Properties of a recombinant beta-glucosidase from polycentric anaerobic fungus Orpinomyces PC-2 and its application for cellulose hydrolysis.
A beta-glucosidase (BglA, EC 3.2.1.21) gene from the polycentric anaerobic fungus Orpinomyces PC-2 was cloned and sequenced. The enzyme containing 657 amino acid residues was homologous to certain animal, plant, and bacterial beta-glucosidases but lacked significant similarity to those from aerobic fungi. Neither cellulose- nor protein-binding domains were found in BglA. When expressed in Saccharomyces cerevisiae, the enzyme was secreted in two forms with masses of about 110 kDa and also found in two forms associated with the yeast cells. Km and Vmax values of the secreted BglA were 0.762 mM and 8.20 micromol/(min x mg), respectively, with p-nitrophenyl-beta-D-glucopyranoside (pNPG) as the substrate and 0.310 mM and 6.45 micromol/(min.mg), respectively, for the hydrolysis of cellobiose. Glucose competitively inhibited the hydrolysis of pNPG with a Ki of 3.6 mM. Beta-glucosidase significantly enhanced the conversion of cellulosic materials into glucose by Trichoderma reesei cellulase preparations, demonstrating its potential for use in biofuel and feedstock chemical production.
Li
Xin-Liang
XL
Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, USDA/ARS,1815 N. University Street, Peoria, IL 61604-3902, USA. lix@ncaur.usda.gov
Ljungdahl
Lars G
LG
Ximenes
Eduardo A
EA
Chen
Huizhong
H
Felix
Carlos R
CR
Cotta
Michael A
MA
Dien
Bruce S
BS
D000595:Amino Acid Sequence; D001709:Biotechnology / Q000379:methods; D002482:Cellulose / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D020085:Neocallimastigales / Q000378:metabolism*; D009598:Nitrophenylgalactosides / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D001617:beta-Glucosidase / Q000737:chemistry*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D009598:Nitrophenylgalactosides; C059200:4-nitrophenylgalactoside; D002482:Cellulose; D004247:DNA; D001617:beta-Glucosidase