Enzyme immobilization using a cellulose-binding domain: properties of a beta-glucosidase fusion protein.
Using molecular genetic techniques, a fusion protein has been produced which contains the cellulose-binding domain (CBD) of an exoglucanase (Cex) from Cellulomonas fimi fused to a beta-glucosidase (Abg) from Agrobacterium sp. The CBD functions as an affinity tag for the simultaneous purification and immobilization of the enzyme on cellulose. Binding to cellulose was stable for prolonged periods at temperatures from 4 degrees C to at least 50 degrees C, at ionic strengths from 10 mM to greater than 1 M, and at pH values below 8. The fusion protein can be desorbed from cellulose with distilled water or at pH greater than 8. Immobilized enzyme columns of the fusion protein bound to cotton fibers exhibited stable beta-glucosidase activity for at least 10 days of continuous operation at temperatures up to 37 degrees C. At higher temperatures, the bound enzyme lost activity. The thermal stability of the fusion protein was greatly improved by immobilization. Immobilization did not alter the pH stability. Except for its ability to bind to cellulose, the properties of the fusion protein were virtually the same as those of the native enzyme.
Department of Microbiology, University of British Columbia, Vancouver, Canada.
E
E
Ong
N R
NR
Gilkes
R C
RC
Miller
A J
AJ
Warren
D G
DG
Kilburn
D000327:Adsorption; D002482:Cellulose; D004800:Enzymes, Immobilized / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D003368:Gossypium; D007700:Kinetics; D010957:Plasmids; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013816:Thermodynamics; D001617:beta-Glucosidase / Q000235:genetics / Q000378:metabolism*
D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; D002482:Cellulose; D001617:beta-Glucosidase