j-Glucosidase was purified from a crude cellulase preparation from Aspergillus niger by affinity chromatography on a methacrylamide-N-methylene-his-methacrylamide copolymer bearing cellobiamine. The purified enzyme was a dimer with an isoelectric point of 4.0. The molecular mass of the enzyme was estimated to be 240 kDa by gel-permeation chromatography. The enzyme hydrolyzed specificically P-glucosidic bonds and catalyzed transglucosylation of the p-glucosyl group of cellobiose to yield 4-0-j-gentiobiosylglucose in the presence of organic solvents or under neutral conditions. The fungus Aspergillus niger excretes a wide range of carbohydrate-hydrolyzing enzymes. Various techniques, including size-exclusion, hydroxyapatite and ion-exchange chromatography, have been applied to the fractionation and purification of each enzyme from the multicomponent enzyme system. However, only limited success has been achieved with the purification of /Y-glucosidase from culture filtrates [l -71. So far, it has been thought that the substrate specificity of A . niger j-glucosidase was not strict for the fl-D-gluco configuration. For Instance, Tavobilov reported that p-glucosidase from A . niger hydrolyzed not only P-D-glucosides, but also j-D-xylosides, P-D-galactosides and r-L-arabinosides [l]. Later, McCleary et al. purified A . niger j-glucosidase 66.7-fold by gel-filtration and ion-exchange chromatography and reported that the enzyme hydrolyzed P-glucosidic bonds almost specifically but contaminating 8-xylosidase and /Igalactosidase activities still remained in the enzyme preparation [2]. Thus, the substrate specificity of A. niger p-glucosidase has differed depending on the extent of purification. Regarding the transglycosylation of this enzyme, Barker et al. reported that culture filtrates of A . niger catalyzed transglycosylation of the /I-glucosyl group of cellobiose to another cellobiose to form trisacchandes, and the principal linkages synthesized were Pl-6. However, Pl-2, j l -3 and pl-4 linkages were also formed, suggesting that j-glucosidase catalyzed the transglucosylation of p-glucosyl group [8]. However, catalytic activities of the purified enzyme have not been reported. In this study, we aimed at characterizing the substrate specificity and transglycosilation of A . niger P-glucosidase using a highly purified enzyme. We have, therefore, developed an affinity chromatography on a methacrylamide-N-methylene-bismethacrylamide copolymer bearing cellobiamine which interacts with subsites of P-glucosidases. Purification and properties of the P-glucosidase are reported here.Correspondence to T. Watanabe, Wood Research Institute, Kyoto University, Gokasho, Uji, Kyoto, Japan, 611Ahhreviutions. pNpp, p-nitrophenyl; CH,CN, acetonitrile; MeOH, methyl alcohol; EtOH, ethyl alcohol; Me2S0, dimethglsulroxide; DMF, N,N-dimethylformamide.Enzyme. &glucosidase (EC 3.2.1.21).
MATERIALS AND METHODS
MaterialsSephadex G-75 and PBE-94 were obtained from Pharmacia. SP-Toyopearl was obtained from Toso. Cellulosin AC-40 was obtained from Ued...