초록
<P>In this study, the coding sequence of the lipase from <I>Proteus</I> sp. SW1 was optimized via codon optimization and subjected to expression in <I>Pichia pastoris</I> GS115. The maximum enzyme yield was 387 mg/L in the supernatants of the shake-flask culture. The purified recombinant lipase exhibited a specific activity of 130 U/mg toward <I>p</I>-nitrophenyl Laurate. Its optimum pH and temperature were 8.0 and 40°C, respectively. It was highly stable and even activated in water-miscible solvents, showing over 102% residual activity after 24 h incubation in ethanol, acetone, isopropanol and acetonitrile. In addition, the enzyme showed promoted activity with the increasing concentrations of methanol/ethanol and exhibited the maximum activity at 80%. In a solvent-free system for biodiesel synthesis with a one-step addition of methanol, the recombinant lipase displayed a 87% conversion rate toward palm oil at the high water content of 80%. The highly improved expression level and activity of the recombinant lipase may contribute to enable its commercial-scale production, and the unique properties would make it a particularly promising biocatalyst for biodiesel production in the future.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The lipase gene was systematically codon-optimized and highly expressed in <I>Pichia pastoris</I>. </LI> <LI> The expression and specific activity of 257- and 18-fold increased, respectively, compared to those in <I>E. coli</I>. </LI> <LI> RLipase displayed a long-term tolerance to high concentrations of methanol or ethanol. </LI> <LI> Instead of being deactivated, RLipase was activated by 70–98% of methanol or ethanol. </LI> <LI> RLipase showed a high conversion rate toward palm oil at the high water content of 80%. </LI> </UL> </P>