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

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