A Novel Cold-Active Lipase from Candida albicans: Cloning, Expression and Characterization of the Recombinant Enzyme |
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Authors: | Lan Dong-Ming Yang Ning Wang Wen-Kai Shen Yan-Fei Yang Bo Wang Yong-Hua |
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Affiliation: | School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China; E-Mails: landongming@yanhoo.com.cn (D.-M.L.); wshyn091011@gmail.com (N.Y.); breezekai@yahoo.com.cn (W.-K.W.); petsyf@126.com (Y.-F.S.). |
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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))(2)SO(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. |
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Keywords: | Candida albicans cold-active lipase Pichia pastoris expression enzyme purification |
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