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1.
Organophosphate hydrolase containing hexahistidine tag at the N-terminus of recombinant protein (His6-OPH) and expressed in Escherichia coli cells was purified using supermacroporous polyacrylamide-based monolith columns with immobilized metal affinity matrices [Me2+-iminodiacetic acid (IDA)–polyacrylamide cryogel (PAA) and Me2+-N,N,N’-tris (carboxymethyl) ethylendiamine (TED)–PAA]. Enzyme preparation with 50% purity was obtained by direct chromatography of nonclarified cell homogenate, whereas the combination of addition of 10 mM imidazole to buffers for cell sonication and sample loading, the use of precolumn with IDA–PAA matrix noncharged with metal ions, and the application of high flow rate provided the 99% purity of enzyme isolated directly from crude cell homogenate. Co2+-IDA–PAA provided the highest level of selectivity for His6-OPH. Comparative analysis of purification using Co2+-IDA–PAA and Ni-nitrilotriacetic acid–agarose showed obvious advantages of the former in process time, specific activity of purified enzyme, and simplicity of adsorbent regeneration.  相似文献   

2.
To express Escherichia coli novablue dipeptidyl carboxypeptidase (EcDCP), the gene was amplified by PCR and cloned into the expression plasmid pQE-31 to yield pQE-EcDCP. His6-tagged EcDCP (His6-EcDCP) was over-expressed in E. coli M15 (pQE-EcDCP) as a soluble and active form under 0.05 mM IPTG induction at 26°C for 12 h. The recombinant enzyme was purified to homogeneity by Ni2+-NTA resin and had a molecular mass of approximately 75 kDa. The temperature and pH optima for His6-EcDCP were 37°C and 7.0, respectively. In the presence of 200 mM NaCl, His6-EcDCP was stimulated by 1.5 fold. The K M and k cat values of the enzyme for N-benzoyl-l-glycyl-l-histidyl-l-leucine were 1.83 mM and 168.3 s−1, respectively. His6-EcDCP activity was dramatically inhibited by 10 mM EDTA, 0.25 mM 1.10-phenanthroline, and 2.5 mM DEPC, but it was not affected by Ser, Asp, Lys, and Trp protease inhibitors. Analysis of His6-EcDCP by circular dichroism revealed that the secondary structures of the enzyme in 30 mM universal buffer (pH 7.0) were 17% α-helix, 35% β-sheet and 47% random coil. Mid point of thermal transition was calculated to be 55°C for the recombinant enzyme.  相似文献   

3.
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   

4.
Phenol hydroxylase that catalyzes the conversion of phenol to catechol in Rhodococcus erythropolis UPV-1 was identified as a two-component flavin-dependent monooxygenase. The two proteins are encoded by the genes pheA1 and pheA2, located very closely in the genome. The sequenced pheA1 gene was composed of 1,629 bp encoding a protein of 542 amino acids, whereas the pheA2 gene consisted of 570 bp encoding a protein of 189 amino acids. The deduced amino acid sequences of both genes showed high homology with several two-component aromatic hydroxylases. The genes were cloned separately in cells of Escherichia coli M15 as hexahistidine-tagged proteins, and the recombinant proteins His6PheA1 and His6PheA2 were purified and its catalytic activity characterized. His6PheA1 exists as a homotetramer of four identical subunits of 62 kDa that has no phenol hydroxylase activity on its own. His6PheA2 is a homodimeric flavin reductase, consisting of two identical subunits of 22 kDa, that uses NAD(P)H in order to reduce flavin adenine dinucleotide (FAD), according to a random sequential kinetic mechanism. The reductase activity was strongly inhibited by thiol-blocking reagents. The hydroxylation of phenol in vitro requires the presence of both His6PheA1 and His6PheA2 components, in addition to NADH and FAD, but the physical interaction between the proteins is not necessary for the reaction.  相似文献   

5.
The lipA gene, a structural gene encoding for protein of molecular mass 48 kDa, and lipB gene, encoding for a lipase-specific chaperone with molecular mass of 35 kDa, of Pseudomonas aeruginosa B2264 were co-expressed in heterologous host Escherichia coli BL21 (DE3) to obtain in vivo expression of functional lipase. The recombinant lipase was expressed with histidine tag at its N terminus and was purified to homogeneity using nickel affinity chromatography. The amino acid sequence of LipA and LipB of P. aeruginosa B2264 was 99–100% identical with the corresponding sequence of LipA and LipB of P. aeruginosa LST-03 and P. aeruginosa PA01, but it has less identity with Pseudomonas cepacia (Burkholderia cepacia) as it showed only 37.6% and 23.3% identity with the B. cepacia LipA and LipB sequence, respectively. The molecular mass of the recombinant lipase was found to be 48 kDa. The recombinant lipase exhibited optimal activity at pH 8.0 and 37°C, though it was active between pH 5.0 and pH 9.0 and up to 45°C. K m and V max values for recombinant P. aeruginosa lipase were found to be 151.5 ± 29 μM and 217 ± 22.5 μmol min−1 mg−1 protein, respectively.  相似文献   

6.
Adenosine 5′-phosphosulfate (APS) reductase is a key enzyme involved in the pathways of sulfate reduction and sulfide oxidation in the biological sulfur cycle. In this study, the gene of APS reductase from Acidithiobacillus ferrooxidans was cloned and expressed in Escherichia coli, the soluble protein was purified by one-step affinity chromatography to apparent homogeneity. The molecular mass of the recombinant APS reductase was determined to be 28 kDa using SDS-PAGE. According to optical and EPR spectra results of the recombinant protein confirmed that the iron–sulfur cluster inserted into the active site of the protein. Site-directed mutation for the enzyme revealed that Cys110, Cys111, Cys193, and Cys196 were in ligation with the iron–sulfur cluster. The [Fe4S4] cluster could be assembled in vitro, and exhibited electron transport and redox catalysis properties. As we know so far, this is the first report of expression in E. coli of APS reductase from A. ferrooxidans.  相似文献   

7.
Genomic DNA was extracted from wild-type Escherichia coli strains ATCC 35321 and 8677. The lac Z gene was amplified and used as a template for in vitro synthesis of β-galactosidase. In addition the enzyme was synthesized in vitro with a C-terminal His6 tag. The enzyme expression was also induced in these strains using isopropyl-β-D-galactoside. Single enzyme molecule assays were performed using a capillary electrophoresis-based protocol on both the in vitro and in vivo synthesized enzyme. In vivo produced enzyme from strains 35321 and 8677 showed average combined turnover numbers for the 4 active sites of the individual enzyme molecules of 53,400 ± 18,400 (N = 139) and 34,300 ± 17,800 min−1 (N = 181) respectively. Average combined turnover numbers of 35,800 ± 20,900 (N = 302) and 31,700 ± 17,700 min−1 (N = 315) were obtained respectively for the in vitro synthesized enzyme from strain 35321 with the absence and presence of a C-terminal His6 tag. For strain 8677, the average combined turnover numbers were 29,000 ± 17,900 (N = 288) and 25,200 ± 12,600 min−1 (N = 240) respectively for the absence and presence of a C-terminal His6 tag. The average combined turnover numbers of the enzyme from both strains synthesized in vivo and in vitro and with the presence and absence of a His6 tag were found to differ significantly. This indicates that the in vivo and in vitro produced enzymes are not identical and the presence of a C-terminal His6 tag alters the activity of β-galactosidase.  相似文献   

8.
The OCTN2 cDNA amplified from human skin fibroblast was cloned in pET-41a(+) carrying the glutathione S-transferase (GST) gene. The construct pET-41a(+)–hOCTN2 was used to express the GST–hOCTN2 fusion protein in Escherichia coli Rosetta(DE3)pLysS. The best over-expression was obtained after 6 h of induction with IPTG at 28°C. The GST–hOCTN2 polypeptide was collected in the inclusion bodies and showed an apparent molecular mass on SDS-PAGE of 85 kDa. After solubilization with a buffer containing 0.8% sarkosyl and 3 M urea, the fusion protein was applied onto a Ni2+-chelating chromatography column. The purified GST–hOCTN2 was treated with thrombin, and the hOCTN2 was separated from the GST by size exclusion chromatography. After the whole procedure, a yield of about 0.2 mg purified protein per liter of cell culture was obtained. To improve the protein yield, hOCTN2 cDNA was subjected to codon bias. The second codon CGG was substituted with AAA; the substitution led to the mutation R2K in the hOCTN2 protein. hOCTN2(R2K) cDNA was cloned in pET-21a(+) carrying a C-terminal 6His tag. The resulting protein was expressed in E. coli Rosetta(DE3)pLysS and purified by Ni2+-chelating chromatography. A yield of about 3.5 mg purified protein per liter of cell culture was obtained with this procedure.  相似文献   

9.
Wang X  Fang B  Luo J  Li W  Zhang L 《Biotechnology letters》2007,29(9):1409-1412
The xylose reductase (XR) gene (xyl1) from Candida shehatae was cloned and expressed in Escherichia coli, and purified as a His6-tagged fusion protein. The recombinant XR had Km values for NADH than NADPH of 150 μM and 20 μM, respectively. The optimal reaction was at pH 6.5 and 35°C. The enzyme was specific for d-xylese.  相似文献   

10.
Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher’s disease, the most common inherited storage disorder. Treatment consists of enzyme replacement therapy by the administration of recombinant GCR produced in Chinese hamster ovary cells. The production of anti-GCR antibodies has already been described with placenta-derived human GCR that requires successive chromatographic procedures. Here, we report a practical and efficient method to obtain anti-GCR polyclonal antibodies against recombinant GCR produced in Escherichia coli and further purified by a single step through nickel affinity chromatography. The purified GCR was used to immunize BALB/c mice and the induction of anti-GCR antibodies was evaluated by enzyme-linked immunosorbent assay. The specificity of the antiserum was also evaluated by western blot analysis against recombinant GCR produced by COS-7 cells or against endogenous GCR of human cell lines. GCR was strongly recognized by the produced antibodies, either as cell-associated or as secreted forms. The detected molecular masses of 59–66 kDa are in accordance to the expected size for glycosylated GCR. The GCR produced in E. coli would facilitate the production of polyclonal (shown here) and monoclonal antibodies and their use in the characterization of new biosimilar recombinant GCRs coming in the near future.  相似文献   

11.
A psychrophilic bacterium Psychrobacter sp. C18 previously isolated from the Southern Okinawa Trough deep-sea sediments showed extracellular lipolytic activity towards tributyrin. A genomic DNA library was constructed and screened to obtain the corresponding lipase gene. The sequenced DNA fragment contains an open reading frame of 945 bp, which was denoted as the lipX gene, from which a protein sequence LipX was deduced of 315 amino acid residues with a molecular mass of 35,028 Da. This protein contained the bacterial lipase GNSMG (GxSxG, x represents any amino acid residue) and HG consensus motifs. The recombinant pET28a(+)/lipX gene was overexpressed in heterologous host Escherichia coli BL21 (DE3) cells to overproduce the lipase protein LipXHis with a 6× histidine tag at its C-terminus. Nickel affinity chromatography was used for purification of the expressed recombinant lipase. The maximum lipolytic activity of the purified recombinant lipase was obtained at temperature of 30°C and pH 8.0 with p-nitrophenyl myristate (C14) as a substrate. Thermostability assay indicated that the recombinant LipXHis is a cold-adapted lipase, which was active in 10% methanol, ethanol, acetone and 30% glycol, and inhibited partially by Zn2+, Co2+, Mn2+, Fe3+ and EDTA. Most non-ionic detergents, such as DMSO, Triton X-100, Tween 60 and Tween 80 enhanced the lipase activity but 1% SDS completely inhibited the enzyme activity. Additionally, the highest lipolytic rate of the recombinant LipXHis lipase was achieved when p-nitrophenyl myristate was used as a substrate, among all the p-nitrophenyl esters tested.  相似文献   

12.
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   

13.
A protocol for the production of transgenic plants was developed for Lotus tenuis via Agrobacterium-mediated transformation of leaf segments. The explants were co-cultivated (for 3 days) with an A. tumefaciens strain harbouring either the binary vector pBi RD29A:oat arginine decarboxylase (ADC) or pBi RD29A:glucuronidase (GUS), which carries the neomycin phosphotransferase II (nptII) gene in the T-DNA region. Following co-cultivation, the explants were cultured in Murashige and Skoog medium supplemented with naphthalenacetic acid (NAA) and benzyladenine (BA) and containing kanamycin (30 μg ml−1) and cefotaxime (400 μg ml−1) for 45 days. The explants were subcultured several times (at 2-week intervals) to maintain the selection pressure during the entire period. About 40% of the explants inoculated with the pBiRD29:ADC strain produced eight to ten adventitious shoots per responsive explant through a direct system of regeneration, whereas 69% of the explants inoculated with the pBi RD29A:GUS strain produced 13–15 adventitious shoots per responsive explant. The selected transgenic lines were identified by PCR and Southern blot analysis. Three ADC transgenic lines were obtained from 30 infected explants, whereas 29 GUS transgenic lines were obtained from 160 explants, corresponding to a transformation efficiency of 10 and 18.1%, respectively. More than 90% of the in vitro plantlets were successfully transferred to the soil. The increase in the activity of arginine decarboxylase from stressed ADC- Lt19 lines was accompanied by a significant rise in the putrescine level. The GUS transgenic line driven by the RD29A promoter showed strong signals of osmotic stress in the leaves and stem tissues. All of the transgenic plants obtained exhibited the same phenotype as the untransformed controls under non-stress conditions, and the stability of the gene introduced into the cloned materials was established.  相似文献   

14.
d-Xylulose-forming d-arabitol dehydrogenase (aArDH) is a key enzyme in the bio-conversion of d-arabitol to xylitol. In this study, we cloned the NAD-dependent d-xylulose-forming d-arabitol dehydrogenase gene from an acetic acid bacterium, Acetobacter suboxydans sp. The enzyme was purified from A. suboxydans sp. and was heterogeneously expressed in Escherichia coli. The native or recombinant enzyme was preferred NAD(H) to NADP(H) as coenzyme. The active recombinant aArDH expressed in E. coli is a homodimer, whereas the native aArDH in A. suboxydans is a homotetramer. On SDS–PAGE, the recombinant and native aArDH give one protein band at the position corresponding to 28 kDa. The optimum pH of polyol oxidation and ketone reduction is found to be pH 8.5 and 5.5 respectively. The highest reaction rate is observed when d-arabitol is used as the substrate (K m = 4.5 mM) and the product is determined to be d-xylulose by HPLC analysis.  相似文献   

15.
Attacin, a 20 kDa antibacterial peptide, plays an important role in immunity. To understand this gene better, gene cloning, expression and biological activity detection of Attacin A was carried out in present study. The full-length open reading frame (ORF) coding for Attacin A gene was generated using RT-PCR which takes total RNA extracted from Drosophila as the template. The gene was inserted directionally into the prokaryotic expression vector pET-32a (+). The resulting recombinant plasmid was transformed into E. coli Rosetta. SDS–PAGE was carried out to detect the expression product which was induced by IPTG. The antimicrobial activity and hemolysis activity were tested in vitro after purification. Agarose gel electrophoresis indicated that the complete ORF of Attacin A gene has been cloned successfully from Drosophila stimulated by E. coli which includes 666 bp and encodes 221 AA. The gene encoding mature Attacin A protein was amplified by PCR from the recombinant plasmid containing Attacin A, which includes 570 bp in all. SDS–PAGE analysis demonstrated that the fusion protein expressed was approximately 39.2 kDa. Biological activities detection showed that this peptide exhibited certain antibacterial activity to several G− bacteria, as well as minor hemolysis activity for porcine red blood cells. In conclusion, Attacin A gene was cloned and expressed successfully. It was the basis for further study of Attacin.  相似文献   

16.
A polyhydroxyalkanote depolymerase gene from Thermobifida sp. isolate BCC23166 was cloned and expressed as a C-terminal His6-tagged fusion in Pichia pastoris. Primary structure analysis revealed that the enzyme PhaZ-Th is a member of a proposed new subgroup of SCL-PHA depolymerase containing a proline–serine repeat linker. PhaZ-Th was expressed as two glycosylated forms with apparent molecular weights of 61 and 70 kDa, respectively. The enzyme showed esterase activity toward p-nitrophenyl alkanotes with V max and K m of 3.63 ± 0.16 μmol min−1 mg−1 and 0.79 ± 0.12 mM, respectively, on p-nitrophenyl butyrate with optimal activity at 50–55°C and pH 7–8. Surface plasmon resonance (SPR) analysis demonstrated that PhaZ-Th catalyzed the degradation of poly-[(R)-3-hydroxybutyrate] (PHB) films, which was accelerated in (R)-3-hydroxyvalerate copolymers with a maximum degradation rate of 882 ng cm−2 h−1 for poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] (12 mol% V). Surface deterioration, especially on the amorphous regions of PHB films was observed after exposure to PhaZ-Th by atomic force microscopy. The use of P. pastoris as an alternative recombinant system for bioplastic degrading enzymes in secreted form and a sensitive SPR analytical technique will be of utility for further study of bioplastic degradation.  相似文献   

17.
Aims: To investigate whether Vibrio vulnificus metalloprotease (VvpE) can induce the production of specific anti‐VvpE antibody to confer effective protection against Vibrio vulnificus infection and to evaluate the possibility of VvpE as a potential vaccine candidate against disease caused by V. vulnificus. Methods and Results: The gene encoding the 65‐kDa VvpE of V. vulnificus was amplified by PCR and cloned into the expression vector pET21(b). The recombinant VvpE of V. vulnificus was expressed in Escherichia coli BL21(DE3). This His6‐tagged VvpE was purified and injected intramuscularly into mice to evaluate its ability to stimulate immune response. Specific antibody levels were measured by ELISA. The 75% protective efficacy of recombinant VvpE was evaluated by active immunization and intraperitoneal challenge with V. vulnificus in mice. Conclusions: The recombinant His6‐tagged VvpE of V. vulnificus is capable of inducing high antibody response in mice to confer effective protection against lethal challenge with V. vulnificus. VvpE might be a potential vaccine candidate to against V. vulnificus infection. Significance and Impact of the Study: This study uses His6‐tagged VvpE to act as vaccine that successfully induces effective and specific anti‐VvpE antibody and offers an option for the potential vaccine candidate against V. vulnificus infection.  相似文献   

18.
Alkaline phosphatase catalyzes the hydrolysis of phosphomonoesters and is widely used in molecular biology techniques and clinical diagnostics. We expressed a recombinant alkaline phosphatase of the marine bacterium, Cobetia marina, in Escherichia coli BL21 (DE3). The recombinant protein was purified with a specific activity of 12,700 U/mg protein, which is the highest activity reported of any bacterial alkaline phosphatase studied to date. The molecular mass of the recombinant protein was 55–60 kDa, as determined by SDS–PAGE, and was observed to be a dimer by gel filtration analysis. The enzyme was optimally active at 45°C and the recombinant alkaline phosphatase efficiently hydrolyzed a phosphoric acid ester in luminescent and fluorescent substrates. Therefore, this enzyme can be considered to be extremely useful as a label conjugated to an antibody.  相似文献   

19.
We found the occurrence of 4-hydroxybenzoate decarboxylase in Enterobacter cloacae P240, isolated from soils under anaerobic conditions, and purified the enzyme to homogeneity. The purified enzyme was a homohexamer of identical 60 kDa subunits. The purified decarboxylase catalyzed the nonoxidative decarboxylation of 4-hydroxybenzoate without requiring any cofactors. Its K m value for 4-hydroxybenzoate was 596 μM. The enzyme also catalyzed decarboxylation of 3,4-dihydroxybenzoate, for which the K m value was 6.80 mM. In the presence of 3 M KHCO3 and 20 mM phenol, the decarboxylase catalyzed the reverse carboxylation reaction of phenol to form 4-hydroxybenzoate with a molar conversion yield of 19%. The K m value for phenol was calculated to be 14.8 mM. The gene encoding the 4-hydroxybenzoate decarboxylase was isolated from E. cloacae P240. Nucleotide sequencing of recombinant plasmids revealed that the 4-hydroxybenzoate decarboxylase gene codes for a 475-amino-acid protein. The amino acid sequence of the enzyme is similar to those of 4-hydroxybenzoate decarboxylase of Clostridium hydroxybenzoicum (53% identity), VdcC protein (vanillate decarboxylase) of Streptomyces sp. strain D7 (72%) and 3-octaprenyl-4-hydroxybenzoate decarboxylase of Escherichia coli (28%). The hypothetical proteins, showing 96–97% identities to the primary structure of E. cloacae P240 4-hydroxybenzoate decarboxylase, were found in several bacterial strains.  相似文献   

20.
An oleandomycin glycosyltransferase (OleD GT) gene from Streptomyces antibioticus was functionally expressed in Escherichia coli BL21 (DE3) with various molecular chaperones. The purified recombinant OleD GT catalyzed glycosylation of various flavonoids: apigenin, chrysin, daidzein, genistein, kaempferol, luteolin, 4-methylumbelliferone, naringenin, quercetin and resveratrol with UDP–glucose. 4.6 μg OleD GT was readily immobilized onto 1 mg hybrid nanoparticles of Fe3O4/silica/NiO on the basis of the affinity between His-tag and NiO nanoparticles with retention of 90% activity. In batch reaction, more than 90% naringenin (20 μM) was converted to its glycoside in 5 h. The immobilized OleD GT was efficiently reused for seven times whilst maintaining >60% of the residual activity in repeated glycosylation of naringenin.  相似文献   

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