Expression And Identification Of Mutants Of Penicillium Expansum Lipase Focused On Oxyanion Hole

In order to explore the effect of the oxyanion hole structure of Penicillium expansum lipase(PEL)on the substrate specificity of the enzyme,the method of in vitro multi-copy vector recombination was used for the expression of the mutants focused on the oxyanion hole structure,which has already been screened out in our lab.Also,the enzymatic properties of the mutants were tested.The research contents are as follows:1.Cloning of the lipase mutant genes.Primer Premier 6.0 was used to design mutation primers:L133M,L133A,H131E/L133A.To do plasmid PCR,pET-47 b(+)-PEL was used as the template.Then,three recombinant plasmids containing the mutant genes were transformed into E.coli competent cells.The transformants were selected and sequenced by Invitrogen.2.Construction of multi-copy recombinant plasmids.In order to obtain the single-copy recombinant expression vector pA0815-m-PEL,the PEL mutant gene fragments were connected with the pA0815 plasmid,and then the recombinant plasmid of pA0815-m-PEL was transferred to the E.coli Top 10.Restriction enzymes Bgl ? and BamH I were used to get single-copy expression cassette.Then,the single-copy expression cassette was connected with the single-copy recombinant expression vector pA0815-m-PEL.The recombinant plasmid was transferred to the E.coli Top10 and the two-copy expression vector of pA0815-m-2PEL can be obtained.Similarly,pA0815-m-3PEL,pA0815-m-4PEL recombinant plasmids were obtained.3.Expression and properties of Penicillium expansum lipase mutantsThe recombinant expression plasmids of pA0815-m-4PEL were transformed into the Pichia pastoris GS115.After screening,transformants of GS-pA0815-m-4PEL can be obtained.The transformants were induced to express lipase mutants.Finally,the enzymatic properties of the lipase mutants were studied.(1)The specific activities of wild-type lipase and its mutants L133M,L133A,H131E/L133A were detected with five substrates of different chain length(4-nitrophenyl butyrate,4-nitrophenyl caproate,4-nitrophenyl octoate,4-nitrophenyl laurate,4-nitrophenyl palmitate).According to the experimental results,the specific activities of the mutants L133M,L133A and H131E/L133A on 4-nitrophenyl butyrate were 0.75 times,2.61 times and 2.25 times of that of the wild-type lipase,respectively.And the specific activities on 4-nitrophenyl caproate were 0.72 times,2.27 times and 1.93 times of that of the wild-type lipase,respectively.The specific activities on 4-nitrophenyl octoate were 0.45 times,1.51 times and 0.96 times of that of the wild-type lipase,respectively.The specific activities on 4-nitrophenyl laurate were 0.84 times,3.93 times and 5.53 times of that of the wild-type lipase,respectively.The specific activities on 4-nitrophenyl palmitate were 0.59 times,9.42 times and 8.82 times of that of the wild-type lipase,respectively.(2)The specific activities of wild-type lipase and its mutants L133M,L133A,H131E/L133A were detected with(R,S)-ethyl mandelate.According to the experimental results,the specific activity of L133M,L133A and H131E/L133A lipase on R-ethyl mandelate are1.89 times,11.1 times and 13.2 times than that of the wild-type lipase,respectively.The specific activity on S-ethyl mandelate are 0.68 times,0.70 and 0.62 times than that of the wild-type lipase,respectively.Compared with wild-type lipase,its mutants,L133A and H131E/L133A brought out a“new”substrate selectivity,which could be able to have a better enantioselectivity for(R,S)-ethyl mandelate.