Enhanced thermostability of a Rhizopus chinensis lipase by in vivo recombination in Pichia pastoris

Microbial Cell Factories

Research

Rui Wang¹  Rong Xiao²  Xiao-Wei Yu³  Yan Xu³  Meng Zhang³ 

[1] Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, 214122, Wuxi, China;School of Medicine and Pharmaceutics, Jiangnan University, 214122, Wuxi, China;State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, 214122, Wuxi, China;Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and Medicine, Rutgers University, 08854, Piscataway, NJ, USA;State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, 214122, Wuxi, China;Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, 214122, Wuxi, China;

关键词: Lipase;    Directed evolution;    Thermostability;    Mutant library construction;    Pichia pastoris;   

DOI  :  10.1186/1475-2859-11-102

 received in 2012-05-03, accepted in 2012-07-28,  发布年份 2012

来源: Springer

【 摘 要 】

BackgroundLipase from Rhizopus chinensis is a versatile biocatalyst for various bioconversions and has been expressed at high-level in Pichia pastoris. However, the use of R. chinensis lipase in industrial applications is restricted by its low thermostability. Directed evolution has been proven to be a powerful and efficient protein engineering tool for improvement of biocatalysts. The present work describes improvement of the thermostability of R. chinensis lipase by directed evolution using P. pastoris as the host.ResultsAn efficient, fast and highly simplified method was developed to create a mutant gene library in P. pastoris based on in vivo recombination, whose recombination efficiency could reach 2.3 × 105 /μg DNA. The thermostability of r27RCL was improved significantly by two rounds of error-prone PCR and two rounds of DNA shuffling in P. pastoris. The S4-3 variant was found to be the most thermostable lipase, under the conditions tested. Compared with the parent, the optimum temperature of S4-3 was two degrees higher, Tm was 22 degrees higher and half-lives at 60°C and 65°C were 46- and 23- times longer. Moreover, the catalytic efficiency kcat/Km of S4-3 was comparable to the parent. Stabilizing mutations probably increased thermostability by increasing the hydrophilicity and polarity of the protein surface and creating hydrophobic contacts inside the protein.ConclusionsP. pastoris was shown to be a valuable cell factory to improve thermostability of enzymes by directed evolution and it also could be used for improving other properties of enzymes. In this study, by using P. pastoris as a host to build mutant pool, we succeeded in obtaining a thermostable variant S4-3 without compromising enzyme activity and making it a highly promising candidate for future applications at high temperatures.

【 授权许可】

Unknown   
© Yu et al.; licensee BioMed Central Ltd. 2012. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.