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 (NH4)2SO4 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 Zn2+, but it was strongly inhibited by Fe2+, Fe3+, Hg2+ 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.
Highlights
Lipases (EC 3.1.1.3) are industrially important lipolytic enzymes which are widely used as biocatalysts in biotechnological applications [1]
Some psychrophilic microorganisms, including Pseudomonas sp., Aeromonas hydrophila, Pseudoalteromonas sp. and Candida antarctica have been found to produce cold-active lipases, most of which were produced by growth of the wild type stains [7]
Sequence analysis of lip5-wt from C. albicans showed no introns in the lip5-wt gene sequence, and a putative N terminus signal sequence includes 14 amino acid residues, which exports the Lip5 outside the cells. lip5-wt gene with the deleted signal sequence was cloned from C. albicans genome DNA and sequenced
Summary
Dong-Ming Lan 1,†, Ning Yang 1,†, Wen-Kai Wang 1, Yan-Fei Shen 1, Bo Yang 1,* and. Key Lab of Fermentation and Enzyme Engineering, College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510641, China. Received: 20 February 2011; in revised form: May 2011 / Accepted: May 2011 /
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