The Research On Specificity And Stability Of Trypsin

Trypsin(EC 3.4.21.4)is a kind of widely used serine protease,as an proteolytic enzyme it can recognize and cleavage the peptide bond at the carboxy terminus of arginine and lysine.In this paper,the difference of specificity between humanized anionic trypsin(Ht2)and porcine trypsin(Pt)were researched.The result showed that Pt own a higher specific activity and low Km than that of Ht2 when BAEE as substrate.In order to investigate the function of the sites G227 and G217 of the trypsin,which located in the recognition of substrate pocket,the mutated recombinant Ht2-G217A,Ht2-G227A,Pt-G217A,Pt-G227A were obtained.Both Pt-G217A and Pt-G227A lost the activity,while a decreased activity of Ht2-G217A and more prone lysine-specific was observed.To study the self-degradation and stability of Pt,the degradation peptides was analyzed by mass spectrometry,the arginine of Pt in the site 122,130 were proved to be the self-degradation sites.The mutational recombinant plasmids of Pt-R122L,Pt-R122H and Pt-R122H-R130T were constructed and the stability of those mutants were investigated.Results further demonstrate that the Pt-R122H is more stable than Pt-R122L.Compared with Pt-R122H,the doble sites mutant Pt-R122H-R130T owned better stability and the RP-HPLC analysis on purity showed less self-digestion,as more than 70%?-trypsin which is 79.3%,and less than 20%?-trypsin which is 14.9%.