Trypsin is an important industrial enzyme, which is widely used in leather bating, food processing and medicine. The production of Streptomyces griseus trypsin (SGT) by recombinant Pichia pastoris was enhanced by strain modification and optimization of fermentation conditions. Firstly, the effects of four chaperones (Cne1, Bmh2, Ssa4 and Vhb) and two transcription factors (Yap1 and Aft1) on the expression of SGT in P. pastoris GS115 were analyzed. The results of shake flask fermentation showed that when co-expressing with transcription factor Aft1, the SGT activity was 19.71% higher than that of the control strain, reaching 217.30 U/mL. The co-expression of other transcription factors or chaperones reduced SGT production. Secondly, the optimal growth pH and induction pH of PP-SGTm/Aft1 were 5.5 and 6.5, respectively. Finally, the ratio of methanol-sorbitol in 5 L fermentation was optimized. When the ratio of methanol to sorbitol was 20∶0.5, the activity of SGT reached 2 667.24 U/mL (144 h), which was 20.1% higher than that of adding methanol only. The results will provide important basic data for industrial fermentation of SGT.
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