title: Enhanced production of leech hyaluronidase by optimizing secretion and cultivation in Pichia pastoris.
abstract: 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 × 10(5) to 1.24 × 10(6) U/ml. Compared with the parental LHAase, the variant AP2-LHAase showed a lower optimum pH (5.0), higher optimum temperature (50 °C), and a broader range of thermal stability (20-60 °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 × 10(6) U/ml, with a high productivity of 1.87 × 10(4) U/ml/h.
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China. zkang@jiangnan.edu.cn.|Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China. zkang@jiangnan.edu.cn.|School of Biotechnology, Jiangnan University, Wuxi, 214122, China. zkang@jiangnan.edu.cn.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Kang
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.|School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
authors.forename: Na
authors.initials: N
authors.lastname: Zhang
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.|School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
authors.forename: Yunfeng
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005818:Genetic Engineering; D006821:Hyaluronoglucosaminidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007865:Leeches / Q000201:enzymology* / Q000502:physiology; D010843:Pichia / Q000201:enzymology* / Q000235:genetics* / Q000502:physiology; D021382:Protein Sorting Signals / Q000235:genetics; D013696:Temperature
chemical_list: D021382:Protein Sorting Signals; D006821:Hyaluronoglucosaminidase
keywords: Amphipathic peptides; Leech hyaluronidase; N-terminal engineering; Pichia pastoris; Signal peptides; Variable temperature control strategy