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.

Biochemical characterization of an isolated 50 kDa beta-glucosidase from the thermophilic fungus Myceliophthora thermophila M.7.7

“…From these data it was also possible to estimate the inhibition constant K i = 1.5 mmol L −1 ; accordingly, glucose has a strong competitive inhibition effect on M. thermophila BGL50. For the yeast Clavispora NRRL Y-50464, K i values were approximately 38 and 62 mmol L −1 for two isoforms (Wang et al, 2016), but K i values for β-glucosidase inhibition from other fungi can reach more than 100 mmol L −1 (Riou et al, 1998). For comparison, kinetic values of an M. thermophila β-glucosidase, expressed when the BGL3a gene was cloned in Pichia pastoris (Karnaouri et al, 2013) were K m = 0.39 ± 0.12 mmol L −1 (close to our estimated value) but K i was 0.28 mmol L −1 , lower than for BGL50.…”

Novel Ethanol- and 5-Hydroxymethyl Furfural-Stimulated β-Glucosidase Retrieved From a Brazilian Secondary Atlantic Forest Soil Metagenome

“…Until now, two native β-glucosidases from the fungus Clavispora have been described as resistant to 5-HMF, but its activity was not enhanced in presence of the compound. Wang et al (2016) showed that 0.12% furfural and 5-HMF did not affect the activity of these two β-glucosidases.…”

Cellulosic Ethanol Production Using a Dual Functional Novel Yeast

“…Expression. When grown on a mixture of glucose and cellobiose, the β-glucosidase activity was induced once glucose was exhausted [17,41]. is indicated that β-glucosidase induction is cellobiose-dependent.…”