibited activities of both dextranase (10.8 U mg−1) and amylase (7.1 U mg−1), which were lower than that of reference dextranase (13.3 U mg−1) and α-amylase (103 U mg−1). The fusion enzyme displayed bifunctional enzyme activity at pH 5–7 at 37°C. These attributes potentially make the fusion
enzyme more convenient for use in sugar processing than a two-enzyme system. 相似文献
16.
Kemel Jellouli Ali Bougatef Laila Manni Rym Agrebi Rayda Siala Islem Younes Moncef Nasri 《Journal of industrial microbiology & biotechnology》2009,36(7):939-948
A protease-producing bacterium was isolated from an alkaline wastewater of the soap industry and identified as Vibrio metschnikovii J1 on the basis of the 16S rRNA gene sequencing and biochemical properties. The strain was found to over-produce proteases
when it was grown at 30°C in media containing casein as carbon source (14,000 U ml−1). J1 enzyme, the major protease produced by V. metschnikovii J1, was purified by a three-step procedure, with a 2.1-fold increase in specific activity and 33.3% recovery. The molecular
weight of the purified protease was estimated to be 30 kDa by SDS-PAGE and gel filtration. The N-terminal amino acid sequence
of the first 20 amino acids of the purified J1 protease was AQQTPYGIRMVQADQLSDVY. The enzyme was highly active over a wide
range of pH from 9.0 to 12.0, with an optimum at pH 11.0. The optimum temperature for the purified enzyme was 60°C. The activity
of the enzyme was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease. The kinetic
constants K
m and K
cat of the purified enzyme using N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide were 0.158 mM and 1.14 × 105 min−1, respectively. The catalytic efficiency (K
cat
/K
m) was 7.23 × 108 min−1 M−1. The enzyme showed extreme stability toward non-ionic surfactants and oxidizing agents. In addition, it showed high stability
and compatibility with some commercial liquid and solid detergents. The aprJ1 gene, which encodes the alkaline protease from V. metschnikovii J1, was isolated, and its DNA sequence was determined. The deduced amino acid sequence of the preproenzyme differs from that
of V. metschnikovii RH530 detergent-stable protease by 12 amino acids, 7 located in the propeptide and 5 in the mature enzyme. 相似文献
17.
Xue-Qin Xie Jie Wang Bao-Fu Huang Sheng-Hua Ying Ming-Guang Feng 《Applied microbiology and biotechnology》2010,86(5):1543-1553
A superoxide dismutase (SOD) was characterized from Beauveria bassiana, a fungal entomopathogen widely applied to insect control. This 209-aa enzyme (BbSod2) showed no more than 71% sequence identity
to other fungal Mn-SODs, sharing all conserved residues with the Mn-SOD family and lacking a mitochondrial signal. The SOD
activity of purified BbSod2 was significantly elevated by Mn2+, suppressed by Cu2+ and Zn2+ but inhibited by Fe3+. Overexpressing the enzyme in a BbSod2-absent B. bassiana strain enhanced its SOD activity (107.2 ± 6.1 U mg−1 protein) by 4–10-fold in different transformants analyzed. The best BbSod2-transformed strain with the SOD activity of 1,157.9 ± 74.7 U mg−1 was 93% and 61% more tolerant to superoxide-generating menadione in both colony growth (EC50 = 2.41 ± 0.03 versus 1.25 ± 0.01 mM) and conidial germination (EC50 = 0.89 ± 0.06 versus 0.55 ± 0.07 mM), and 23% more tolerant to UV-B irradiation (LD50 = 0.49 ± 0.02 versus 0.39 ± 0.01 J cm−2). Its virulence to Spodoptera litura larvae was enhanced by 26% [LT50 = 4.5 (4.2–4.8) versus 5.7 (5.2–6.4) days]. Our study highlights for the first time that the Mn2+-cofactored, cytosolic BbSod2 contributes significantly to the virulence and stress tolerance of B. bassiana and reveals possible means to improving field persistence and efficacy of a fungal formulation by manipulating the antioxidant
enzymes of a candidate strain. 相似文献
18.
As a common pollutant, nitrite concentrations can approach 15 mg NO2−-N L−1 in some aquatic systems. Microcystis aeruginosa blooms are common and widespread in eutrophic freshwater bodies. In this study, M. aeruginosa was exposed to nitrite concentrations ranging from 0 to 15 mg NO2−-N L−1, and the responses of M. aeruginosa were investigated. The specific growth rates, maximum cell densities, light-saturated photosynthetic rates (Pm
chla
), dark respiration rates (Rd
chla
), and apparent photosynthetic efficiencies (αchla
) showed a significant decline with nitrite concentrations increasing. Electrical conductivity and malondialdehyde contents
investigation revealed cell membrane damage and apparent leakage of intracellular contents under high nitrite level conditions
due to oxidative stress enhancement. Intracellular microcystin (MC)-LR content reached the highest value at 10 mg NO2−-N L−1; however, extracellular MC-LR contents showed a continuous increase until 15 mg NO2−-N L−1 owing to the increasing leakage of intracellular contents. These results elucidated that the high-level nitrite inhibited
M. aeruginosa growth by rising oxidative stress, damaging cell membrane, and reducing photosynthesis. However, the moderate increase in
nitrite concentrations promoted toxin production and release of toxin. 相似文献
19.
Bacillus coagulans has been of great commercial interest over the past decade owing to its strong ability of producing optical pure l-lactic acid from both hexose and pentose sugars including l-arabinose with high yield, titer and productivity under thermophilic conditions. The l-arabinose isomerase (L-AI) from Bacillus coagulans was heterologously over-expressed in Escherichia coli. The open reading frame of the L-AI has 1,422 nucleotides encoding a protein with 474 amino acid residues. The recombinant
L-AI was purified to homogeneity by one-step His-tag affinity chromatography. The molecular mass of the enzyme was estimated
to be 56 kDa by SDS-PAGE. The enzyme was most active at 70°C and pH 7.0. The metal ion Mn2+ was shown to be the best activator for enzymatic activity and thermostability. The enzyme showed higher activity at acidic
pH than at alkaline pH. The kinetic studies showed that the K
m, V
max and k
cat/K
m for the conversion of l-arabinose were 106 mM, 84 U/mg and 34.5 mM−1min−1, respectively. The equilibrium ratio of l-arabinose to l-ribulose was 78:22 under optimal conditions. l-ribulose (97 g/L) was obtained from 500 g/l of l-arabinose catalyzed by the enzyme (8.3 U/mL) under the optimal conditions within 1.5 h, giving at a substrate conversion
of 19.4% and a production rate of 65 g L−1 h−1. 相似文献
20.
Kim HT Ko HJ Kim N Kim D Lee D Choi IG Woo HC Kim MD Kim KH 《Biotechnology letters》2012,34(6):1087-1092
A gene, alg7D, from Saccharophagus degradans, coding for a putative alginate lyase belonging to the family of polysaccharide lyase-7, was overexpressed in Escherichia coli. The properties of the recombinant Alg7D were characterized. The enzyme endolytically depolymerized alginate by β-elimination
into oligo-alginates with degrees of polymerization of 2–5. Its activity was maximal at 50°C and pH 7 and was slightly increased
in the presence of Na+. The K
M
, V
max
, k
cat
, and k
cat
/K
M
values were: 3 mg ml−1, 6.2 U mg−1, 1.9 × 10−2 s−1, and 6.3 × 10−3 mg−1 ml s−1, respectively. 相似文献