Monday, April 30, 2012
Napoleon Ballroom C-D, 3rd fl (Sheraton New Orleans)
Lipase (EC 3.1.1.3) is a popular enzyme used as a biocatalyst for many biochemical reactions. Lipase is usually expressed in the Escherichia coli cytoplasm as an inactive inclusion body and at a low level. CalB was fused with various sizes of polyanionic amino acid tags and expressed in E. coli BL21 star (DE3) in order to improve the expression level of CalB and biological activity. The five aspartate tag (D5-CalB) resulted in the highest lipase activity of 3.3 U/ml in the medium, corresponding to a 8.2-fold enhancement compared with the authentic CalB. A fed-batch fermentation done with the recombinant E. coli grown in a pH-stat mode resulted in 2.6 g/L active CalB titer. Enzymatic characterization revealed that a fusion of short anion tags at the N-terminal of CalB changed its quaternary structure from hexamer to trimer. Fusion of the ten aspartate tag with the C-terminal of D5-CalB for purification (D5-CalB-D10) led to simple and effective purification of CalB with 89% purity and 72% purification yield.