The Research On Stability Of Recombinant Human Trypsin Mutants

Trypsin(EC 3.4.21.4)is a kind of widely used proteolytic enzyme,by identifying the positive charge on the arginine and lysine side chain,to hydrolyse the peptide bond as one kind of endopeptidase.There are three types of the known human trypsin.In order to increase the stability of human anionic trypsin(hT2),series of mutants of hT2,such as mhT2-S139C,mhT2-S139C-S206C-R122L,mhT2-S139C-S206C-S121A,mhT2-S121A-R122L,mhT2-S139C-S206C-S121A-R122L,mhT2-S121A mutants were successful expressed and their purification and stability and enzyme characters were further investigated in this paper.Comparing the characteristics and stabilities of wild-type human cationic trypsin(hT1),hT2 and their mutants,the disulfide bonds S139C-S206C was shown to be a special factor to stabilize human trypsin.And a mutant mhT2-S139C-S206C-R122L showed high thermo-stability,for example,while kept at 50?,12h,and 60?,12h,82%and 60%residual activity was still remained,respectively.Contrary to the self-cleavage sites R122,another site S121 was selected to study its effect on the stability of hT2.By comparing the mutant mhT2-S139C-S206C-R122Land mhT2-S139C-S206C-S121A,almost the same effect to increase the stability of hT2 was found.A composite mutant mhT2-S 13 9C-S206C-S121A-R122L was finally obtained.the enzyme activities could be kept above 80%when kept hT2 mutant at different pH3-11,25? for 2h.Further study showed that the enzyme activities could be kept above 70%when kept hT2 mutant at pH3,60? for 12h.Its high thermo-stability and wide-range pH stability provide a guarantee for its wide usage in the future.