Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 15, 1996
Publication Date: N/A
Interpretive Summary: More than one billion gallons of ethanol are produced annually in the United States, with approximately 95% derived from fermentation of corn starch. Various agricultural residues such as corn stover, straw and bagasse can also serve as low-value and abundant feedstocks for production of fuel alcohol. Currently, the utilization of biomass to produce fuel ethanol presents significant technical and economic challenges, and its success depends largely on the development of highly efficient and cost effective enzymes for conversion of pretreated biomass to fermentable sugars. One particular enzyme is sensitive to the simple sugar, glucose and controls cellulose breakdown. We have discovered a novel enzyme from a yeast which shows very high glucose tolerance. The enzyme has been purified and characterized. The properties of the enzyme shows great potential for industrial application in the enzymatic breakdown of biomass to glucose.
Technical Abstract: The enzyme, beta-glucosidase, is sensitive to substrate (cellobiose) and product (glucose) inhibition and is rate limiting during enzymatic hydrolysis of cellulose to glucose. Candida peltata (NRRL Y-6888) produced beta-glucosidase when grown in liquid culture on various substrates. An extracellular beta-glucosidase was purified 1800-fold to homogeneity from the culture supernatant. The enzyme was a monomeric glycoprotein with an apparent molecular weight of 43,250. It was optimally active at pH 5.0 and 50 deg C and had a specific activity of 108 umol.min**-1.mg**-1 protein against p-nitrophenyl beta-D-glucoside (pNPbetaG). The purified beta-glucosidase readily hydrolyzed pNPbetaG and cellobiose with Km values of 2.3 and 66 mM, respectively. The enzyme was highly tolerant to glucose inhibition with a Ki of 1.4 M (252 mg/ml). Substrate inhibition was not observed with 40 mM pNPbetaG or 15% cellobiose. The enzyme did not require divalent cations for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), ethylenediaminetetraacetate (10 mM) or dithiothreitol (10 mM). Ethanol, at optimal concentration (0.75%, v/v), stimulated the initial enzyme activity by only 11%. Cellobiose (10%, w/v) was almost completely hydrolyzed to glucose by the purified beta-glucosidase (1.5 U/ml) both in the absence and presence of glucose (6%). Glucose production was enhanced by 8.3% when microcrystalline cellulose (2%, w/v) was treated for 24 h with a commercial cellulase preparation (5 U/ml) that was supplemented with purified beta-glucosidase (0.4 U/ml). This is the first report of a novel beta-glucosidase from a yeast having such a high tolerance to glucose.