Expression in Pichia pastoris of Candida antarctica lipase B and lipase B fused to a cellulose-binding domain

Candida antarctica lipase B (CALB) and C. antarctica lipase B fused to a ce llulose-binding domain (CBD-CALB) were expressed functionally in the methyl otrophic yeast Pichia pastoris. The cellulose-binding domain originates fro m cellulase A of the anaerobic rumen fungus Neocallimastix patriciarum. The genes were fused to the a-factor secretion signal sequence of Saccharomyce s cerevisiae and placed under the control of the alcohol oxidase gene (AOX1 ) promoter. The recombinant proteins were secreted into the culture medium reaching levels of approximately 25 mg/L. The proteins were purified using hydrophobic interaction chromatography and gel filtration with an overall y ield of 69%. Results from endoglycosidase H digestion of the proteins showe d that CALB and CBD-CALB were N-glycosylated. The specific hydrolytic activ ities of recombinant CALB and CBD-CALB were identical to that reported for CALB isolated from its native source. The fusion of the CBD to the lipase r esulted in a greatly enhanced binding toward cellulose for CBD-CALB compare d with that for CALB. (C) 2001 Academic Press.