Purification and biochemical characteristics of β‐D‐glucosidase from a thermophilic fungus, Thermomyces lanuginosus–SSBP

Abstract

The β‐D‐glucosidase produced by Thermomyces lanuginosus‐SSBP was purified to apparent homogeneity. The purified enzyme consisted of two identical subunits with a native molecular mass of 200 kDa. The purified β‐D‐glucosidase only hydrolysed the glucoside substrates containing a terminal, non‐reducing β‐D‐glucose residue and was active on both aryl‐β‐glucoside and cellobiose. This enzyme also exhibited less, but significant α‐D‐glucosidase activity and was capable of hydrolysing β‐1,6‐linked diglucosides and gentiobiose. The K appm, Vmax and kcat values for p‐nitrophenyl‐β‐D‐glucopyranoside were calculated to be 0.075 mM, 12.12 units/mg of protein and 44.44 glucose molecules released/s respectively. The β‐D‐glucosidase retained its full activity after a 30 min incubation at 50 °C but was inactive after the same treatment at 70 °C. The enzyme appeared to be stable when the pH of the storage buffer was above 5.0. Maximal β‐D‐glucosidase activity occurred at 65 °C and pH 6.0. This enzyme was competitively inhibited by glucose, cellobiose and salicin with Ki values of 0.55, 0.52 and 0.81 mM respectively. The presence of Hg2+ and N‐bromosuccinimide inhibited the enzyme activity completely at 2 mM, while cysteine enhanced β‐D‐glucosidase activity.