The Effect Of The ?-loop Which Is Next To The Active Site On The Stability And Activity Of Trypsin

As an important tool enzyme,trypsin has been used for many fields including pharmaceutical and scientific research.In industry,extraction form pancreas juice is the conventional means for trypsin production for its low cost and easy process.While inhomogeneity between different batch of products and residual pathogenic microorganisms make it in urgent need of developping a mature approach to achieve trypsin with high safty and quality.In MS assay of novel proteins,denaturants usually complicated subsequent analysis.While in high temperature,denatured protein could be digested by trypsin which avoids the applification of denaturants.Thus there is a need to engineering thermostability of trypsin by rational design.In this research,we attempted to improve the thermostability and activity of porcine trypsin by deleting?-loop which is next to the active site.Molecular dynamics software Gromacs v4.5.5 was used to predict the flexibility profile of the porcine trypsin structures.The?-loop which is next to the active site was confirmed to represent the flexible region.Subsequently,according to the difference in the amount of deleted amino acids in the?-loop,three mutations of R1Q,K3Q and C5Q were determined,and glycosylation site-directed mutation were introduced in?-loop and its wing chain which contained active site Asp84 and two mutants M77,M81 were obtained.The results demonstrated that the activity of R1Q,K3Q and C5Q was improved to some extent.The maximal activity of R1Q mutant enzyme was improved by 1.42 times compared with that of wild type.At the same time,the inactivation half-life period and the optimal reaction temperature were increased by213.3min and 10~oC respectively.The half-life period of M77 and M81 increased156.9min and 189.5min compared with wild type.In this study,a trategy to improve thermostability of trypsin were proposed:through its native glycosylation modification of Pichia pastoris,oligosaccharides were added in?-loop and its wing chains which contained the active site.With this strategy,we successfully improved the thermostability of trypsin without its activity changed.The strategy provided a referential method to engineer thermostability of other industrial enzymes and had the potential for an extensive application.