Enhanced production of leech hyaluronidase by optimizing secretion and cultivation in Pichia pastoris

Leech hyaluronidase (LHAase) was recently cloned and successfully expressed in Pichia pastoris. To increase its secretory expression level, four signal peptides (nsB, YTP1, SCS3, and HKR1) and six amphipathic peptides (APs) were comparatively investigated. After substitution with nsB and fusion with AP2, the production of LHAase was significantly increased, from 8.42 x 10(5) to 1.24 x 10(6) U/ml. Compared with the parental LHAase, the variant AP2-LHAase showed a lower optimum pH (5.0), higher optimum temperature (50 A degrees C), and a broader range of thermal stability (20-60 A degrees C). To further promote fermentative production of the variant AP2-LHAase, the cultivation temperature was systematically optimized according to cell viability and alcohol oxidase activity. Eventually, through a combination of N-terminal engineering and optimization of cultivation, the production of LHAase was improved to 1.68 x 10(6) U/ml, with a high productivity of 1.87 x 10(4) U/ml/h.