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Author Meng, Zhang ♦ Xiaowei, Yu ♦ Yan, Xu ♦ Rui, Wang ♦ Rong, Xiao
Source Directory of Open Access Journals (DOAJ)
Content type Text
Publisher BioMed Central
File Format HTM / HTML
Date Created 2013-03-12
Copyright Year ©2012
Language English
Subject Domain (in LCC) QR1-502
Subject Keyword Thermostability ♦ Science ♦ Lipase ♦ Microbiology ♦ Pichia pastoris ♦ Mutant library construction ♦ Directed evolution
Abstract Abstract Background Lipase 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. Results An 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 × 10 <sup>5</sup> /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, T <sub>m</sub> was 22 degrees higher and half-lives at 60°C and 65°C were 46- and 23- times longer. Moreover, the catalytic efficiency k <sub>cat</sub>/K <sub>m</sub> 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. Conclusions P. 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.
ISSN 14752859
Age Range 18 to 22 years ♦ above 22 year
Educational Use Research
Education Level UG and PG ♦ Career/Technical Study
Learning Resource Type Article
Publisher Date 2012-08-01
e-ISSN 14752859
Journal Microbial Cell Factories
Volume Number 11
Issue Number 1
Starting Page 102

Source: Directory of Open Access Journals (DOAJ)