Isolation and Characterization of a β-Glucosidase from a Clavispora Strain with Potential Applications in Bioethanol Production from Cellulosic Materials

We previously reported on a new yeast strain of Clavispora sp. NRRL Y-50464 that is capable of utilizing cellobiose as sole source of carbon and energy by producing sufficient native beta-glucosidase enzyme activity without further enzyme supplementation for cellulosic ethanol production using simultaneous saccharification and fermentation. Eliminating the addition of external beta-glucosidase reduces the cost of cellulosic ethanol production. In this study, we present results on the isolation and identification of a beta-glucosidase protein from strain Y-50464. Using Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and blast search of the NCBInr database (National Center for Biotechnology Information nonredundant), the protein from Y-50464 was identified as a beta-glucosidase (BGL1) with a molecular weight of 93.3 kDa. The BGL1 protein was purified through multiple chromatographic steps to a 26-fold purity (K (m) = 0.355 mM [pNPG]; K (i) = 15.2 mM [glucose]), which has a specific activity of 18.4 U/mg of protein with an optimal performance temperature at 45 A degrees C and pH of 6.0. This protein appears to be intracellular although other forms of the enzyme may exist. The fast growth rate of Y-50464 and its capability to produce sufficient beta-glucosidase activity for ethanol conversion from cellobiose provide a promising means for low-cost cellulosic ethanol production through a consolidated bioprocessing development.