Properties of an intracellular beta-glucosidase purified from the cellobiose-fermenting yeast Candida wickerhamii.
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 a medium containing cellobiose as a carbohydrate source. The molecular mass of the purified protein was approximately 94 KDa. It appeared to exist as a dimeric structure with a native molecular mass of about 180 KDa. The optimal pH ranged from 6.0 to 6.5 with p-nitrophenyl beta-D-glucopyranoside (NpGlc) as a substrate. The optimal temperature for short-term (15-min) assays was 35 degrees C, while temperature-stability analysis revealed that the enzyme was labile at temperatures of 28 degrees C and above. Using NpGlc as a substrate, the enzyme was estimated to have a Km of 0.28 mM and a Vmax of 525 mumol product min-1 mg protein-1. Similar to the extracellular beta-glucosidase produced by C. wickerhamii, this enzyme resisted end-product inhibition by glucose, retaining 58% of its activity at 100 mM glucose. The activity of the enzyme was highest against aryl beta-1,4-glucosides. However, p-nitrophenyl 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. The amino acid sequence, obtained by Edman degradation analysis, suggests that this beta-glucosidase is related to the family-3 glycosyl hydrolases.
Skory
C D
CD
National Center for Agricultural Utilization Research, USDA/Agricultural Research Utilization, Peoria, IL 61604-3902, USA. skorycd@ncaur1.ncaur.gov
Freer
S N
SN
Bothast
R J
RJ
D000332:Aerobiosis; D000438:Alcohols / Q000494:pharmacology; D000595:Amino Acid Sequence; D002175:Candida / Q000201:enzymology*; D002241:Carbohydrates / Q000494:pharmacology; D002475:Cellobiose / Q000378:metabolism*; D004795:Enzyme Stability; D005285:Fermentation; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017421:Sequence Analysis; D013379:Substrate Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D000438:Alcohols; D002241:Carbohydrates; D002475:Cellobiose; D001617:beta-Glucosidase