首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 62 毫秒
1.
The methylcobalamin (MeB12) homocysteine transmethylase activity and the B12-dependent 5-methyltetrahydrofolate (5-MeH4-folate) homocysteine transmethylase activity in cell-free extracts of E. coli B are catalytic functions of separate sites on a single enzyme-protein. Whether these two transmethylases exactly co-purify from extracts, and are protected against p-chloromercuribenzoate (pCMB), however, depends on whether or not the cells were previously cultured in the presence of approximately 1 × 10?8 m cyanocobalamin (CNB12). E. coli B (met H?) contains a defective 5-MeH4-folate apoenzyme which does not tightly bind B12 as a prosthetic group. While the folate-inactive apoenzyme from the mutant strain still catalyzes MeB12 homocysteine transmethylation, this second site on the defective protein is not protected by media CNB12 against pCMB inactivation. Both transmethylase activities are repressed 50% by growth in the presence of 10 m l-methionine.  相似文献   

2.
The thermostable bifunctional CMCase and xylanase encoding gene (rBhcell-xyl) from Bacillus halodurans TSLV1 has been expressed in Escherichia coli. The recombinant E. coli produced rBhcell-xyl (CMCase 2272 and 910 U L?1 xylanase). The rBhcell-xyl is a ~62-kDa monomeric protein with temperature and pH optima of 60 °C and 6.0 with T1/2 of 7.0 and 3.5 h at 80 °C for CMCase and xylanase, respectively. The apparent K m values (CMC and Birchwood xylan) are 3.8 and 3.2 mg mL?1. The catalytic efficiency (k cat/K m ) values of xylanase and CMCase are 657 and 171 mL mg?1 min?1, respectively. End-product analysis confirmed that rBhcell-xyl is a unique endo-acting enzyme with exoglucanase activity. The rBhcell-xyl is a GH5 family enzyme possessing single catalytic module and carbohydrate binding module. The action of rBhcell-xyl on corn cobs and wheat bran liberated reducing sugars, which can be fermented to bioethanol and fine biochemicals.  相似文献   

3.
Diol synthase from Aspergillus nidulans was cloned and expressed in Escherichia coli. Recombinant E. coli cells expressing diol synthase from A. nidulans converted linoleic acid to a product that was identified as 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The recombinant cells and the purified enzyme showed the highest activity for linoleic acid among the fatty acids tested. The optimal reaction conditions for the production of 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid from linoleic acid using whole recombinant E. coli cells expressing diol synthase were pH 7.5, 35°C, 250 rpm, 5 g l?1 linoleic acid, 23 g l?1 cells, and 20% (v/v) dimethyl sulfoxide in a 250-ml baffled flask. Under these optimized conditions, whole recombinant cells expressing diol synthase produced 4.98 g l?1 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid for 150 min without detectable byproducts, with a conversion yield of 99% (w/w) and a productivity of 2.5 g l?1 h?1. This is the first report on the biotechnological production of dihydroxy fatty acid using whole recombinant cells expressing diol synthase.  相似文献   

4.
Purified enzymes and cell-free homogenates encapsulated by liquid-surfactant membrane have been shown to retain their catalytic activity (see previously published literature). This paper describes the preparation and properties of liquid-surfactant membrane-encapsulated whole cells of Micrococcus denitrificansATCC 21909. Batch and continuous studies with this model system have demonstrated that encapsulated viable cells reduce nitrates and retain their catalytic activity over anextended period of time. In batch operation, the reactivity of the encapsulated whole cells has been investigated under a variety of experimental conditions. The system is capable of reducing NO3? or NO2?. Data obtained indicate that encapsulated live cells have a broad pH and temperature optimum range. The encapsulated cells remain viable and do not “escape” into the external aqueous phase, even after five days of constant stirring with nitrate-containing simulated wastewater. Pulsed substrate addition experiments have demonstrated that the encapsulated cells also effectively reduce NO2? with no significant reduction in activity, even after 5.5 days of incubation at 30°C. The membrane selectivity for ion transfer has been achieved by incorporating oil-soluble ion exchangers in the membrane. Because of the protection of the liquid membranes, the catalytic reduction of NO2? by the encapsulated whole cells is not inhibited by 1 × 10?4 M mercuric chloride, which is otherwise extremely toxic to the cells, when present in the external aqueous phase. Continuous reduction of 20 ppm of NO2? by liquid membrane-encapsulated whole cells has been demonstrated in a constantly stirred reactor over a test period of about one week. In this paper we will discuss the reduction of NO3?and NO2? by the liquid membrane-encapsulated whole cells of M. denitrificansATCC 21909 mainly in batch runs undera variety of experimental conditions, such as cell and substrate concentrations, product and inhibitor permeation, pH and temperature, effect of oil-soluble ion exchangers on the substrate diffusion, etc.  相似文献   

5.
The cDNA gene coding for formate dehydrogenase (FDH) from Ogataea parapolymorpha DL-1 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by nickel affinity chromatography and was characterized as a homodimer composed of two identical subunits with approximately 40 kDa in each monomer. The enzyme showed wide pH optimum of catalytic activity from pH 6.0 to 7.0. It had relatively high optimum temperature at 65 °C and retained 93, 88, 83, and 71 % of its initial activity after 4 h of exposure at 40, 50, 55, and 60 °C, respectively, suggesting that this enzyme had promising thermal stability. In addition, the enzyme was characterized to have significant tolerance ability to organic solvents such as dimethyl sulfoxide, n-butanol, and n-hexane. The Michaelis–Menten constant (K m), turnover number (k cat), and catalytic efficiency (k cat/K m) values of the enzyme for the substrate sodium formate were estimated to be 0.82 mM, 2.32 s?1, and 2.83 mM?1 s?1, respectively. The K m for NAD+ was 83 μM. Due to its wide pH optimum, promising thermostability, and high organic solvent tolerance, O. parapolymorpha FDH may be a good NADH regeneration catalyst candidate.  相似文献   

6.
Rhizobium sp. strain TAL1145 catabolizes mimosine, which is a toxic non-protein amino acid present in Leucaena leucocephala (leucaena). The objective of this investigation was to study the biochemical and catalytic properties of the enzyme encoded by midD, one of the TAL1145 genes involved in mimosine degradation. The midD-encoded enzyme, MidD, was expressed in Escherichia coli, purified and used for biochemical and catalytic studies using mimosine as the substrate. The reaction products in the enzyme assay were analyzed by HPLC and mass spectrometry. MidD has a molecular mass of ~45 kDa and its catalytic activity was found to be optimal at 37 °C and pH 8.5. The major product formed in the reaction had the same retention time as that of synthetic 3-hydroxy-4-pyridone (3H4P). It was confirmed to be 3H4P by MS/MS analysis of the HPLC-purified product. The K m, V max and K cat of MidD were 1.27 × 10?4 mol, 4.96 × 10?5 mol s?1 mg?1, and 2,256.05 s?1, respectively. Although MidD has sequence similarities with aminotransferases, it is not an aminotransferase because it does not require a keto acid as the co-substrate in the degradation reaction. It is a pyridoxal-5′-phosphate (PLP)-dependent enzyme and the addition of 50 μM hydroxylamine completely inhibited the reaction. However, the supplementation of the reaction with 0.1 μM PLP restored the catalytic activity of MidD in the reaction containing 50 μM hydroxylamine. The catalytic activity of MidD was found to be specific to mimosine, and the presence of its structural analogs including l-tyrosine, l-tryptophan and l-phenylalanine did not show any competitive inhibition. In addition to 3H4P, we also identified pyruvate and ammonia as other degradation products in equimolar quantities of the substrate used. The degradation of mimosine into a ring compound, 3H4P with the release of ammonia indicates that MidD of Rhizobium sp. strain TAL1145 is a C–N lyase.  相似文献   

7.
Ensifer (Sinorhizobium) meliloti is a nitrogen-fixing α-proteobacterium able to biosynthesize the osmoprotectant glycine betaine from choline sulfate through a metabolic pathway that starts with the enzyme choline-O-sulfatase. This protein seems to be widely distributed in microorganisms and thought to play an important role in their sulfur metabolism. However, only crude extracts with choline sulfatase activity have been studied. In this work, Ensifer (Sinorhizobium) meliloti choline-O-sulfatase was obtained in a high degree of purity after expression in Escherichia coli. Gel filtration and dynamic light scattering experiments showed that the recombinant enzyme exists as a dimer in solution. Using calorimetry, its catalytic activity against its natural substrate, choline-O-sulfate, gave a kcat=2.7×10?1 s?1 and a KM=11.1 mM. For the synthetic substrates p-nitrophenyl sulfate and methylumbelliferyl sulfate, the kcat values were 3.5×10?2 s?1 and 4.3×10?2 s?1, with KM values of 75.8 and 11.8 mM respectively. The low catalytic activity of the recombinant sulfatase was due to the absence of the formylglycine post-translational modification in its active-site cysteine 54. Nevertheless, unmodified Ensifer (Sinorhizobium) meliloti choline-O-sulfatase is a multiple-turnover enzyme with remarkable catalytic efficiency.  相似文献   

8.
The gene coding for d-psicose 3-epimerase (DPEase) from Clostridium sp. BNL1100 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by Ni-affinity chromatography. It was a metal-dependent enzyme and required Co2+ as optimum cofactor. It displayed catalytic activity maximally at pH 8.0 and 65 °C (as measured over 5 min). The optimum substrate was d-psicose, and the K m, turnover number (k cat), and catalytic efficiency (k cat/K m) for d-psicose were 227 mM, 32,185 min?1, and 141 min?1 mM?1, respectively. At pH 8.0 and 55 °C, 120 g d-psicose l?1 was produced from 500 g d-fructose l?1 after 5 h.  相似文献   

9.
An endo-1,4-β-xylanase gene, xylcg, was cloned from Chaetomium globosum and successfully expressed in Escherichia coli. The complete gene of 675 bp was amplified, cloned into the pET 28(a) vector, and expressed. The optimal conditions for the highest activity of the purified recombinant XylCg were observed at a temperature of 40 °C and pH of 5.5. Using oat-spelt xylan, the determined K m, V max, and k cat/K m values were 0.243 mg?ml?1, 4,530 U?mg?1 protein, and 7,640 ml?s?1?mg?1, respectively. A homology model and sequence analysis of XylCg, along with the biochemical properties, confirmed that XylCg belongs to the GH11 family. Rice straw pretreated with XylCg showed 30 % higher conversion yield than the rice straw pretreated with a commercial xylanase. Although xylanases have been characterized from fungal and bacterial sources, C. globosum XylCg is distinguished from other xylanases by its high catalytic efficiency and its effectiveness in the pretreatment of lignocellulosic biomass.  相似文献   

10.
The industrial-scale production of phenylalanine ammonia-lyase (PAL) mainly uses strains of Rhodotorula. However, the PAL gene from Rhodotorula has not been cloned. Here, the full-length gene of PAL from Rhodotorula glutinis was isolated. It was 2,121 bp, encoding a polypeptide with 706 amino acids and a calculated MW of 75.5 kDa. Though R. glutinis is an anamorph of Rhodosporium toruloides, the amino acid sequences of PALs them are not the same (about 74 % identity). PAL was expressed in E. coli and characterized. Its specific activity was 4.2 U mg?1 and the k cat/K m was 1.9 × 104 mM?1 s?1, exhibiting the highest catalytic ability among the reported PALs. The genetic and biochemical information reported here should facilitate future application in industry.  相似文献   

11.
The Sphingopyxis sp. 113P3 gene oph, encoding oxidized polyvinyl alcohol hydrolase (OPH), was optimized with the preferred codons of Pichia pastoris and ligated into the pPIC9K vector behind the α-factor signal sequence. The vector was then transfected into P. pastoris GS115 and genomic integration was confirmed. Large-scale production of recombinant protein was performed by induction with 14.4 g/L methanol at 22 °C in a 3-L bioreactor. The maximal OPH activity obtained was 68.4 U/mL, which is the highest activity reported. The optimal pH and temperature of recombinant OPH were 8.0 and 45 °C, respectively. OPH activity was stable over a pH range of 5.0–8.5, and at a maximal temperature of 45 °C. The K cat /K m of recombinant OPH was 598 mM?1 s?1, which was 4.27-fold higher than that of recombinant OPH derived from Escherichia coli. The improved catalytic efficiency of OPH expressed in recombinant P. pastoris makes it favorable for industrial applications.  相似文献   

12.
A novel esterase gene, estB, was cloned from the marine microorganism Alcanivorax dieselolei B-5(T) and overexpressed in E. coli DE3 (BL21). The expressed protein EstB with a predicted molecular weight of 45.1 kDa had a distinct catalytic triad (Ser211-Trp353-Gln385) and the classical consensus motif conserved in most lipases and esterases Gly209-X-Ser211-X-Gly213. EstB showed very low similarity to any known proteins and displayed the highest similarity to the hypothetical protein (46 %) from Rhodococcus jostii RHA1. EstB showed the optimal activity around pH 8.5 and 20 °C and was identified to be extremely cold-adaptative retaining more than 95 % activity between 0 and 10 °C. The values of kinetic parameters on p-NP caproate (K m, K cat and K cat/K m) were 0.15 mM, 0.54 × 103 s?1 and 3.6 × 103 s?1 mM?1, respectively. In addition, EstB showed remarkable stability in several studied organic solvents and detergents of high concentrations with the retention of more than 70 % activity after treatment for 30 min. The cold activity and its tolerance towards organic solvents made it a promising biocatalyst for industrial applications under extreme conditions.  相似文献   

13.
Escherichia coli has four hydrogenases (Hyd), three genes of which are encoded by the hya, hyb, and hyc operons. The proton-reducing and hydrogen-oxidizing activities of Hyd-2 (hyb) were analyzed in whole cells grown to stationary phase and cell extracts, respectively, during glycerol fermentation using novel double mutants. H2 production rate at pH 7.5 was decreased by ~3.5- and ~7-fold in hya and hyc (HDK 103) or hyb and hyc (HDK 203) operon double mutants, respectively, compared with the wild type. At pH 6.5, H2 production decreased by ~2- and ~5-fold in HDK103 and HDK203, respectively, compared with the wild type. At pH 5.5, H2 production was reduced by ~4.5-fold in the mutants compared with the wild type. The total hydrogen-oxidizing activity was shown to depend on the pH of the growth medium in agreement with previous findings and was significantly reduced in the HDK103 or HDK203 mutants. At pH 7.5, Hyd-2 activity was 0.26 U (mg protein)?1 and Hyd-1 activity was 0.1 U (mg protein)?1. As the pH of the growth medium decreased to 6.5, Hyd-2 activity was 0.16 U (mg protein)?1, and Hyd-1 was absent. Surprisingly, at pH 5.5, there was an increase in Hyd-2 activity (0.33 U mg protein)?1 but not in that of Hyd-1. These findings show a major contribution of Hyd-2 to H2 production during glycerol fermentation that resulted from altered metabolism which surprisingly influenced proton reduction.  相似文献   

14.
A codon-optimized 2-deoxyribose-5-phosphate aldolase (DERA) gene was newly synthesized and expressed in Escherichia coli to investigate its biochemical properties and applications in synthesis of statin intermediates. The expressed DERA was purified and characterized using 2-deoxyribose-5-phosphate as the substrate. The specific activity of recombinant DERA was 1.8 U/mg. The optimum pH and temperature for DERA activity were pH 7.0 and 35 °C, respectively. The recombinant DERA was stable at pH 4.0–7.0 and at temperatures below 50 °C. The enzyme activity was inhibited by 1 mM of Ni2+, Ba2+ and Fe2+. The apparent K m and V max values of purified enzyme for 2-deoxyribose-5-phosphate were 0.038 mM and 2.9 μmol min?1 mg?1, for 2-deoxyribose were 0.033 mM and 2.59 μmol min?1 mg?1, respectively, which revealed that the enzyme had similar catalytic efficiency towards phosphorylated and non-phosphorylated substrates. To synthesize statin intermediates, the bioconversion process for production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose from chloroacetaldehyde and acetaldehyde by the recombinant DERA was developed and a conversion of 94.4 % was achieved. This recombinant DERA could be a potential candidate for application in production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose.  相似文献   

15.
The gene coding for ribose-5-phosphate isomerase (Rpi) from Thermotoga lettingae TMO was cloned and expressed in E. coli. The recombinant enzyme was purified by Ni-affinity chromatography. It converted d-psicose to d-allose maximally at 75 °C and pH 8.0 with a 32 % conversion yield. The k m, turnover number (k cat), and catalytic efficiency (k cat k m ?1 ) for substrate d-psicose were 64 mM, 6.98 min?1 and 0.11 mM?1 min?1 respectively.  相似文献   

16.
The kinetics of chromium(VI) reduction by Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli) was studied under both pure and mixed cultures. Initially, the study of kinetics was performed in pure culture. It was observed that the growth of the two bacteria was both inhibited in the presence of chromium(VI). The maximum specific growth rate (μ m ) of P. aeruginosa decreased from 2.3942 h?1 (without Cr(VI)) to 1.8551 h?1 (with Cr(VI)). Under the mixed culture, the growth of E. coli was inhibited by P. aeruginosa. The maximum specific growth rate (μ m ) of E. coli decreased form 0.871 h?1 (in pure culture) to 0.153 h?1 (in mixed culture). When the concentration of each bacterium was 4.5 × 108 cells ml?1, the half-velocity reduction rate constant (K C) and the maximum specific reduction rate constant (v max) of chromium(VI) were 80.05 mg chromium(VI) l?1 and 3.674 mg chromium(VI) cells?1 h?1, respectively. The results showed that the simulation appeared in good agreement with the experimental data, supporting the series of mathematical models represented the bacteria growth and chromium(VI) reduction in both pure and mixed cultures usefully.  相似文献   

17.
A potential novel fumarate reductase gene designated frd1A was isolated by screening a marine metagenomic library through a sequence-based strategy. Sequence analyses indicated that Frd1A and other putative fumarate reductases were closely related. The putative fumarate reductase gene was subcloned into a pETBlue-2 vector and expressed in Escherichia coli Tuner(DE3)pLac? cells. The recombinant protein was purified to homogeneity. Functional characterization by high-performance liquid chromatography demonstrated that the recombinant Frd1A protein could catalyze the hydrogenation of fumarate to succinate acid. The Frd1A protein displayed an optimal activity at pH 7.0 and 28 °C, which could be stimulated by adding metal ions such as Zn2+ and Mg2+. The Frd1A enzyme showed a comparable affinity and catalytic efficiency under optimal reaction conditions: k m?=0.227 mmol/L, v max= 29.9 U/mg, and k cat/k m=5.44?×?104 per mol/s. The identification of Frd1A protein underscores the potential of marine metagenome screening for novel biomolecules.  相似文献   

18.
Nisin A and polymyxin B were tested alone and in combination in order to test their antagonism against Listeria innocua HPB13 and Escherichia coli RR1, respectively. While the combination of both antibacterial substances was synergistically active against both target bacteria, nisin A alone did not show any inhibition of E. coli RR1. The nisin A/polymyxin B combination at 1.56/2.5 μg ml?1 caused lysis of about 35.86 ± 0.35 and 73.36 ± 0.14% of L. innocua HPB13 cells after 3 and 18 h, respectively. Polymyxin B at 0.12 μg ml?1 and nisin A/polymyxin B at 4.64/0.12 μg ml?1 decreased the numbers of viable E. coli RR1 cells by about 0.23 and 0.65 log10 CFU ml?1, respectively, compared to the control. Our data suggest that the concentration of nisin A required for the effective control of pathogenic strains Listeria spp. could be lowered considerably by combination with polymyxin B. The use of lower concentrations of nisin A or polymyxin B should slow the emergence of bacterial populations resistant to these agents.  相似文献   

19.
The trihemic bacterial cytochrome c peroxidase from Escherichia coli, YhjA, is a membrane-anchored protein with a C-terminal domain homologous to the classical bacterial peroxidases and an additional N-terminal (NT) heme binding domain. Recombinant YhjA is a 50?kDa monomer in solution with three c-type hemes covalently bound. Here is reported the first biochemical and spectroscopic characterization of YhjA and of the NT domain demonstrating that NT heme is His63/Met125 coordinated. The reduction potentials of P (active site), NT and E hemes were established to be ?170?mV, +133?mV and +210?mV, respectively, at pH?7.5. YhjA has quinol peroxidase activity in vitro with optimum activity at pH?7.0 and millimolar range KM values using hydroquinone and menadiol (a menaquinol analogue) as electron donors (KM?=?0.6?±?0.2 and 1.8?±?0.5?mM H2O2, respectively), with similar turnover numbers (kcat?=?19?±?2 and 13?±?2?s?1, respectively). YhjA does not require reductive activation for maximum activity, in opposition to classical bacterial peroxidases, as P heme is always high-spin 6-coordinated with a water-derived molecule as distal axial ligand but shares the need for the presence of calcium ions in the kinetic assays. Formation of a ferryl Fe(IV)?=?O species was observed upon incubation of fully oxidized YhjA with H2O2. The data reported improve our understanding of the biochemical properties and catalytic mechanism of YhjA, a three-heme peroxidase that uses the quinol pool to defend the cells against hydrogen peroxide during transient exposure to oxygenated environments.  相似文献   

20.
Isomaltulose is a structural isomer of sucrose commercially used in food industries. In this work, recombinant Escherichia coli producing sucrose isomerase (SIase) was used to convert sucrose into isomaltulose. To develop an economical industrial medium, untreated cane molasses (10.63 g l?1), yeast extract (25.93 g l?1), and corn steep liquor (10.45 g l?1) were used as main culture compositions for SIase production. The relatively high SIase activity (14.50 ± 0.11 U mg DCW?1) was obtained by the recombinant cells. To the best of our knowledge, this is the first investigation on SIase production by engineered E. coli using untreated cane molasses. The recombinant E. coli cells expressing the SIase gene were immobilized in calcium alginate gel in order to improve the efficiency of recycling. The immobilization was most effective with 2 % (w/v) sodium alginate and 3 % (w/v) calcium chloride. The optimal initial biomass for immobilization was 20 % (w/v, wet wt.), with a hardening time of 8 h for cell immobilization. The immobilized E. coli cells exhibited good stability for 30 batches with the productivity of 0.45 g isomaltulose g pellet?1 h?1. A continuous isomaltulose formation process using a column reactor remained stable for 40 days with 83 ± 2 % isomaltulose yield, which would be beneficial for economical production of isomaltulose.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号