Improving production of Streptomyces griseus trypsin for its application in processing insulin precursor

Abstract

Background: Trypsin has a plenty application in food and pharmaceutical manufacture. While, the commercial trypsin is usually extracted from pork pancreas, which has the risk of infectious and immunogenicity. Therefore, the microbial Streptomyces griseus trypsin (SGT) is a prior alternation because it processes efficient hydrolysis activity without the aforementioned risk. The remarkable hydrolysis efficiency of SGT caused its autolysis, and five autolysis sites R21, R32, K122, R153, and R201 were identified from its' autolysate. Results: The tbcf (K101A, R201V) mutant was screened by directed selection approach for improved activity in flask culture (60.85 ± 3.42 U·mL -1 , increased 1.5-fold). From the molecular dynamics simulation, the K101A/R201V mutation shortened the distant between catalytical residues D102 and H57 from 6.5 Å vs 7.0 Å, which afforded the improved specific activity 1527.96 ± 62.81 U·mg -1 . Further, the production of trypsin was increased 302.8% (689.47 ± 6.78 U·mL −1 ) in 3-L bio-reactor, with co-overexpression of chaperones SSO2 and UBC1 in Pichia pastoris. Conclusions: The SGT protein could be an adequate trypsin for insulin production. When working with hydrolysates analysis and direction selection, the tbcf (K101A, R201V) mutant increased 1.5-fold activity. Further, the production of trypsin was improved 3-fold by overexpressing chaperone protein in Pichia pastoris . The future study should be emphasized on the application of SGT in insulin manufacture and pharmaceutical.