A novel cold-active lipase from Candida albicans: Cloning, expression and characterization of the recombinant enzyme

Abstract

A novel lipase gene lip5 from the yeast Candida albicans was cloned and sequenced. Alignment of amino acid sequences revealed that 86-34% identity exists with lipases from other Candida species. The lipase and its mutants were expressed in the yeast Pichia pastoris, where alternative codon usage caused the mistranslation of 154-Ser and 293-Ser as leucine. 154-Ser to leucine resulted in loss of expression of Lip5, and 293-Ser to leucine caused a marked reduction in the lipase activity. Lip5-DM, which has double mutations that revert 154 and 293 to serine residues, showed good lipase activity, and was overexpressed and purified by (NH 4) 2SO 4 precipitation and ion-exchange chromatography. The pure Lip5-DM was stable at low temperatures ranging from 15-35°C and pH 5-9, with the optimal conditions being 15-25°C and pH 5-6. The activation energy of recombinant lipase was 8.5 Kcal/mol between 5 and 25°C, suggesting that Lip5-DM was a cold-active lipase. Its activity was found to increase in the presence of Zn 2+, but it was strongly inhibited by Fe 2+, Fe 3+, Hg 2+ and some surfactants. In addition, the Lip5-DM could not tolerate water-miscible organic solvents. Lip5-DM exhibited a preference for the short- and medium-chain length p-nitrophenyl (C4 and C8 acyl group) esters rather than the long chain length p-nitrophenyl esters (C12, C16 and C18 acyl group) with highest activity observed with the C8 derivatives. The recombinant enzyme displayed activity toward triacylglycerols, such as olive oil and safflower oil. © 2011 by the authors; licensee MDPI, Basel, Switzerland.