title: 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))(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.
authors: 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.).
authors: Dong-Ming
authors: DM
authors: Lan
authors: Ning
authors: N
authors: Yang
authors: Wen-Kai
authors: WK
authors: Wang
authors: Yan-Fei
authors: YF
authors: Shen
authors: Bo
authors: B
authors: Yang
authors: Yong-Hua
authors: YH
authors: Wang
mesh_terms: D000595:Amino Acid Sequence; D002176:Candida albicans / Q000201:enzymology*; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism; D055550:Protein Stability; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D012997:Solvents / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D012997:Solvents; D008049:Lipase