Pseudomonas putida KT2440 株由来リパーゼ遺伝子のクローニングと大腸菌での発現

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Triglyceride lipase is an enzyme that catalyzes the hydrolyzation of triglyceride to glycerin and fatty acid. Bacterial lipase is a useful enzyme in the clinical diagnosis field.Most bacterial lipase needs a chaperon which constructs enzyme in the active formal structure.Recently,a kind of bacterial strain,Pseudomonas putida KT2440,was determined for full chromosomal DNA sequence,and the lipase gene was included for it.It is known that gene of a lipase-chaperon which mostly exists in the downstream with a lipase gene on bacterial chromosomal DNA,but doesn't exist for P.putida KT2440.This study aims at cloning and expression of the lipase gene for the strain.The lipase gene was amplified by using PCR,prerared in the plasmind vector pUC118 and pUC119.There were transformed into E.coli TOP10 strain,but the transformant did not show an enzymatic activity of lipase.In order to get activated lipase,the lipaes gene and some multi-chaperon genes from E.coli(gro gene etc.)were co-transformed into E.coli.Some of the transformant showed a lipase activity by the dna,gro,and tig genes.We compared the chaperon genes between E.coli and P.putida in the DNA database(DDBJ),then,the multi-chaperon genes also found in the P.putida chromosome.It supposed that the lipase protein from P.putida could be activated by its own multi-chaperons.Thus,we have demonstrated a production of the active lipase by using an E.coli host vector system.
2010-03-01