Saturation Mutation At The Site Of 131/133 Of Penicillium Expansum Lipase And Properties Of The Mutant L133M

Penicillium expansum Lipase(PEL)belongs to the serine protease family,whose active center is Ser-Asp-His,there is a conserved pentapeptide domain-GX₁SX₂G-nearby.And the S(Ser)of the domain is the same amino acid as that forms the active center,the two amino acids of X₁ and X₂vary among the different lipases.In order to explore the relationship between the amino acids at the sites of X₁ and X₂ in this structure and the catalytic properties of PEL,site-directed saturation mutations were carried out at the site 131/133 of PEL in this paper,and the enzymatic properties of wild-type lipase and the mutant were compared.The main results are as follows:1.Saturation mutation at the site 131/133 and screening of mutantsSite-directed saturation mutation was carried out at the 131/133 site of Penicillium expansum lipase gene,and the mutant library was built.A mutant L133M whose activity might be larger than that of the wild-type was selected out.2.Construction of mutant high-expression strain and expression inductionThe coding gene fragment of the mutant L133M was cloned into the p AO815expression vector,a four-copy recombinant plasmid p AO815-m-4PEL-L133M was constructed,and electrotransformed into Pichia pastoris GS115.Yeast strains that could express mutant L133M efficiently were selected out.The selected yeast strains were induced to express with 2%methanol,and the results showed that the concentrations of lipase mutant L133M reached 9.015 g/L in the obtained fermentation broth.Compared with the reported lipase-producing strains,its expression level is higher---which indicated that the construction and induced expression strategy of lipase high-producing strains used in this experiment is more reasonable and effective.3.Comparative study on the catalytic properties of the mutant L133M and wild-type lipase.The catalytic properties of the mutant L133M and wild-type lipase were compared and the results showed that:(1)With the increase of carbon chain length of the substrates,the hydrolysis activities of the two lipases to the substrates gradually decrease,and the two lipases showed the same preference for short-chain substrate.At 3-10 min,the activities of the mutant L133M towards nitrophenyl butyrate,nitrophenyl octanoate,nitrophenyl palmitate,tributyrin,tricaprylin and olive oil were lower than those of wild type lipase.However,the hydrolysis activity of the mutant L133M towards olive oil was 1.61 times of that of wild-type lipase after 48 h reaction,which was consistent with the result of substrate plate screening.(2)The change trend of the thermal stability of mutant lipase is similar to that of wild-type lipase,and there is no obvious difference between the two.This shows that it is not the difference in thermal stability that causes the huge difference in the long-term(48h)and short-term(3-10 min)catalytic results of wild-type and mutant lipases.(3)Wild-type lipase exhibited interface activation when the concentration of p-nitrophenyl butyrate was greater than 3 m M;While mutant L133M needed a higher concentration of the substrate micelles and the interfacial activation only appeared when the concentration of p-nitrophenyl butyrate was greater than 5m M,and the activation process seemed to be slower and more difficult.After further analysis with bioinformatics software,it can be seen that the change in the properties of the mutant L133M is not caused by the change in the number of hydrogen bonds or the change in the length of hydrogen bonds.A new hydrophobic interaction between the methionine introduced by the mutation at position 133 and the lid of the enzyme increases the resistance to be overcome during the interface activation of the enzyme.Therefore,in case that the enzyme activity is measured for a short period of 3-10 minutes,the mutant showed less activity than that of the wild-type lipase.However,after the enzyme is activated(lid opened),the binding energy between the mutant and the substrate tributyrin molecule is lower,showing stronger substrate binding ability and higher catalytic activity,which may be the reason why the mutant showed higher catalytic activity than wild-type lipase after48 h of catalytic reaction.