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1.
Lin ZJ Zheng RC Wang YJ Zheng YG Shen YC 《Journal of industrial microbiology & biotechnology》2012,39(1):133-141
A novel enzymatic route for the synthesis of 2-amino-2,3-dimethylbutyramide (ADBA), important intermediate of highly potent
and broad-spectrum imidazolinone herbicides, from 2-amino-2,3-dimethylbutyronitrile (ADBN) was developed. Strain Rhodococcus boritolerans CCTCC M 208108 harboring nitrile hydratase (NHase) towards ADBN was screened through a sophisticated colorimetric screening
method and was found to be resistant to cyanide (5 mM). Resting cells of R. boritolerans CCTCC M 208108 also proved to be tolerant against high product concentration (40 g l−1) and alkaline pH (pH 9.3). A preparative scale process for continuous production of ADBA in both aqueous and biphasic systems
was developed and some key parameters of the biocatalytic process were optimized. Inhibition of NHase by cyanide dissociated
from ADBN was successfully overcome by temperature control (at 10°C). The product concentration, yield and catalyst productivity
were further improved to 50 g l−1, 91% and 6.3 g product/g catalyst using a 30/70 (v/v) n-hexane/water biphasic system. Furthermore, cells of R. boritolerans CCTCC M 208108 could be reused for at lease twice by stopping the continuous reaction before cyanide concentration rose to
2 mM, with the catalyst productivity increasing to 12.3 g product/g catalyst. These results demonstrated that enzymatic synthesis
of ADBA using whole cells of R. boritolerans CCTCC M 208108 showed potential for industrial application. 相似文献
2.
Yuko Ishimoto Shiho Kato Sumio Maeda 《World journal of microbiology & biotechnology》2008,24(11):2731-2735
Freeze-thaw treatment of condensed suspensions of mixed Escherichia coli strains in natural waters and food extracts caused in situ lateral transfer of non-conjugative plasmids. This phenomenon
also occurred in distilled water and LB broth, and after 1–2 months of preservation at −20°C. The sensitivity of lateral transfer
towards DNase activity suggested the involvement of in situ transformation. There were no clear correlations between transformation
frequency and the chemical characteristics (Ca2+, Mg2+, and pH) of the samples. These results suggest the possibility that freeze–thaw-induced lateral gene transfer between bacterial
cells occurs in natural and human environments. 相似文献
3.
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
4.
A Bacillus sp., capable of degrading chloroform, was immobilized in calcium alginate. The beads in 20 g alginate l−1 (about 2 × 108 cells/bead) could be re-used nine times for degradation of chloroform at 40 μM. The immobilized cells had a higher range
of tolerance (pH 6.5–9 and 20–41°C) than free cells (pH 7–8.5 and 28–32°C). At 5 g alginate l−1, leakage of the cells from the beads was 0.51 mg dry wt ml−1. This species is the first reported Bacillus that can degrade chloroform as the sole carbon source. 相似文献
5.
6.
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
7.
Daniel F. Visser Fritha Hennessy Konanani Rashamuse Maureen E. Louw Dean Brady 《Extremophiles : life under extreme conditions》2010,14(2):185-192
A purine nucleoside phosphorylase from the alkaliphile Bacillus halodurans Alk36 was cloned and overexpressed in Escherichia coli. The enzyme was purified fivefold by membrane filtration and ion exchange. The purified enzyme had a V
max of 2.03 × 10−9 s −1 and a K
m of 206 μM on guanosine. The optimal pH range was between 5.7 and 8.4 with a maximum at pH 7.0. The optimal temperature for
activity was 70°C and the enzyme had a half life at 60°C of 20.8 h. 相似文献
8.
Two 60-day experiments were conducted to study the influence of photon flux density (PFD) and temperature on the attachment
and development of Gloiopeltis tenax and Gloiopeltis furcata tetraspores. In the first experiment, tetraspores of the two Gloiopeltis species were incubated at five temperature ranges (8°C, 12°C, 16°C, 20°C, 24°C) under a constant PFD of 80 μmol photons m−2 s−1 with a photoperiod of 12:12. In a second experiment, tetraspores were incubated under five PFD gradients (30, 55, 80, 105,
130 μmol photons m−2 s−1) at a constant temperature of 16°C with a photoperiod of 12:12. Maximum density of attached tetraspores was observed at 16°C
for both species. Maximum per cent of spore germinating into disc was recorded at 12–16°C for G. tenax and 8–12°C for G. furcata. Maximum per cent of discs producing erect axes for G. tenax and G. furcata were recorded at 24°C and 20°C, respectively. Light had no significant effect on tetraspore attachment and developing into
disc, but it affected the growth, sprouting and survival of its discs. Under 30–55 μmol photons m−2 s−1, the discs of the two species of Gloiopeltis did not form thallus until the end of the experiment. Optimum PFD range for G. tenax discs was 80–105 μmol photons m−2 s−1, whilst it was 80–130 μmol photons m−2 s−1 for G. furcata. Results presented in this study are expected to assist the progress of artificial seeding of Gloiopeltis. 相似文献
9.
Cell free extracts of Galactomyces reessii contain a hydratase as the key enzyme for the transformation of 3-methylcrotonic acid to 3-hydroxy-3-methylbutyric acid.
Highest levels of hydratase activity were obtained during growth on isovaleric acid. The enzyme, an enoyl CoA hydratase, was
purified 147-fold by precipitation with ammonium sulphate and successive chromatography over columns of DE-52, Blue Sepharose
CL-6B and Sephacryl S-200. During purification, hydratase activity was measured spectrophotometrically (OD change at 263 nm)
for 3-methylcrotonyl CoA and crotonyl CoA as substrates. The enzyme displayed highest activity with crotonyl CoA with a K
cat of 1,050,000 min−1. The ratio of crotonyl CoA to 3-methylcrotonyl CoA activities was constant (20:1) during all steps of purification. The K
cat for crotonyl CoA was also about 20 times greater than the K
cat for 3-methylcrotonyl CoA (51,700 min−1). The enzyme had pH and temperature optima at 7.0 and 35°C, a native M
r of 260±4.5 kDa and a subunit M
r of 65 kDa, suggesting that the enzyme was a homotetramer. The pI of the purified hydratase was 5.5, and the N-terminal amino acid sequence was VPEGYAEDLLKGKMMRFFDS. Hydratase activity for
3-methylcrotonyl CoA was competitively inhibited by acetyl CoA, propionyl CoA and acetoacetyl CoA. Journal of Industrial Microbiology & Biotechnology (2002) 28, 81–87 DOI: 10.1038/sj/jim/7000215
Received 27 June 2001/ Accepted in revised form 17 September 2001 相似文献
10.
Fábio C. Sampaio Janaína T. de Faria Flávia M. Lopes Passos Attilio Converti Luis Antônio Minin 《Journal of industrial microbiology & biotechnology》2009,36(2):293-300
Xylose reductase (XR) is the enzyme that catalyzes the first step of xylose metabolism. Although XRs from various yeasts have
been characterized, little is known about this enzyme in Debaryomyces hansenii. In the present study, response surface analysis was used to determine the optimal conditions for D. hansenii UFV-170 XR activity. The influence of pH and temperature, ranging from 4.0 to 8.0 and from 25 to 55°C, respectively, was
evaluated by a 22 central composite design face-centered. The F-test (ANOVA) and the Student’s t test were performed to evaluate the statistical significance of the model and the regression coefficients, respectively.
The NADPH-dependent XR activity varied from 0.502 to 2.53 U mL−1, corresponding to 0.07–0.352 U mg−1, whereas the NADH-dependent one was almost negligible. The model predicted with satisfactory correlation (R
2 = 0.940) maximum volumetric activity of 2.27 U mL−1 and specific activity of 0.300 U mg−1 at pH 5.3 and 39°C, which were fairly confirmed by additional tests performed under these conditions. The enzyme proved very
stable at low temperature (4°C), keeping its activity almost entirely after 360 min, which corresponded to the half-time at
39°C. On the other hand, at temperatures ≥50°C it was lost almost completely after only 20 min. 相似文献
11.
Chellappan S Jasmin C Basheer SM Kishore A Elyas KK Bhat SG Chandrasekaran M 《Journal of industrial microbiology & biotechnology》2011,38(6):743-752
An alkaline protease from marine Engyodontium album was characterized for its physicochemical properties towards evaluation of its suitability for potential industrial applications.
Molecular mass of the enzyme by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) analysis was calculated
as 28.6 kDa. Isoelectric focusing yielded pI of 3–4. Enzyme inhibition by phenylmethylsulfonyl fluoride (PMSF) and aprotinin
confirmed the serine protease nature of the enzyme. K
m, V
max, and K
cat of the enzyme were 4.727 × 10−2 mg/ml, 394.68 U, and 4.2175 × 10−2 s−1, respectively. Enzyme was noted to be active over a broad range of pH (6–12) and temperature (15–65°C), with maximum activity
at pH 11 and 60°C. CaCl2 (1 mM), starch (1%), and sucrose (1%) imparted thermal stability at 65°C. Hg2+, Cu2+, Fe3+, Zn2+, Cd+, and Al3+ inhibited enzyme activity, while 1 mM Co2+ enhanced enzyme activity. Reducing agents enhanced enzyme activity at lower concentrations. The enzyme showed considerable
storage stability, and retained its activity in the presence of hydrocarbons, natural oils, surfactants, and most of the organic
solvents tested. Results indicate that the marine protease holds potential for use in the detergent industry and for varied
applications. 相似文献
12.
Madanala R Gupta V Deeba F Upadhyay SK Pandey V Singh PK Tuli R 《Biotechnology letters》2011,33(10):2057-2063
A gene from Withania somnifera (winter cherry), encoding a highly stable chloroplastic Cu/Zn superoxide dismutase (SOD), was cloned and expressed in Escherichia coli. The recombinant enzyme (specific activity of ~4,200 U mg−1) was purified and characterized. It retained ~90 and ~70% residual activities after 1 h at 80 and 95°C, respectively. At
95°C, thermal inactivation rate constant (K
d) of the enzyme was 2.46 × 10−3 min−1 and half-life of heat inactivation was 4.68 h. The enzyme was stable against a broad pH range (2.5–11.0). It also showed
a high degree of resistance to detergent, ethanol and protease digestion. This recombinant Cu/Zn SOD could therefore have
useful applications. 相似文献
13.
Halophilic archaeal strains R26T and R22 were isolated from the brown alga Laminaria produced at Dalian, Liaoning Province, China. Cells from the two strains were pleomorphic rods and Gram negative, and colonies
were red pigmented. Strains R26T and R22 were able to grow at 20–50°C (optimum 37°C) in 1.4–5.1 M NaCl (optimum 3.1–4.3 M) at pH 5.5–9.5 (optimum pH 8.0–8.5)
and neither strain required Mg2+ for growth. Cells lyse in distilled water and the minimum NaCl concentration required to prevent cell lysis was 8% (w/v)
for strain R26T and 12% (w/v) for strain R22. The major polar lipids of the two strains were phosphatidylglycerol, phosphatidylglycerol phosphate
methyl ester and minor phosphatidylglycerol sulfate; glycolipids were not detected. Phylogenetic analyses based on 16S rRNA
genes and rpoB′ genes revealed that strains R26T and R22 formed a distinct clade with the closest relative, Natronoarchaeum mannanilyticum. The DNA G+C content of strains R26T and R22 was 65.8 and 66.4 mol%, respectively. The DNA–DNA hybridization value between strains R26T and R22 was 89%. The phenotypic, chemotaxonomic and phylogenetic properties suggest that the strains R26T and R22 represent a novel species in a new genus within the family Halobacteriaceae, for which the name Salinarchaeum laminariae gen. nov., sp. nov. is proposed. The type strain is R26T (type strain R26T = CGMCC 1.10590T = JCM 17267T, reference strain R22 = CGMCC 1.10589). 相似文献
14.
Sumra Afzal Mahjabeen Saleem Riffat Yasmin Mamoona Naz Muhammad Imran 《Molecular biology reports》2010,37(4):1717-1723
Consistent with its precloning characterization from the cellulolytic Bacillus sp., β-1,4-endoglucanase purified from the recombinant E. coli exhibited maximum activity at 60°C and pH 7.0. It was highly specific for CMC hydrolysis, with stability up to 70°C and over
a pH range of 6.0–8.0. The K
m and V
max values for CMCase activity of the enzyme were 4.1 mg/ml and 25 μmole/ml min−1, respectively. The purified enzyme was a monomer of 65 kDa, as determined by SDS-PAGE. The presence of sucrose and IPTG in
fermentation media increased the endoglucanase activity of the recombinant enzyme to 5.2-folds as compared with that of the
actual one. 相似文献
15.
The endochitinase DNA and cDNA from Trichoderma sp. were cloned, sequenced and expressed. The cloned DNA and cDNA sequences were 1,476 and 1,275 bp in length, respectively.
There were three introns in DNA sequence in comparison with the cDNA sequence. The endochitinase protein contained three regions:
the signal peptide, the prepro-region and the mature protein region. The gene fragment encoding the mature endochitinase was
ligated into the expression vector pET-28a+, yielding pET-1. The plasmid pET-1 was transformed into the Escherichia coli BL21 (DE3). The clone bearing pET-1 was picked and cultured at 30°C for the expression of endochitinase. SDS-PAGE analysis
showed that the endochitinase was expressed in the periplasmic space and the purified protein showed a single band. The activity
of 70.2 U/mg was obtained from the cellular extract of the recombinant strain. The activity of endochitinase was 2.5-fold
higher at 24 h than at 16 h in the periplasmic space. The optimal pH and temperature of the recombinant endochitinase were
determined to be 7.0 and 35°C, respectively. It was relatively stable within the pH range of 5–8. Significant activity stimulation
by 1 mM Mg2+ and 5 mM Fe2+ and inhibition by 5 mM Co2+ and 5 mM Hg2+ were observed. The kinetic constants Km, Vmax and Kcat for the hydrolysis of the colloidal chitin were 1.5 mM, 1.37 μmol min−1 and 6.23 min−1, respectively. 相似文献
16.
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
17.
A novel endo-type β-agarase gene, agaA, was cloned from a newly isolated marine bacterium, Agarivorans sp. LQ48. It encodes a protein of 457 amino acids with a calculated molecular mass of 51.2 kDa. The deduced protein contains
a typical N-terminal signal peptide of 25 amino acid residues, followed by a catalytic module, which is homologous to that
of glycoside hydrolase family 16. A sequence similar to a carbohydrate-binding module is found in the C-terminal region of
the enzyme. The overall amino acid sequence shares a highest identity of 73% with the sequence of beta-agarase AgaB from Pseudoalteromonas sp. strain CY24. The mature agarase was highly expressed extracellularly in Escherichia coli. At pH 7.0 and 40°C, the purified recombinant AgaA had a high specific activity of 349.3 μmol min−1 mg−1, a K
m of 3.9 mg ml−1, and a V
max of 909.1 μmol min−1 mg−1 for agarose. The recombinant enzyme hydrolyzed the β-1,4-glycosidic linkages of agarose, yielding neoagarotetraose and neoagarohexaose
as the main products. Enzyme activity analysis revealed that the optimal temperature and pH of the recombinant AgaA were 40°C
and 7.0, respectively. Notably, AgaA still retained more than 95% activity after incubation at pH 3.0–11.0 for 1 h, a characteristic
much different from other agarases reported. It is the first agarase identified to have so wide a pH range stability. This
favorable property could make AgaA to be attractive to the food, cosmetic, and medical industrial applications. 相似文献
18.
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
19.
Jeyagowri Kiddinamoorthy Alfredo J. Anceno Gulelat D. Haki Sudip K. Rakshit 《World journal of microbiology & biotechnology》2008,24(5):605-612
Production of extracellular xylanase from Bacillus sp. GRE7 using a bench-top bioreactor and solid-state fermentation (SSF) was attempted. SSF using wheat bran as substrate
and submerged cultivation using oat-spelt xylan as substrate resulted in an enzyme productivity of 3,950 IU g−1 bran and 180 IU ml−1, respectively. The purified enzyme had an apparent molecular weight of 42 kDa and showed optimum activity at 70°C and pH
7. The enzyme was stable at 60–80°C at pH 7 and pH 5–11 at 37°C. Metal ions Mn2+ and Co2+ increased activity by twofold, while Cu2+ and Fe2+ reduced activity by fivefold as compared to the control. At 60°C and pH 6, the K
m for oat-spelt xylan was 2.23 mg ml−1 and V
max was 296.8 IU mg−1 protein. In the enzymatic prebleaching of eucalyptus Kraft pulp, the release of chromophores, formation of reducing sugars
and brightness was higher while the Kappa number was lower than the control with increased enzyme dosage at 30% reduction
of the original chlorine dioxide usage. The thermostability, alkali-tolerance, negligible presence of cellulolytic activity,
ability to improve brightness and capacity to reduce chlorine dioxide usage demonstrates the high potential of the enzyme
for application in the biobleaching of Kraft pulp. 相似文献
20.
Bao W Peng R Zhang Z Tian Y Zhao W Xue Y Gao J Yao Q 《Molecular biology reports》2012,39(4):3871-3877
A novel laccase gene from Monilinia fructigena was synthesized chemically according to the yeast bias codon and integrated into the genome of Pichia pastoris GS115 by electroporation. The expressed enzyme was recovered from the culture supernatant and purified. The result of enzyme
activity assay and SDS-PAGE demonstrated that the recombinant laccase was induced and extracellularly expressed in P. pastoris. Main biochemical properties of this laccase, such as thermodependence and thermostability, optimal pH and pH stability,
and the effect of metal ions and inhibitors, were characterized. With 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate (ABTS)
as the substrate, MfLcc had its optimal pH at 3.5 and optimal temperature at 45°C. The Km values of the ABTS, guaiacol were 0.012 and 0.016 Mm, respectively, and the corresponding V
max values are 243.9 and 10.55 Um min−1 mg−1, respectively. The recombinant laccase degraded 80% 2,4,6-trichlorophenol after 8 h under the optimal conditions. The recombinant
strain and its laccase can be considered as candidate for treating waste water polluted with trichlorophenols. 相似文献