High-level expression of Candida parapsilosis lipase/acyltransferase in Pichia pastoris |
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Authors: | Brunel Laetitia Neugnot Virginie Landucci Laure Boze Hélène Moulin Guy Bigey Frédéric Dubreucq Eric |
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Affiliation: | UMR Ingénierie des réactions biologiques, bioproductions (IR2B), INRA--ENSA.M.--Université Montpellier II, Equipe de Génie Microbiologique et Enzymatique, ENSA.M.--INRA, Place Viala, F-34060 Montpellier Cedex 1, France. |
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Abstract: | Candida parapsilosis has been previously shown to produce a lipase/acyltransferase (EC 3.1.1.3) that preferentially catalyses transfer reactions such as alcoholysis over hydrolysis in the presence of suitable nucleophiles other than water, even in aqueous media (aw > 0.9 ). This enzyme has been shown to belong to a new family of lipases. The present work describes the cloning of the gene coding for this lipase/acyltransferase in the yeast Pichia pastoris and the heterologous high-level expression of the recombinant enzyme. The lipase/acyltransferase gene, in which the sequence encoding the signal peptide was replaced by that of the alpha-factor of Saccharomyces cerevisiae, was placed under the control of the methanol inducible promoter of the alcohol oxidase 1 gene (AOX1). A transformed P. pastoris clone, containing five copies of the lipase/acyltransferase gene, was selected for the production of recombinant enzyme. The fed-batch culture supernatant contained 5.8 gl(-1) (weighted) of almost pure recombinant lipase/acyltransferase displaying the same catalytic behavior as the original enzyme. |
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