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| High-efficient expression of Thermomyces dupontii lipase in Pichia pastoris based on combinatorial optimization strategy |
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| DOI： |
| KeyWord:Thermomyces dupontii lipase (LIP1) combinatorial optimization strategy Pichia pastoris high-density expression substrate specificity |
| FundProject: |
| Author Name | Affiliation | | WANG Buqing1, CHEN Zhou1, WANG Yasen1, XU Xiangyang2, GAO Xiaodong1, FUJITA Morihisa1, LI Zijie1 | 1.School of Bioengineering, Jiangnan University, Wuxi 214122, Jiangsu, China
2.Zaozhuang Jienuo Biological Enzyme Co. , Ltd. , Zaozhuang 277100, Shandong,China |
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| Abstract: |
| Thermomyces dupontii lipase (LIP1) is an alkaline lipase, which posses good application potential in washing industry, biodiesel preparation, oil modification, etc. However, the natural productivity of LIP1 is extremely too low to meet the requirements of industrial application. A combinatorial optimization strategy was applied for the screening of Pichia pastoris strain highly expressing LIP1. Firstly, a Pichia pastoris prefered codon optimized LIP1 gene was constructed, and the recombinant strain with higher LIP1 gene copy numbers was screened through high-concentration G418 plates and BMMY-rhodamine B qualitative plates. Moreover, through signal peptides optimization and chaperone co-expression optimization, a high-productivity recombinant Pichia postoris strain GS115/pPIC9K-Mss-SSA4-LIP1 was obtained.The enzyme activity was determined by alkali titration,and the substrate specificity was determined by colorimetric assay. The results showed that the highest secretive activity of the strain after combinatorial optimization strategy could reach1 136 U/mL in flask fermentation and 12 150 U/mL in 5 L fermenter, The substrate specificity result showed that the most suitable substrate for Pichia pastoris recombinanted LIP1 was p-nitrophenol ester with eight carbons.In conclusion, the high-efficient expression of LIP1 in Pichia pastoris can be realized based on the combinatorial optimization strategy, which will lay the foundation for the future industrialization of LIP1. |
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