Cell Surface Display Of CALB In Saccharomyces Cerevisiae With A-agglutinin And Optimization Of Fermentation Conditions

1 Background and ObjectiveCandida antarctic lipase B is one of the most widely used lipase. It has a strong catalytic activity in non-water-soluble and water-soluble systems. CALB shows greater superiority than other lipases in a variety of reactions because of its stability and specificity. It has already been expressed in E.coli and S. cerevisiae. But the application of CALB is still restricted by the high cost of commercial enzymes and the process of separation, purification and immobilization.2 Methods and ResultsThe artificially synthetic gene calb(only mature peptide sequence) was combined with AGA2, and inserted into the plasmid pYD1 for surface display of CALB. Recombinant plasmid pYD1-CALB was successfully transformed into Saccharomyces cerevisiae EBY100 and analysis on genetic stability showed that the enzyme activity of recombinant yeast stayed steady.Optimization of recombinant yeast fermentation in industrial medium showed: Under the conditions of temperature 28℃, pH 5.0, inoculum volume 10%, dissolved oxygen >30%, the optical density (OD600) was 36.5 after 22 hours during the growth period of S.cerevisiae, During the expression period, the optical density (OD600) was 53.3 after 82 hours under the conditions of temperature 25℃, pH 5.0, dissolved oxygen >30%, concentration of galactose 2%. The activity of CALB in fomenters achieved 4.25U/ml after 48 hours induction.3 ConclusionImmobilizing CALB using cell surface display system in S. cerevisiae with a-agglutinin as carrier protein was viable. Saccharomyces cerevisiae had already been widely used in pharmaceutical, food industry and was safe for human beings and environment. The new cell-enzymes dramatically reduced the cost of enzyme production by immobilizing on the cell instead of separating and purifying. Industrial fermentation medium also brought economic benefit.