Enhanced catalytic efficiency and substrate specificity of Streptomyces griseus trypsin by evolution-guided mutagenesis

Streptomyces griseus trypsin (SGT) is a bacteria-sourced that could be potentially applied to industrial insulin productions. However, SGT produced by microbial hosts displayed low catalytic efficiency and undesired preference lysine residue. In this study, engineering the α signal peptide in Pichia pastoris, we increased amidase activity 67.91 U mL−1 shake flask cultures. Afterwards, engineered evolution-guided mutagenesis obtained three variants A45S, V177I E180M with efficiencies. On basis, performed iterative combinatorial constructed mutant A45S/V177I/E180M which reached 98 flasks 2506 3-L fed-batch Moreover, single mutation T190 S190 substrate of R K R/K value was improved 7.5, 2 times better than animal-sourced trypsin.