High-level expression of Candida parapsilosis lipase/acyltransferase in Pichia pastoris.
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.
Brunel
Laetitia
L
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.
Neugnot
Virginie
V
Landucci
Laure
L
Boze
Hélène
H
Moulin
Guy
G
Bigey
Frédéric
F
Dubreucq
Eric
E
D000595:Amino Acid Sequence; D002175:Candida / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D008049:Lipase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis
D011994:Recombinant Proteins; D008049:Lipase