扩展青霉脂肪酶热稳定性的分子突变-微生物学专业论文.docx

AbstractAbstract Abstract Abstract In order to improve the enzyme properties of the Penicillum expaasum lipase(PEL)， the PEL gene was mutated by site-mutagenesis and random mutagenesis． 1．Site—directed mutagenesis of PEL In order to improve the thermostability of Penicillium expansum lipase(PEL)， Eleven mutations were created by site-directed mutagenesis．Mutations were generated by overlap extension PCR using the eDNA of wild-type lipase gene(1ip07)or a single site mutant lipase ep8 as the template and two special primers that corresponding to mutations． Recombinant vector which contain mutant genes was constructed and electroporated into Pichia pasteris GS 1 1 5． (1)PEL—P 1 97E single mutant and PEL-ep8-P 1 97E double mutant Double mutant PEL-·ep8··P 1 97E was created by mutate Pro to Glu at 1 97 based on the single mutant PEL—ep8．The double mutant shows a good improvement in thermostability with a Tm of 41．50C，which is 2．80C higher than the wild-type PEL and 2．20C higher than single mutant PEL-ep8． PEL-P 1 97E is a single mutant of PEL at amino acid 1 97．The Tm of this mutant is 39．50C which is 0．90C higher the PEL． After three days methanol induced expression，the enzyme activity of single mutant and double mutant are 556U／mL and 592U／mL respectly． In addition，the substrate specificity of PEL-ep8一P 1 97E is changed． (2)PEL-K1 52R single mutant and PEL-ep8一K1 52R double mutant Double mutant PEL-·ep8—-K1 52R was created by mutate Lys to Arg at 1 52 based on the signal mutant PEL—ep8．The Tm of this mutant is 38．90C which is similar to that of the wild-type enzyme． PEL-K1 52R is a single mutant of PEL at amino acid 1 52．The Tm of this mutant is 38．90C which is similar to that ofthe wild—type enzyme． After three days methanol induced expression，the enzyme activity of single mutant and double mutant are 530U／mL and 416U／mL respectly． (3)PEL-P 1 02L single mutant and PEL-ep8一P 1 02L double mutant III 福建师范大学刘燕雅硕士学位论文Double 福建师范大学刘燕雅硕士学位论文 Double mutant PEL—ep8一P 1 02L WaS