CHARACTERIZATION AND CLONING OF LIPASE GENE(S) FROM CANDIDA BOMBICOLA, A BIOSURFACTANT-PRODUCING YEAST
Biobased and Other Animal Co-Products
2010 Annual Report
1a.Objectives (from AD-416)
The objective of the collaborative work is to isolate and express the lipase gene(s), and then proceed to characterize the enzymatic properties of the gene-products from Candida bombicola yeast used in the production of the biosurfactant sophorolipid.
1b.Approach (from AD-416)
Using a polymerase-chain-reaction gene-fishing approach developed and routinely used in our laboratory, the lipase gene(s) will be cloned from Candida bombicola – a yeast species that produces sophorolipid biosurfactant. The genes will be expressed in E.coli or other suitable host, and the enzymes will be purified and characterized by following common methodologies. Their application potential in the synthesis of nutraceuticals through interesterification reactions and in the formulation of washing mixes will be assessed.
Project researchers (PRs) carried out extensive computer program-aided alignment of known lipase sequences and the design of a number of degenerative oligonucleotide-primers for use in PCR-cloning of Candida bombicola lipase genes. PRs also constructed hygromycin-resistance cassette for use in selection of C. bombicola transformants by joining known C. bombicola promoters with a mutated hygromycin-resistance gene previously optimized for increased activity in yeast. Shotgun cloning approach is currently attempted to clone lipase genes of C. bombicola by screening an expression genomic DNA library of this yeast in E. coli using rhodamine assay plates. A grant award from Organisation for Economic Cooperation and Development (OECD) was secured to fund the research visit to ARS lab by a visiting scientist from Chungnam National University.
Progress of the research project was monitored through frequent face-to-face meetings with the visiting scientist while on-site, and e-mail contacts and teleconference calls.