期刊
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
卷 100, 期 2, 页码 707-717出版社
SPRINGER
DOI: 10.1007/s00253-015-7056-5
关键词
Leech hyaluronidase; Pichia pastoris; N-terminal engineering; Signal peptides; Amphipathic peptides; Variable temperature control strategy
资金
- Key Technologies R & D Program of Jiangsu Province, China [BE2014607]
- National High Technology Research and Development Program of China (863 Program) [2012AA022005]
- Program for Changjiang Scholars and Innovative Research Team in University [IRT1135]
- National Science Foundation for Post-doctoral Scientists of China [2013M540414]
- Jiangsu Planned Projects for Postdoctoral Research Funds [1301010B]
- 111 Project
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.
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