Extracellular expression of glucose inhibition-resistant Cellulomonas flavigena PN-120 β-glucosidase by a diploid strain of Saccharomyces cerevisiae
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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.
KeywordsCellulomonas flavigena β-Glucosidase Inhibition resistant Saccharomyces cerevisiae
This work was supported by Consejo Nacional de Ciencia y Tecnología México (CONACYT) (Grant 104333). D. J. Mendoza-Aguayo received a scholarship number 204305 from CONACYT México. María Isabel Pérez- Montfort corrected the English version of the manuscript.
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