The lipase/acyltransferase from Candida parapsilosis: molecular cloning and characterization of purified recombinant enzymes.
Candida parapsilosis has been previously shown to produce a lipase (i.e. able to catalyze efficiently the hydrolysis of insoluble lipid esters such as triacylglycerols) that preferentially catalyses transfer reactions such as alcoholysis in the presence of suitable nucleophiles other than water, even in aqueous media with high (> 0.9) water thermodynamic activity. The present work describes the cloning and the overexpression of the gene coding for this enzyme. Two ORFs (CpLIP1 and CpLIP2) were isolated. The deduced 465-amino-acid protein sequences contained the consensus motif (G-X-S-X-G) which is conserved among lipolytic enzymes. Only one of the two deduced proteins (CpLIP2) contained peptide sequences obtained from the purified lipase/acyltransferase. Homology investigations showed that CpLIP2 has similarities principally with 11 lipases produced by C. albicans (42-61%) and the lipase A from Candida antarctica (31%) but not with the other lipases sequenced so far. Both CpLIP1 and CpLIP2 were expressed in Saccharomyces cerevisiae, but only CpLIP2 coded for an active protein. The substrate specificity and the catalytic behavior of purified recombinant CpLIP2, with or without a C-terminal histidine tag, were not changed compared to those of the native lipase.
Neugnot
Virginie
V
Ecole Nationale Supérieure Agronomique de Montpellier, Institut National de la Recherche Agronomique, UFR de Microbiologie Industrielle et de Génétique des Microorganismes, Montpellier, France.
Moulin
Guy
G
Dubreucq
Eric
E
Bigey
Frédéric
F
D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002175:Candida / Q000201:enzymology*; D003001:Cloning, Molecular; D017931:DNA Primers; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D000217:Acyltransferases; D008049:Lipase