Extracellular expression of glucose inhibition-resistant Cellulomonas flavigena PN-120 β-glucosidase by a diploid strain of Saccharomyces cerevisiae.
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.
Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, AV. IPN 2508, 07300, Zacatenco, DF, México.
David J
DJ
Mendoza-Aguayo
Héctor M
HM
Poggi-Varaldo
Jaime
J
García-Mena
Ana C
AC
Ramos-Valdivia
Luis M
LM
Salgado
Mayra
M
de la Torre-Martínez
Teresa
T
Ponce-Noyola
D040143:Cellulomonas / Q000201:enzymology* / Q000235:genetics*; D004171:Diploidy; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D006868:Hydrolysis; D009844:Oligosaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D001617:beta-Glucosidase / Q000235:genetics* / Q000378:metabolism*
D004791:Enzyme Inhibitors; D009844:Oligosaccharides; D011994:Recombinant Proteins; D001617:beta-Glucosidase; D005947:Glucose