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 cellulose-binding domain (CBD-CALB) were expressed functionally in the methylotrophic yeast Pichia pastoris. The cellulose-binding domain originates from cellulase A of the anaerobic rumen fungus Neocallimastix patriciarum. The genes were fused to the alpha-factor secretion signal sequence of Saccharomyces 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 yield of 69%. Results from endoglycosidase H digestion of the proteins showed that CALB and CBD-CALB were N-glycosylated. The specific hydrolytic activities 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 resulted in a greatly enhanced binding toward cellulose for CBD-CALB compared with that for CALB.
Department of Biotechnology, Royal Institute of Technology, SE-100 44 Stockholm, Sweden.
J C
JC
Rotticci-Mulder
M
M
Gustavsson
M
M
Holmquist
K
K
Hult
M
M
Martinelle
D000327:Adsorption; D001665:Binding Sites; D015153:Blotting, Western; D002175:Candida / Q000201:enzymology* / Q000235:genetics; D002482:Cellulose / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005818:Genetic Engineering; D006031:Glycosylation; D006868:Hydrolysis; D008049:Lipase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors; D014280:Triglycerides / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D014280:Triglycerides; D002482:Cellulose; D008049:Lipase; C005830:tributyrin