2012 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. 3.Progress Report: Researchers conclusively verified the biological function of the previously cloned Pseudomonas resinovorans lipase (lip) and its modulator (lif) genes. For this purpose, a lipase gene knockout strain of P. resinovorans was constructed and shown to have lost its lipase activity. Project researchers then constructed a recombinant plasmid expressing the lipase gene, introduced the recombinant plasmid into the knockout strain, and showed that the lipase activity was restored. A manuscript describing the study is now in press in a peer-reviewed journal. Project researchers had also performed an in vitro assay study to characterize the substrate specificity of the lipase enzyme coded by the lip gene.