Molecular Improvement Of Expandase From Streptomyces Clavuligerus And Study Of A Thermostable Lipase From Bacillus Subtilis FS1403

Deacetoxycephalosporin C synthetase(expandase)is an iron(Ⅱ)andα-oxoglutarate dependent oxygenase.The objective of the study was to generate expandase mutants capable of converting unnatural substrates of penicillin G.via molecular enzyme evolution,and the expandase mutants would be used as an important industrial enzyme in the production of 7-aminodeacetoxycephalosporanic acid(7-ADCA).The site-directed mutagenesis and direction evolution methods were used to alter the substrate specificity of expandase.Based on the previous studies,C155,V275,C281,Y184,C37,Q121 and A61 of scDAOCS were chosen as targets for site-directed mutagenesis.The abilities of converting unnatural substrates penicillin G.of all mutants were assayed after site-directed mutageneis.Among mutants constructed,C155Y,V275I,C281Y,Y184H and Q121M were identified as improved mutants with expanded substrate specificity and enhanced activity against penicillin G..C37S and A61E showed significant reduced in their ability to convert penicillin G..The mutant of C155Y+V275I+C281Y+Y184H+C37S+Q121M showed significant improvement over the wild type enzyme in their ability to convert penicillin G,The ability to convert penicillin G is 7.43 times higher than that of wild type enzyme.To further change the substrate specificity of expandase,scDAOCS genes from Streptomyces clavuligerus was randomly mutagenized by error-prone PCR,and two different homologous DAOCS genes were randomly mutagenized by family shuffling.30 putative mutant expandase clones were analyzed by bioassay using penicillin G as substrate,no clone with improved ability relative to scDAOCS was found.To find new enzyme resource from extreme environmental microorganism,A lipase-producing heat-resistant bacterium FS1403 was isolated from the volcanic vent soil of Philippine.The strain was identified as Bacillus subtilis based on the analysis of homology and phylogenetic of 16S rDNA.The lipase gene was further cloned by PCR.And functional expressed in E.coli.BL21 judged by both SDS-PAGE and tributyrin plate.The recombinant lipase showed the highest activity in pH of 8.0 and temperature of 50℃.The results provide a new lipase gene resource for improvement the existing lipase by molecular directed evolution.