Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 3, 1996
Publication Date: N/A
Interpretive Summary: Agricultural biomass such as plant stalks, grasses, wood chips, and paper refuse represents an abundant renewable resource that can be used for the production of liquid fuels. The first requirement before these products can be utilized is that they be broken down into simple sugars that can be fermented to ethanol. One particular enzyme important in this breakdown scheme is called beta-glucosidase. The yeast Candida wickerhamii not only makes several forms of this enzyme, but is also able to convert the resulting sugars directly to ethanol. In this paper, we describe the isolation and purification of one of these beta-glucosidases. The properties of the enzyme are thoroughly examined to determine the industrial potential. Characteristics of particular interest include such things as the optimal temperature and the efficiency of the enzyme to perform desired reactions. Also discussed in the paper is the potential to use this enzyme for enzymatically releasing aroma compounds in food and beverages, thereby making the food more pleasing to the consumer.
Technical Abstract: An intracellular beta-glucosidase was isolated from the cellobiose fermenting yeast, Candida wickerhamii. Production of the enzyme was stimulated under aerobic growth, with the highest level of production in medium containing cellobiose as a carbohydrate source. The molecular mass of the purified protein was approximately 94,000 Da. It appeared to exist as a dimeric structure with a native molecular mass of approximately 180,000 Da. The optimal pH ranged from 6.0 to 6.5 using p-nitrophenyl-beta-D-glucopyranoside (pNPG) as a substrate. The optimal temperature for short term (15 min) assays was 35 deg C, while temperature stability analysis revealed that the enzyme was labile at temperatures of 28 deg C and above. Using pNPG as a substrate, the enzyme was estimated to have a Km of 0.28mM and a Vmax of 525 umole product/min/mg protein. Similar to the extracellular beta-glucosidase produced by C. wickerhamii, this enzyme was fairly resistant to end-product inhibition by glucose, retaining 58% of its activity at 100 mM glucose. Activity of the enzyme was highest against aryl beta-1,4-glucosides. However, pNP-xylopyranoside, lactose, cellobiose, and trehalose also served as substrates for the purified protein. Activity of the enzyme was stimulated by long chain n-alkanols and inhibited by ethanol, 2-propanol, and 2-butanol. Amino acid sequence obtained by Edman degradation analysis suggests that this beta-glucosidase is related to the family-3 glycosyl hydrolases.