High-level expression of extracellular lipase Lip2 from Yarrowia lipolytica in Pichia pastoris and its purification and characterization |
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Authors: | Yu Mingrui Lange Stefan Richter Sven Tan Tianwei Schmid Rolf D |
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Institution: | Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, PR China. |
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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). |
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Keywords: | Lipase Yarrowia lipolytica Pichia pastoris Fed-batch culture Purification |
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