Construction Of Gene Engineering Bacterium That Can Express Urotoxin Clastic Enzyme

Objective:Cloning urate oxidase gene from candida utilis to Lactobacillus delbrueckii subsp bulgaricus.the urate oxidase was expressed in the recombinant Lactobacillus delbrueckii subsp bulgaricus.Methods:First candida utilis chromosomal DNA was isolated then according to the urate oxidase of candida utilis gene encoding sequences a pair of primers was designed and the urate oxidase gene was cloned by PCR amplification with primers.the urate oxidase gene fragment was digested with restriction endonucleases Xhaâ… and Hindâ…¢, and linked with the vector plasmid PMG36e disgested with the same enzyme digestion to construct the recombinant expression vector PMG36e-U,the recombinant plasmid PMG36e-U was transformed into Escherichia coli(DH5a), screening and identity, extracting recombinant plasmid PMG36e-U from DH5a,then recombinant plasmid was transformed into Lactobacillus delbrueckii subsp bulgaricus by electroportion. SDS-PAGE identity the urate oxidase and the enzymatic activity was assay. Result:1. Cloning urate oxidase gene from candida utilis.Getting 0.9kb oxidase gene fragment2. The recombinant Bacillus coli containing of plasmid pMD-18T-U was digested with Xbaâ… and Hindâ…¢. Getting 0.9kb urate oxidase gene fragment and having the same sequence with urate oxidase gene in genebank3. The recombinant Lactobacillus delbrueckii subsp bulgaricus that containing of recombinant plasmid PMG36e-U was constructed screening and verified,digesting with Xbaâ… and Hindâ…¢and Getting 0.9kb urate oxidase gene fragment4. The recombinant Lactobacillus delbrueckii subsp bulgaricuswas analyze by SDS-PAGE and Getting the recombinant protein with molecular weight about 34KD5. Recombinant uricase was purified by DEAE DE-52 cellulose, the uricase have about 0.39u/ml activityConclusions:Construction of gene engineering Lactobacillus delbrueckii subsp bulgaricus that can Express urate oxidase Objective:Cloning creatinine amidohydrolase gene from Pseudomonas putida to Lactobacillus delbrueckii subsp bulgaricus, the creatinine amidohydrolase was expressed in the recombinant Lactobacillus delbrueckii subsp bulgaricus.Methods:First Pseudomonas putida chromosomal DNA was isolated then according to the creatinine amidohydrolase of Pseudomonas putida gene encoding sequences a pair of primers was designed and the creatinine amidohydrolase gene was cloned by PCR amplification with primers.the creatinine amidohydrolase gene fragment was digested with restriction endonucleases Xhaâ… and Hindâ…¢, and linked with the vector plasmid PMG36e disgested with the same enzyme digestion to construct the recombinant expression vector PMG36e-C,the recombinant plasmid PMG36e-C was transformed into Escherichia coli(DH5a), screening and identity, extracting recombinant plasmid PMG36e-C from DH5a,then recombinant plasmid was transformed into LactobaciUus delbrueckii subsp bulgaricus by electroportion. SDS-PAGE identity the creatinine amidohydrolase and the enzymatic activity was assay. Result:1. Cloning creatinine amidohydrolase gene from Pseudomonas putida.Getting 0.78kb creatinine amidohydrolase gene fragment2. The recombinant Bacillus coli containing of plasmid pMD-18T-C was digested with Xbaâ… and Hindâ…¢. Getting 0.78kb creatininase gene fragment and having the same sequence with creatininase gene in genebank3. The recombinant Lactobacillus delbrueckii subsp bulgaricus that containing of recombinant plasmid PMG36e-C was constructed screening and verified,digesting with Xbaâ… and Hindâ…¢and Getting 0.78kb creatinine amidohydrolase gene fragment4. The recombinant Lactobacillus delbrueckii subsp bulgaricus was analyze by SDS-PAGE and Getting the recombinant protein with molecular weight about 28KD5. Recombinant creatinine amidohydrolase was purified by DEAE DE-52 cellulose, the creatininase have about 0.19u/ml activityConclusions:Construction of gene engineering Lactobacillus delbrueckii subsp bulgaricus that can Express creatinine amidohydrolase...