title: High-level expression of extracellular lipase Lip2 from Yarrowia lipolytica in Pichia pastoris and its purification and characterization.
abstract: The extracellular lipase gene from Yarrowia lipolytica (YlLip2) was cloned into the pPICZalphaA and integrated into the genome of the methylotrophic yeast Pichia pastoris X-33. The lipase was successfully expressed and secreted with an apparent molecular weight of 39kDa using Saccharomyces cerevisiae secretion signal peptide (alpha-factor) under the control of the methanol inducible promoter of the alcohol oxidase 1 gene (AOX1). The lipase activity of 12,500,000U/l (2.10g total protein and 0.63g lipase per liter) was obtained in a fed-batch cultivation, where methanol feeding was linked to the dissolved oxygen content after initial glycerol culture. After fermentation, the supernatant was concentrated by ultrafiltration with a 10kDa cut off membrane and purified with ion exchange chromatography using Q Sepharose FF. Deglycosylation showed that the recombinant lipase is a glycoprotein which contains the same content of sugar (about 12%) as the native lipase from Y. lipolytica. The optimum temperature and pH of the recombinant lipase was 40 degrees C and 8.0, respectively. The lipase showed high activity toward long-chain fatty acid methyl esters (C12-C16).
authors.affiliation: Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, PR China.
authors.forename: Mingrui
authors.initials: M
authors.lastname: Yu
authors.forename: Stefan
authors.initials: S
authors.lastname: Lange
authors.forename: Sven
authors.initials: S
authors.lastname: Richter
authors.forename: Tianwei
authors.initials: T
authors.lastname: Tan
authors.forename: Rolf D
authors.initials: RD
authors.lastname: Schmid
mesh_terms: D019149:Bioreactors; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D015870:Gene Expression; D005800:Genes, Fungal; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D010802:Phylogeny; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D025062:Yarrowia / Q000201:enzymology* / Q000235:genetics*
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; C408971:LIP2 protein, Yarrowia lipolytica; D008049:Lipase