title: Two New Native β-Glucosidases from Clavispora NRRL Y-50464 Confer Its Dual Function as Cellobiose Fermenting Ethanologenic Yeast.
abstract: Yeast strain Clavispora NRRL Y-50464 is able to produce cellulosic ethanol from lignocellulosic materials without addition of external β-glucosidase by simultaneous saccharification and fermentation. A β-glucosidase BGL1 protein from this strain was recently reported supporting its cellobiose utilization capability. Here, we report two additional new β-glucosidase genes encoding enzymes designated as BGL2 and BGL3 from strain NRRL Y-50464. Quantitative gene expression was analyzed and the gene function of BGL2 and BGL3 was confirmed by heterologous expression using cellobiose as a sole carbon source. Each gene was cloned and partially purified protein obtained separately for direct enzyme assay using varied substrates. Both proteins showed the highest specific activity at pH 5 and relatively strong affinity with a Km of 0.08 and 0.18 mM for BGL2 and BGL3, respectively. The optimum temperature was found to be 50°C for BGL2 and 55°C for BGL3. Both proteins were able to hydrolyze 1,4 oligosaccharides evaluated in this study. They also showed a strong resistance to glucose product inhibition with a Ki of 61.97 and 38.33 mM for BGL2 and BGL3, respectively. While BGL3 was sensitive showing a significantly reduced activity to 4% ethanol, BGL2 demonstrated tolerance to ethanol. Its activity was enhanced in the presence of ethanol but reduced at concentrations greater than 16%. The presence of the fermentation inhibitors furfural and HMF did not affect the enzyme activity. Our results suggest that a β-glucosidase gene family exists in Clavispora NRRL Y-50464 with at least three members in this group that validate its cellobiose hydrolysis functions for lower-cost cellulosic ethanol production. Results of this study confirmed the cellobiose hydrolysis function of strain NRRL Y-50464, and further supported this dual functional yeast as a candidate for lower-cost cellulosic ethanol production and next-generation biocatalyst development in potential industrial applications.
authors.affiliation: Sichuan Agricultural University, Chengdu, China.|Bioenergy Research Unit, National Center for Agricultural Utilization Research, USDA-ARS, Peoria, Illinois, 61604, United States of America.
authors.forename: Xu
authors.initials: X
authors.lastname: Wang
authors.affiliation: Bioenergy Research Unit, National Center for Agricultural Utilization Research, USDA-ARS, Peoria, Illinois, 61604, United States of America.
authors.forename: Z Lewis
authors.initials: ZL
authors.lastname: Liu
authors.affiliation: Bioenergy Research Unit, National Center for Agricultural Utilization Research, USDA-ARS, Peoria, Illinois, 61604, United States of America.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Weber
authors.affiliation: Sichuan Agricultural University, Chengdu, China.
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D002475:Cellobiose / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D000431:Ethanol / Q000378:metabolism*; D005285:Fermentation; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005947:Glucose / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D013379:Substrate Specificity; D015003:Yeasts / Q000235:genetics / Q000378:metabolism*; D001617:beta-Glucosidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D002475:Cellobiose; D000431:Ethanol; D001617:beta-Glucosidase; D005947:Glucose