Isolation and properties of an extracellular β-glucosidase from the polycentric rumen fungus Orpinomyces sp. strain PC-2

Abstract

An extracellular β-glucosidase (EC 3.2.1.21) was purified from culture filtrate of the anaerobic rumen fungus Orpinomyces sp. strain PC-2 grown on 0.3% (wt vol-1) Avicel by using Q Sepharose anion-exchange chromatography, ammonium sulfate precipitation, chromatofocusing ion-exchange chromatography, and Superose 12 gel filtration. The enzyme is monomeric with a M(r) of 85,400, as estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, has a pI of 3.95, and contains about 8.5% (wt vol-1) carbohydrate. The N terminus appears to be blocked. The enzyme catalyzes the hydrolysis of cellobiose and p-nitrophenyl-β-D-glucoside (PNPG). The K(m) and V(max) values with cellobiose as the substrate at pH 6.0 and 40°C are 0.25 mM and 27.1 μmol · min-1 · mg-1, respectively; with PNPG as the substrate, the corresponding values are 0.35 mM and 27.7 μmol · min-1 · mg-1. Glucose (K(i) = 8.75 mM, with PNPG as the substrate) and gluconolactone (K(i) = 1.68 x 10-2 and 2.57 mM, with PNPG and cellobiose as the substrates, respectively) are competitive inhibitors. Optimal activity with PNPG and cellobiose as the substrates is at pH 6.2 and 50°C. The enzyme has high activity against sophorose (β-1,2-glucobiose) and laminaribiose (β-1,3-glucobiose) but has no activity against gentiobiose (β-1,6- glucobiose). The activity of the β-glucosidase is stimulated by Mg2+, Mn2+, Co2+, and Ni2+ and inhibited by Ag+, Fe2+, Cu2+, Hg2+, SDS, and p-chloromercuribenzoate.