Kinetic characterization of a beta-glucosidase from a yeast, Candida wickerhamii.
The extracytoplasmic, cell-bound beta-1,4-glucosidase of Candida wickerhamii was characterized kinetically. The enzyme was found to produce glucose from cellobiose and cellodextrins (degree of polymerization from three to six) by catalyzing the removal of the terminal glucose moiety from the nonreducing end of these beta-glucans. The Km values for the series, cellobiose through cellohexaose, were 210.7, 106.6, 106.3, 105.9, and 79.8 mM, respectively, whereas the kcat values were 14.79, 13.24, 13.78, 15.13, and 7.66 mumol of glucose.min-1.mg-1 of protein, respectively. A computer program was developed to estimate the integrated rate equation. When the above kinetic constants were used in the computer model, the predicted rates of glucose formation agreed well with the experimental data. Saccharomyces cerevisiae, which is unable to ferment cellobiose or cellodextrins, ferments glucose about twice as fast as C. wickerhamii. If S. cerevisiae is cultured on cellobiose or cellodextrins and the purified C. wickerhamii beta-glucosidase is added to the S. cerevisiae culture at levels that mimic the production of beta-glucosidase by a C. wickerhamii culture with time, the two cultures produce ethanol at equivalent rates. This suggests that the rate-limiting step in the fermentation of cellobiose/cellodextrins by C. wickerhamii is the production of beta-glucosidase.
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Fermentation Biochemistry Research Unit, United States Department of Agriculture, Peoria, Illinois 61604.
D002175:Candida / Q000201:enzymology*; D002475:Cellobiose / Q000378:metabolism; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003912:Dextrins / Q000378:metabolism; D005285:Fermentation; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D012984:Software; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000378:metabolism*
D003912:Dextrins; D002475:Cellobiose; D002482:Cellulose; C068994:cellodextrin; D001617:beta-Glucosidase