title: [High expression of LIP1 in Pichia pastoris].
abstract: The gene of LIP1, the most important isoenzyme of Candida rugosa lipase (CRL), was artificially synthesized according to its mature peptide sequence. It consisted of 20 codons of preference in Pichia pastoris. The artificial gene was cloned into methanol-inducible expression vector pPICZalphaA, and constitutive expression vector pGAPZalphaA, respectively. The linearized recombinant plasmids were transformed into chromosome of Pichia pastoris SMD1168H strain by electroporation. The abilities of expressing LIP1 in both transformed yeasts had been compared, and the yeast transformed with pGAPZalphaA was more efficient than pPICZalphaA. A recombinant yeast strain named CHT-II expressed LIP1 constitutively, and was the most efficient one. Some enzymatic properties of the recombinant LIP1 were also determined. CHT-II secreted LIP1 into supernate at a level of 2.00x10(5) u/L after 72 h (the cells had been transferred to fresh culture medium at the 24th h). After optimizing the conditions for high cell-density fermentation, the selected yeast strain could secrete LIP1 into supernate at a level of 1.395x10(6) u/L after 72 h. The results indicated that this modification of lip1 gene was successful.
authors.affiliation: The State Key Laboratory for Biological Control, Biopharmaceutical Center, Zhongshan University, Guangzhou 510275, China. wxz@zsu.edu.cn
authors.forename: Jin-Long
authors.initials: JL
authors.lastname: Bei
authors.forename: Jin-Wen
authors.initials: JW
authors.lastname: Wang
authors.forename: Xun-Zhang
authors.initials: XZ
authors.lastname: Wang
authors.forename: Qin-Xing
authors.initials: QX
authors.lastname: Long
authors.forename: Lin
authors.initials: L
authors.lastname: Yang
authors.forename: Ying-Yu
authors.initials: YY
authors.lastname: Deng
mesh_terms: D000595:Amino Acid Sequence; D002175:Candida / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D005813:Genes, Synthetic / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014170:Transformation, Genetic; D014304:Triolein / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D014304:Triolein; D008049:Lipase