Molecular cloning and functional expression of a novel extracellular lipase from the thermotolerant yeast Candida thermophila.
The thermotolerant yeast Candida thermophila SRY-09 isolated from Thailand produces an extracellular lipase that hydrolyses various triglycerides. To clone the gene encoding the lipase, Saccharomyces cerevisiae was transformed with a C. thermophila genomic library and screened for lipase activity on medium containing olive oil emulsion and rhodamine B. One C. thermophila lipase gene (CtLIP) was found that contained an ORF of 1317 bp encoding a deduced polypeptide of 438 amino acids. Candida thermophila lipase contained a Gly-Asp-Ser-Gln-Gly motif which matched the consensus Gly-X-Ser-X-Gly conserved among lipolytic enzymes. Heterologous expression of the cloned CtLIP under the control of the alcohol oxidase gene (AOX1) promoter in the methylotrophic yeast Pichia pastoris, and enzymatic measurements confirmed the function of the respective protein as a lipase. The recombinant CtLIP could hydrolyse various substrates at high temperature (55 degrees C) with higher efficiency than at 37 or 45 degrees C and preferentially hydrolysed two-positional ester bonds. As with C. thermophila, the heterologously expressed lipase was secreted into the medium by Pichia pastoris.
Thongekkaew
Jantaporn
J
Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand.
Boonchird
Chuenchit
C
D002175:Candida / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000502:physiology; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D006358:Hot Temperature; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D005656:Fungal Proteins; D011994:Recombinant Proteins; D008049:Lipase