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...

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Bibliographic Details
Published in:Protein expression and purification 2001-04, Vol.21 (3), p.386-392
Main Authors: Rotticci-Mulder, Johanna C., Gustavsson, Malin, Holmquist, Mats, Hult, Karl, Martinelle, Mats
Format: Article
Language:English
Subjects:
2 lipases
Adsorption
Binding Sites
Blotting, Western
Candida - enzymology
Candida - genetics
Cellulose - metabolism
Electrophoresis, Polyacrylamide Gel
endoglucanase
enzyme
Gene Expression
Genetic Engineering
Glycosylation
high-level production
Hydrolysis
immobilization
inhibitors
Lipase - biosynthesis
Lipase - genetics
Lipase - isolation & purification
Lipase - metabolism
Molecular Weight
oryzae
Pichia - genetics
Protein Binding
Protein Structure, Tertiary
purification
Recombinant Fusion Proteins - biosynthesis
Recombinant Fusion Proteins - chemistry
Recombinant Fusion Proteins - isolation & purification
Recombinant Fusion Proteins - metabolism
Time Factors
trichoderma-reesei
Triglycerides - metabolism
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Summary: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 α-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.
ISSN:1046-5928
1096-0279
1096-0279
DOI:10.1006/prep.2000.1387