Extracellular expression of glucose inhibition-resistant Cellulomonas flavigena PN-120 β-glucosidase by a diploid strain of Saccharomyces cerevisiae

Abstract

The catalytic fraction of the Cellulomonas flavigena PN-120 oligomeric β-glucosidase (BGLA) was expressed both intra- and extracellularly in a recombinant diploid of Saccharomyces cerevisiae, under limited nutrient conditions. The recombinant enzyme (BGLA¹⁵) expressed in the supernatant of a rich medium showed 582 IU/L and 99.4 IU/g dry cell, with p-nitrophenyl-β-D-glucopyranoside as substrate. BGLA¹⁵ displayed activity against cello-oligosaccharides with 2-5 glucose monomers, demonstrating that the protein is not specific for cellobiose and that the oligomeric structure is not essential for β-D-1,4-bond hydrolysis. Native β-glucosidase is inhibited almost completely at 160 mM glucose, thus limiting cellobiose hydrolysis. At 200 mM glucose concentration, BGLA¹⁵ retained more than 50 % of its maximal activity, and even at 500 mM glucose concentration, more than 30 % of its activity was preserved. Due to these characteristics of BGLA¹⁵ activity, recombinant S. cerevisiae is able to utilize cellulosic materials (cello-oligosaccharides) to produce bioethanol.

Kallar grass (Leptochloa fusca L. Kunth) as a feedstock for ethanol fermentation with the aid of response surface methodology

“…The adjusted-R 2 value for both ethanol and SCE represent the significance of the model. However, in saccharification, the accumulation of glucose to a certain extent causes the feedback inhibition of the cellulolytic enzyme, and shuts down the saccharification process [31,32]. Results from ANOVA demonstrate that all the four factors were found significant, based on their P values <0.05, for ethanol production.…”

Enhanced soluble expression of a thermostble β-glucosidase from Thermotoga maritima in Escherichia coli and its applicaton in immobilization

“…Chen et al [12] reported expression of a secretory β glucosidase from Trichoderma reesei in P. pastoris that the maximum recombinant β glucosidase activity achieved 60 U/mL. Mendoza Aguayo with coworkers [13] revealed extra cellular expression of β glucosidase of Cellulomonas fla vigena PN 120 in the supernatant of a rich medium showing 582 U/L. On the other hand, there are a few reports on expression and extracellular secretion of β glucosidase from the thermophilic bacteria in E. coli [9][10][11][12][13].…”

“…Mendoza Aguayo with coworkers [13] revealed extra cellular expression of β glucosidase of Cellulomonas fla vigena PN 120 in the supernatant of a rich medium showing 582 U/L. On the other hand, there are a few reports on expression and extracellular secretion of β glucosidase from the thermophilic bacteria in E. coli [9][10][11][12][13]. The advantages of thermostable enzymes in industrial processes include reduced risk of contamina tion, increased substrate solubility, higher stable of enzymes against denaturing agents and proteolytic enzymes, and reduced cost of external cooling [14].…”