| Abstract |
High-molecular-weight β-glucosidase (EC 3.2.1.21) was purified from the culture filtrate of Trichoderma koningii through a four-step procedure including chromatography on Bio-Gel P-150, DEAE-Sephadex A-50 and SP-Sephadex C-50; and chromatofocusing on Polybuffer exchanger PBE 94. The molecular weight of the enzyme was determined to be about 101,000 by SDS-polyacrylamide gel electrophoreses, and the isoelectric point was estimated to be 4.96 by analytical isoelectric focusing. The temperature optimum for activity was about 55℃, and the pH optimumwas 3.5. The enzyme was considerably thermostable, for no loss of activity was observed when the enzyme was preincubated at 60℃ for 5h. Km values for cellobiose, gentiobiose, sophorose, salicin and p-nitrophenyl-β-D-glucoside were 99.2, 14.7, 7.09, 3.15 and 0.70 mM, respectively, which indicates that the enzyme has much higher affinity towards p-nitrophenyl-β-D-glucoside than towards the other substrates, especially cellobiose. Substrate inhibition by p-nitrophenyl-β-D-glucoside and salicin was observed at the conecntrations exceeding 5mM. Gluconolactone was a powerful inhibitor against the action of the enzyme on p-nitrophenyl-β-D- glucoside(K_i 37.9 uM), wherease glucose was much less effective (K_i 1.95 mM). Inhibition was of the competitive type in each case. Transglucosylation activity was detected shen the readtion products formed from p-nitrophenyl-β-D-glucoside by the enzyme were analysed using high-performance liquid chromatography. |
