| A 190 kDa β-glucosidase purified from commercial Aspergillus niger cellulase was shown to be a dimer and to be N-glycosylated (comprised mainly of mannose). It had unique pH dependent kinetics at high substrate concentrations. This was explained by the finding that the β-glucosidase is both a transglucosylase that is pH independent between pH 3 and 7, with the substrate itself being a transglucosidic acceptor at high substrate concentrations, and a glycosyl hydrolase with a pH optimum between 4 and 4.5. The main transglucosidic products of the β-glucosidase reaction at high pNPGlc and cellobiose concentrations were shown to be p-nitrophenyl-β-D-glucopyranosyl-β(1→6)-glucopyranose and 4-O-gentiobiosyl-glucose, respectively. Gentiobiose was formed at high glucose concentrations. The enzyme is an exo-β-D-glucosidase and has at least 5 glucose subsites.; The gene for a β-glucosidase from A. niger was integrated into the genome of Pichia pastoris. Sequencing of a CNBr peptide and studies of the physical and kinetic properties of the recombinant enzyme showed that it was identical to the commercial β-glucosidase.; The roles of some conserved Trp and a Leu were investigated. Based on sequence alignments of glycosyl hydrolase family 3 members and on the only available family 3 structure, eight Trp (21, 49, 139, 262, 430, 551, 806, 813) and 1 Leu (426) were chosen for mutagenesis.; Analyses of the substituted β-glucosidases showed that major changes from wild type only occurred upon substituting for Trp-49 and Trp-262. Substitutions of Trp-49 caused large decreases of transglucosidic activity. Trp-49 seems to preserve the configuration at the non-reducing binding site, but adversely affects binding at other subsites, and thus the acceptor action is decreased. Substitutions of Trp-262 resulted in large decreases of the hydrolytic activity. This results from a loss of binding interactions with the non-reducing end glucose while the acceptor subsites are unaffected. Glycosidic cleavage then only takes place at very high concentrations and only transglucosylis occurs. Other more minor effects found at other positions may merit further studies. Surprisingly, Trp-430 and Leu-426, which are known to be very important at the active site of another family 3 glycosidase (H. vulgare β-D-glucan exohydrolase), did not seem to be important for this β-glucosidase. |
