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United States Department of Agriculture

Agricultural Research Service

Research Project: GENOMICS AND ENGINEERING OF STRESS TOLERANT MICROBES FOR LOWER COST PRODUCTION OF ETHANOL FROM LIGNOCELLULOSE

Location: Bioenergy Research Unit

Title: Isolation and characterization of a ß-glucosidase from a Clavispora strain with potential applications in bioethanol production from cellulosic materials

Authors
item Liu, Zonglin
item Weber, Scott
item Cotta, Michael

Submitted to: BioEnergy Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 16, 2012
Publication Date: July 3, 2012
Citation: Liu, Z., Weber, S.A., Cotta, M.A. 2013. Isolation and characterization of a ß-glucosidase from a Clavispora strain with potential applications in bioethanol production from cellulosic materials. Bioenergy Research. 6:65-74.

Interpretive Summary: Renewable bioethanol production for transportation fuels provides a promising means of reduced dependence on petroleum and a more environmentally friendly energy system. However, the necessary deconstruction of cellulosic polymers and enzymatic hydrolysis require additional processing procedures that increase the cost of lignocellulose-to-ethanol conversion. Cellobiose derived from cellulose needs to be further degraded into the simple sugar glucose by ß-glucosidase before it can be utilized by conventional yeast for growth and subsequent ethanol fermentation. Previously, we discovered a new yeast NRRL Y-50464 producing sufficient ß-glucosidase activity to complete a cellulose-to-ethanol conversion without exogenous ß-glucosidase. This research isolated, purified, and identified a protein from Y-50464 as ß-glucosidase (BGL1). Strain Y-50464 was highly efficient with regard to growth and ethanol conversion on cellobiose when compared among ß-glucosidase producing strains which is desirable for economic cellulosic ethanol production. This research provided additional evidence to support the function of cellobiose utilization by strain Y-50464. These results will advance the development of consolidated bioprocesses for lower-cost cellulosic ethanol production.

Technical Abstract: 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 ß-glucosidase enzyme activity without further enzyme supplementation for cellulosic ethanol production using simultaneous saccharification and fermentation (SSF). Eliminating the addition of external ß-glucosidase reduces the cost of cellulosic ethanol production. In this study, we present results on the isolation and identification of a ß-glucosidase protein from strain Y-50464. Using MALDI-TOF (Matrix-assisted laser desorption/ionization-Time-of-Flight) mass spectrometry and blast search of the NCBInr database (National Center for Biotechnology Information non-redundant), the protein from Y-50464 was identified as a ß-glucosidase (BGL1) with a molecular weight of 93.3 kD. The BGL1 protein was purified through multiple chromatographic steps to a 26-fold purity (Km = 0.355 mM; Ki = 15.2 mM) which has a specific activity of 18.4 U/mg of protein with an optimal performance temperature at 45°C and pH of 6.0. The protein appears to be intercellular and membrane associated. The fast growth rate of Y-50464 and its capability to produce sufficient ß-glucosidase activity for ethanol conversion from cellobiose provide a promising means for low-cost cellulosic ethanol production through a consolidated bioprocessing development.

Last Modified: 11/21/2014
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