Research Of Engineering Trypsin Thermostability By Rational Design

As an essential tool enzyme,trypsin has been widely applied in variety of fields,such as industrial or scientific research fields.Digestion with trypsin is usually carried out at 20-40°C,while most reactions in industrial or scientific research fields require high temperature.Trypsin can not maintain activity under high temperature conditions,which limits its industrial applications.In recent years,protein engineering has been proven to be a powerful approach to increase thermostability of enzymes.Therefore,improving the thermostability of trypsin by using protein engineering could be better for satisfying the industrial applications.In this research,based on the strategy of rational design,the thermostability of trypsin was improved by the method of truncating the flexible region.At first,molecular dynamic simulation,Fold Unfold,and Flex Pred were used to predict the flexible regions of trypsin.The Fragment 78-90 was identified through the combination of three methods.Secondly,the Fragment 78-90 was truncated for rigidifying the protein.Five variants(D3,D5,D7,D9 and D11)were generated by truncating amino acids from the N-terminal of Asp89.The gene sequences of variants in this study were achieved by overlapping extension PCR,and then plasmids of wild-type trypsin and five variants were transformed into pichia pastoris GS115 for expression.The wild-type trypsin and five variants could be obtained after purified by a SP-Sepharose column.Finally,the protein yield of wild-type trypsin was 43.32 mg/L,while the yields of D3,D5,D7,D9and D11 were 25.82 mg/L,29.92 mg/L,53.44 mg/L,40.58 mg/L and 33.49 mg/L,respectively.We investigated the thermostability,specific activity,kinetic properties,secondary structures,and tertiary structures of wild-type and five variants.The variant D9 showed higher thermostability,with a 4.5°C rise in T_m,a 5°C increase in T_opt,and a 5.8°C rise in T₅₀¹⁰,compared to the wild-type.Moreover,the half-life value of the variant D9 was also found to be dramatically improved by 46 min.Meanwhile,the specific activity and catalytic efficiency of variant D9 had no decrease.Circular dichroism and intrinsic fluorescence indicated that the structures had no significant change between the variant D9 and the wild-type.In addition,the surface hydrophobicity of the variant D9 was measured to be lower than that of wild-type,indicating the increased hydrophobic interaction,which could have contributed to the improved thermostability of D9.In this study,the thermostability of trypsin has been improved without compromising enzymatic activity through the method of truncating the flexible region.This method has the potential to be used for enhancing the thermostability of other tool enzymes.