title: Comparison of catalytic properties of multiple β-glucosidases of Trichoderma reesei.
abstract: Ten putative Trichoderma reesei β-glucosidase (BGL) isozymes were heterologously expressed in Escherichia coli and Aspergillus oryzae and purified to homogeneity. Catalytic properties of nine enzymes which showed hydrolytic activity on cellobiose and p-nitrophenyl-β-D-glucopyranoside (pNPG) were investigated. Three BGLs, encoded by the genes cel3A, cel3B, and cel3E, contained a predicted signal peptide, showed higher hydrolytic activity on cello-oligosaccharides than on pNPG, and preferred longer oligosaccharides. Another three putative extracellular BGLs, Cel3B, Cel3F, and Cel3G, and two intracellular enzymes, Cel3C and Cel3D, exhibited preference for pNPG. Intracellular Cel1A showed the highest affinity for cellobiose as a typical cellobiase. Four BGLs, Cel3A, Cel3B, Cel3E, Cel1A, that showed high activity against cello-oligosaccharides were capable of catalyzing transglycosylation reactions from cellobiose, leading to formation of cellotriose and isomeric glucobioses. While Cel3A, Cel3B, and Cel3E synthesized mainly gentiobiose, glycosyl transfer reactions of Cel1A led mainly to sophorose and laminaribiose. Conversion of cellobiose to sophorose by Cel1A reached about 3.6 and 10 % at 1 and 10 % cellobiose concentration, respectively. The formation and persistence of individual cellobiose isomers in incubation mixtures of four BGLs (Cel3A, Cel3B, Cel3E, and Cel1A) with cellobiose correlated well with the k cat values for isomeric glucobioses. Cel1A also showed the lowest sensitivity to inhibition by glucose. Based on all studied catalytic properties, Cel1A appears to be unambiguously the best candidate for site-directed mutations or directed evolution toward improvement of activity, thermostability, and, eventually, efficiency of sophorose synthesis.
authors: Department of Bioscience and Textile Technology, Interdisciplinary Graduate School of Science and Technology, Shinshu University, 4-17-1 Wakasato, Nagano, 380-8553, Japan.
authors: Boyang
authors: B
authors: Guo
authors: Department of Bioscience and Textile Technology, Interdisciplinary Graduate School of Science and Technology, Shinshu University, 4-17-1 Wakasato, Nagano, 380-8553, Japan.
authors: Nobuaki
authors: N
authors: Sato
authors: Institute of Chemistry, Slovak Academy of Sciences, 845 38, Bratislava, Slovakia.
authors: Peter
authors: P
authors: Biely
authors: Department of Bioscience and Textile Technology, Interdisciplinary Graduate School of Science and Technology, Shinshu University, 4-17-1 Wakasato, Nagano, 380-8553, Japan.
authors: Yoshihiko
authors: Y
authors: Amano
authors: Department of Bioscience and Textile Technology, Interdisciplinary Graduate School of Science and Technology, Shinshu University, 4-17-1 Wakasato, Nagano, 380-8553, Japan. knoza@shinshu-u.ac.jp.
authors: Kouichi
authors: K
authors: Nozaki
mesh_terms: D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000201:enzymology / Q000235:genetics; D002475:Cellobiose / Q000378:metabolism; D044602:Cellulases / Q000096:biosynthesis* / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D004187:Disaccharides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics; D005936:Glucans / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D005960:Glucosides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D004271:DNA, Fungal; D004187:Disaccharides; D005656:Fungal Proteins; D005936:Glucans; D005960:Glucosides; D011994:Recombinant Proteins; C014637:laminaribiose; D002475:Cellobiose; C025193:4-nitrophenyl beta-D-glucoside; D044602:Cellulases; C100052:gentiobiose; D005947:Glucose; C003328:sophorose
keywords: Cellulose degradation; Transglycosylation; Trichoderma reesei; β-glucosidase