Open AccessTwo New Native β-Glucosidases from Clavispora NRRL Y-50464 Confer Its Dual Function as Cellobiose Fermenting Ethanologenic Yeast
Author(s) -
Xu Wang,
Z. Lewis Liu,
Scott A. Weber,
Xiaoping Zhang
Publication year - 2016
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0151293
Subject(s) - cellobiose , fermentation , biochemistry , chemistry , yeast , heterologous expression , enzyme , ethanol fermentation , beta glucosidase , enzyme assay , ethanol , gene , recombinant dna , cellulase
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 K m 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 K i 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.