The extracellular lipase gene from Yarrowia lipolytica (YILip2) was cloned into the pPICZ alpha A 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 39 kDa using Saccharomyces cerevisiae secretion signal peptide (alpha-factor) under the control of the methanol inducible promoter of the alcohol oxidase I gene (AOX1). The lipase activity of 12,500,000 U/1 (2.10 g total protein and 0.63 g 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 10 kDa 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). (C) 2006 Elsevier Inc. All rights reserved.