초록
<P><B>Background</B></P><P>Lipase from <I>Rhizopus chinensis</I> is a versatile biocatalyst for various bioconversions and has been expressed at high-level in <I>Pichia pastoris</I>. However, the use of <I>R. chinensis</I> 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 <I>R. chinensis</I> lipase by directed evolution using <I>P. pastoris</I> as the host.</P><P><B>Results</B></P><P>An efficient, fast and highly simplified method was developed to create a mutant gene library in <I>P. pastoris</I> based on <I>in vivo</I> 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 <I>P. pastoris</I>. 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, <I>T</I><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 <I>k</I><SUB>cat</SUB>/<I>K</I><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.</P><P><B>Conclusions</B></P><P><I>P. pastoris</I> 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 <I>P. pastoris</I> 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.</P>