The gene encoding fibrinolytic enzyme from Bacillus sp. zlw-2 was cloned and sequenced (accession no. EU734749), which was 1146 bp, encoded 381 amino acids and had 99% homology with Nattokinase YF308 and NAT. The ge...The gene encoding fibrinolytic enzyme from Bacillus sp. zlw-2 was cloned and sequenced (accession no. EU734749), which was 1146 bp, encoded 381 amino acids and had 99% homology with Nattokinase YF308 and NAT. The genes encoding pre-pro-fibrinolytic enzyme (including signal peptide, propeptide, and mature peptide) and fibrinolytic enzyme (including mature peptide) were cloned into pET28a vector respectively and then transformed into Escherichia coli BL21 (DE3). The recombinant ofpre-pro-fibrinolytic enzyme showed enzyme activity of 183 U mL^-1, while no detectable enzyme activity could be found from the recombinant of the mature peptide.展开更多
A strain WL-11 with high laccase activity was isolated from activated sludge collected from the effluent treatment plant of a textile and dyeing industry. It was identified as Aeromonas hydrophila by physiological tes...A strain WL-11 with high laccase activity was isolated from activated sludge collected from the effluent treatment plant of a textile and dyeing industry. It was identified as Aeromonas hydrophila by physiological test and 16S rDNA sequence analysis. A gene encoding of laccase from a newly isolated Aeromonas hydrophila WL-11 was cloned and characterized. Nucleotide sequence analysis showed an open reading frame of 1605 bp encoding a polypeptide comprised of 534 amino acids. The primary structure of the enzyme predicted the structural features characteristic of other laccases, including the conserved regions of four histidine-rich copper-binding sites. The predicted amino acid sequence showed a high homology (more than 60%) with bacterial laccases in the genome and protein databases and the highest degree of similarity (61% identity) was observed with the multicopper oxidase of KlebsieUa sp. 601. When expressed in Escherichia coli, the recombinant enzyme was overproduced in the cytoplasm as soluble and active form. The purified enzyme had an optimum pH of 2.6 and 8.0 for ABTS (2,2'-azino-bis(3-ethylbenzthiazolinesulfonic acid) and DMP (2,6-dimethoxyphenol), respectively. The kinetic study on ABTS revealed a higher affinity of this enzyme to this substrate than DMP.展开更多
Spatial and temporal expression patterns of Sbel and Sbe2 that encode starch branching enzyme (SBE) Ⅰ and Ⅱ, respectively, in sweet potato (Ipomoea batatas L.) were analyzed. Expression of both genes in Escheric...Spatial and temporal expression patterns of Sbel and Sbe2 that encode starch branching enzyme (SBE) Ⅰ and Ⅱ, respectively, in sweet potato (Ipomoea batatas L.) were analyzed. Expression of both genes in Escherichia coli indicate that both genes encoded active SBE. Analysis with real-time quantitative polymerase chain reaction technique indicates that IbSbel mRNA was expressed at very low levels in leaves but was the predominant isoform in tuberous root while the reverse case was found for lbSbe2. The expression pattern of IbSbel, closely resembles that of AGPase S, a gene coding for one of the subunits ofADP-glucose pyrophosphorylase, which is the key regulatory enzyme in the starch biosynthetic pathway. Western analysis detected at least two isoforms of SBE I in tuberous roots, those two isoforms showed adverse expression patterns with the development of the tuberous roots. Expression of the two IbSbe genes exhibited a diurnal rhythm during a 12-h cycle when fed a continuous solution of sucrose. Abscisic acid (ABA) was aother potent inducer of IbSbe expression, but bypassed the semidian oscillator.展开更多
Extracellular xylanase XYNB from Streptomy-ces olivaceoviridis A1 has been purified and characterized. The optimal pH value and temperature of XYNB for its ac-tivity are 5.2 and 60℃, respectively. The specific activi...Extracellular xylanase XYNB from Streptomy-ces olivaceoviridis A1 has been purified and characterized. The optimal pH value and temperature of XYNB for its ac-tivity are 5.2 and 60℃, respectively. The specific activity of XYNB is as high as 2869.78 U/mg. Metal cations, EDTA and SDS have no effects on enzyme activity of XYNB. The gene xynB coding mature protein of XYNB has been cloned by PCR. The forward oligonucleotide primer used in the PCR reaction was synthesized based on the N-terminal amino acid sequence of XYNB mature protein, and the reverse oligonu-cleotide primers are random oligonucleotide. The cloned gene xynB is 576 bp long and its G+C content is 64.3%. The xynB encodes 191 amino acid residues, and the putative mo-lecular weight of XYNB is 20.839 kD. The xynB has been expressed in E. coli, and the expressed xylanase has normal bioactivity.展开更多
[ Objective ] This study aimed to obtain the recombinant Pichia yeast strain which can efficiently degrade guar gum. The properties of the recombinant enzyme were studied preliminarily. [ Method ] A positive clone tha...[ Objective ] This study aimed to obtain the recombinant Pichia yeast strain which can efficiently degrade guar gum. The properties of the recombinant enzyme were studied preliminarily. [ Method ] A positive clone that could hydrolyze guar gum was obtained through the construction and functional screening of a soil genome library. Sequence analysis indicated that the 1485-bp clone encodes a 494-amino acid protein with a relative molecular mass of 53 949 kD, containing a cellulose-binding domain. The recombinant plasmid pHBM731 was generated by inserting the optimized target gene into a Pichia pastoris expression vector pHBMg05 that was transformed into three Pichia pastoris strains, GS115, KM71 and SMD1168. The biochemical properties of the enzyme were assessed. [ Result] The cloned galactonumnan (GM)-degrading enzyme was expressed and secreted by Pichia pastoris GSll5. High cell density fermentation was induced in recombi- nant Pichia pastoris at 25 and 28 ~C ; a higher enzyme activity was observed at an induction temperature of 28 ~C. The optimal temperature for the recombinant en- zyme is 60 ~C, and the optimal pH is 6.6. The enzyme activity was 38.61 U under optimal conditions. Over 50% of the enzyme activity was maintained under the optimal conditions after 9 h. Under the optimal conditions, the effect of metal ions on enzyme activity was analyzed. Ca2 + , Fe2 + and Li ~ slightly enhanced enzyme activity, while Mn2+ and Co2+ had little effect. Enzyme activity was modestly suppressed by Mg2~ , K~ and Na+ , but considerably suppressed by Ag2~ and Zn2~ , with Cu2 + showing the strongest inhibitory effects. [ Conclusion] A novel GM-degrading enzyme expressed by soil yeast was cloned, which can potentially be used in industrial applications to obtain eommereially useful guar gum-degradation products.展开更多
Objective:To clone feruloyl esterase gene C from Penicillium chrysogenum and characterize the general properties of the enzyme.Methods:The feruloyl esterase C gene was amplified by PCR based on the Penicillium chrysog...Objective:To clone feruloyl esterase gene C from Penicillium chrysogenum and characterize the general properties of the enzyme.Methods:The feruloyl esterase C gene was amplified by PCR based on the Penicillium chrysogenum feruloyl esterase C gene sequence and cloned into the expression vector p PIC9K,resulting the recombinant plasmid p PIC9K-Pcfae C.The recombiant plasmid was linerized and transformed into P.pastoris by electroporation.The transformants was screened based on the transparent zone technology.The screened transformants was then induced by methanol.the enzymatic properties of the protein were then measured.Results:SDS-PAGE analysis showed that the molecular mass of the enzyme was about 30 k D.The length of the gene was 762 bp.It comprised one open reading framwork(ORF)and annotated to encode 249 amino acid.The optimal temperature and p H was found to be 40℃and 6,respectively.Moreover,the recombinant enzyme was stable at 40-50℃and p H 5-7.Conclusion:The enzyme successfully expressed in P.pastoris could laid theoretical foundation in food,fodder and paper making industry.展开更多
The violaxanthin de-epoxidase gene was cloned from rice (Oryza sativa subsp. japonica). The full length of the cDNA is 1887 bp, encoding a 446-amino acids protein with the transit peptide of 98 amino acids. The bacter...The violaxanthin de-epoxidase gene was cloned from rice (Oryza sativa subsp. japonica). The full length of the cDNA is 1887 bp, encoding a 446-amino acids protein with the transit peptide of 98 amino acids. The bacterial expression vector pET-Rvde was constructed and the expression quantity of the exogenous protein increased with the induction time by 0.4 mmol/L IPTG. Its molecular weight was similar with that of the native VDE. Western blotting indicated that the expressed protein has immu-nological reaction with the VDE polyclonal antibody. The absorbance spectrum together with xanthophyll pigments quantification by HPLC demonstrated that the expressed VDE has its enzyme activity, which can de-epoxidate violaxanthin into antheraxanthin and zeaxanthin in vitro.展开更多
Starch branching enzyme (SBE) catalyzes the biosynthesis of amylopectin. We described the isolation and characterization of SBEIIb promoter and their expression patterns in transgenic tobacco. Using the genomic DNA ...Starch branching enzyme (SBE) catalyzes the biosynthesis of amylopectin. We described the isolation and characterization of SBEIIb promoter and their expression patterns in transgenic tobacco. Using the genomic DNA of maize cultivar Lunuo 1 as template, the SBEIIb promoter was isolated by PCR and was cloned into pMD18-T vector. To study SEBIIb gene regulation at the cellular level, SBEIIb promoter was fused to the ^-glucuronidase (GUS) report gene. The results of the fluorometric GUS assays indicate that the sbeⅡb-GUS fusion directed a seed-specific expression. Four series of constructs were made with the promoter and the GUS reporter gene to investigate the cis-acting analysis, showing that the four different constructs all can drive expression of the GUS gene in seed plumule and cotyledon and the GUS activity was apparently decreased with the progressive loss of promoter 5' end.展开更多
基金funded by the Science and Technology Research Project during the 11th Five-Year Plan period of Hebei Province, China (06220106D)
文摘The gene encoding fibrinolytic enzyme from Bacillus sp. zlw-2 was cloned and sequenced (accession no. EU734749), which was 1146 bp, encoded 381 amino acids and had 99% homology with Nattokinase YF308 and NAT. The genes encoding pre-pro-fibrinolytic enzyme (including signal peptide, propeptide, and mature peptide) and fibrinolytic enzyme (including mature peptide) were cloned into pET28a vector respectively and then transformed into Escherichia coli BL21 (DE3). The recombinant ofpre-pro-fibrinolytic enzyme showed enzyme activity of 183 U mL^-1, while no detectable enzyme activity could be found from the recombinant of the mature peptide.
基金supported by the Korea Research Foundation Grant funded by the Korean Government(MOEHRD,Basic Research Promotion Fund) (No.KRF-2007-313-D00402)
文摘A strain WL-11 with high laccase activity was isolated from activated sludge collected from the effluent treatment plant of a textile and dyeing industry. It was identified as Aeromonas hydrophila by physiological test and 16S rDNA sequence analysis. A gene encoding of laccase from a newly isolated Aeromonas hydrophila WL-11 was cloned and characterized. Nucleotide sequence analysis showed an open reading frame of 1605 bp encoding a polypeptide comprised of 534 amino acids. The primary structure of the enzyme predicted the structural features characteristic of other laccases, including the conserved regions of four histidine-rich copper-binding sites. The predicted amino acid sequence showed a high homology (more than 60%) with bacterial laccases in the genome and protein databases and the highest degree of similarity (61% identity) was observed with the multicopper oxidase of KlebsieUa sp. 601. When expressed in Escherichia coli, the recombinant enzyme was overproduced in the cytoplasm as soluble and active form. The purified enzyme had an optimum pH of 2.6 and 8.0 for ABTS (2,2'-azino-bis(3-ethylbenzthiazolinesulfonic acid) and DMP (2,6-dimethoxyphenol), respectively. The kinetic study on ABTS revealed a higher affinity of this enzyme to this substrate than DMP.
基金supported by funds from the National Science & Technology Pillar Program of China(2007BAD78B03)the 11th Five-Year Plan Key Project of Sichuan Province, China (07SG111-003-1)
文摘Spatial and temporal expression patterns of Sbel and Sbe2 that encode starch branching enzyme (SBE) Ⅰ and Ⅱ, respectively, in sweet potato (Ipomoea batatas L.) were analyzed. Expression of both genes in Escherichia coli indicate that both genes encoded active SBE. Analysis with real-time quantitative polymerase chain reaction technique indicates that IbSbel mRNA was expressed at very low levels in leaves but was the predominant isoform in tuberous root while the reverse case was found for lbSbe2. The expression pattern of IbSbel, closely resembles that of AGPase S, a gene coding for one of the subunits ofADP-glucose pyrophosphorylase, which is the key regulatory enzyme in the starch biosynthetic pathway. Western analysis detected at least two isoforms of SBE I in tuberous roots, those two isoforms showed adverse expression patterns with the development of the tuberous roots. Expression of the two IbSbe genes exhibited a diurnal rhythm during a 12-h cycle when fed a continuous solution of sucrose. Abscisic acid (ABA) was aother potent inducer of IbSbe expression, but bypassed the semidian oscillator.
基金This work was supported by the State"863"High-Tech Programs(Grant No.2001AA214041).
文摘Extracellular xylanase XYNB from Streptomy-ces olivaceoviridis A1 has been purified and characterized. The optimal pH value and temperature of XYNB for its ac-tivity are 5.2 and 60℃, respectively. The specific activity of XYNB is as high as 2869.78 U/mg. Metal cations, EDTA and SDS have no effects on enzyme activity of XYNB. The gene xynB coding mature protein of XYNB has been cloned by PCR. The forward oligonucleotide primer used in the PCR reaction was synthesized based on the N-terminal amino acid sequence of XYNB mature protein, and the reverse oligonu-cleotide primers are random oligonucleotide. The cloned gene xynB is 576 bp long and its G+C content is 64.3%. The xynB encodes 191 amino acid residues, and the putative mo-lecular weight of XYNB is 20.839 kD. The xynB has been expressed in E. coli, and the expressed xylanase has normal bioactivity.
基金Supported by Yantai Municipal Science and Technology Development Plan(2013ZH097)Scientific and Technological Innovation Fund for Students in Binzhou Medical University(BY2013DKCX122)
文摘[ Objective ] This study aimed to obtain the recombinant Pichia yeast strain which can efficiently degrade guar gum. The properties of the recombinant enzyme were studied preliminarily. [ Method ] A positive clone that could hydrolyze guar gum was obtained through the construction and functional screening of a soil genome library. Sequence analysis indicated that the 1485-bp clone encodes a 494-amino acid protein with a relative molecular mass of 53 949 kD, containing a cellulose-binding domain. The recombinant plasmid pHBM731 was generated by inserting the optimized target gene into a Pichia pastoris expression vector pHBMg05 that was transformed into three Pichia pastoris strains, GS115, KM71 and SMD1168. The biochemical properties of the enzyme were assessed. [ Result] The cloned galactonumnan (GM)-degrading enzyme was expressed and secreted by Pichia pastoris GSll5. High cell density fermentation was induced in recombi- nant Pichia pastoris at 25 and 28 ~C ; a higher enzyme activity was observed at an induction temperature of 28 ~C. The optimal temperature for the recombinant en- zyme is 60 ~C, and the optimal pH is 6.6. The enzyme activity was 38.61 U under optimal conditions. Over 50% of the enzyme activity was maintained under the optimal conditions after 9 h. Under the optimal conditions, the effect of metal ions on enzyme activity was analyzed. Ca2 + , Fe2 + and Li ~ slightly enhanced enzyme activity, while Mn2+ and Co2+ had little effect. Enzyme activity was modestly suppressed by Mg2~ , K~ and Na+ , but considerably suppressed by Ag2~ and Zn2~ , with Cu2 + showing the strongest inhibitory effects. [ Conclusion] A novel GM-degrading enzyme expressed by soil yeast was cloned, which can potentially be used in industrial applications to obtain eommereially useful guar gum-degradation products.
文摘Objective:To clone feruloyl esterase gene C from Penicillium chrysogenum and characterize the general properties of the enzyme.Methods:The feruloyl esterase C gene was amplified by PCR based on the Penicillium chrysogenum feruloyl esterase C gene sequence and cloned into the expression vector p PIC9K,resulting the recombinant plasmid p PIC9K-Pcfae C.The recombiant plasmid was linerized and transformed into P.pastoris by electroporation.The transformants was screened based on the transparent zone technology.The screened transformants was then induced by methanol.the enzymatic properties of the protein were then measured.Results:SDS-PAGE analysis showed that the molecular mass of the enzyme was about 30 k D.The length of the gene was 762 bp.It comprised one open reading framwork(ORF)and annotated to encode 249 amino acid.The optimal temperature and p H was found to be 40℃and 6,respectively.Moreover,the recombinant enzyme was stable at 40-50℃and p H 5-7.Conclusion:The enzyme successfully expressed in P.pastoris could laid theoretical foundation in food,fodder and paper making industry.
基金This work was supported by the State Key Basic Research Development Plan of China (Grant No. 1998010100)the Innovation Foundation of Laboratory of Photosynthesis Basic Research, Institute of Botany, the Chinese Academy of Sciences.
文摘The violaxanthin de-epoxidase gene was cloned from rice (Oryza sativa subsp. japonica). The full length of the cDNA is 1887 bp, encoding a 446-amino acids protein with the transit peptide of 98 amino acids. The bacterial expression vector pET-Rvde was constructed and the expression quantity of the exogenous protein increased with the induction time by 0.4 mmol/L IPTG. Its molecular weight was similar with that of the native VDE. Western blotting indicated that the expressed protein has immu-nological reaction with the VDE polyclonal antibody. The absorbance spectrum together with xanthophyll pigments quantification by HPLC demonstrated that the expressed VDE has its enzyme activity, which can de-epoxidate violaxanthin into antheraxanthin and zeaxanthin in vitro.
基金supported by the projects in the National Key Technologies R&D Program during the 11th Five-Year Plan period of China (2006BAD01A03)
文摘Starch branching enzyme (SBE) catalyzes the biosynthesis of amylopectin. We described the isolation and characterization of SBEIIb promoter and their expression patterns in transgenic tobacco. Using the genomic DNA of maize cultivar Lunuo 1 as template, the SBEIIb promoter was isolated by PCR and was cloned into pMD18-T vector. To study SEBIIb gene regulation at the cellular level, SBEIIb promoter was fused to the ^-glucuronidase (GUS) report gene. The results of the fluorometric GUS assays indicate that the sbeⅡb-GUS fusion directed a seed-specific expression. Four series of constructs were made with the promoter and the GUS reporter gene to investigate the cis-acting analysis, showing that the four different constructs all can drive expression of the GUS gene in seed plumule and cotyledon and the GUS activity was apparently decreased with the progressive loss of promoter 5' end.