Abstract
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.
Highlights
Lipase from Rhizopus chinensis is a versatile biocatalyst for various bioconversions and has been expressed at high-level in Pichia pastoris
We developed a faster and simplified mutant library production method, based on which, the thermostability of the lipase r27RCL from R. chinensis CCTCC M201021 was improved by directed evolution
Library construction A novel approach for lipase mutant library construction in P. pastoris was established based on the formation of a recombination cassette in vivo (Figure 1)
Summary
Lipase from Rhizopus chinensis is a versatile biocatalyst for various bioconversions and has been expressed at high-level in Pichia pastoris. The present work describes improvement of the thermostability of R. chinensis lipase by directed evolution using P. pastoris as the host. Lipases (triacylglycerol ester hydrolases EC 3.1.1.3) are enzymes that hydrolyze the ester bonds of waterinsoluble substrates at the interface between substrate and water. They are well known because of their remarkable levels of activity and stability in non-aqueous environments, which makes them as attractive enzyme for use in industrial applications, such as in food, pharmaceutical, chemical and detergent industries [1,2,3]. The Tm value of RNL from R. niveus expressed in Saccharomyces cerevisiae was improved from 47°C to 57°C [17]
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