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

• DOI: 10.1128/aem.60.1.64-70.1994
• Publication Date: 2020-01-01T04:52:25Z
• Authors: H Chen; X Li; L G Ljungdahl
• Keywords: Chromatofocusing; Ammonium sulfate precipitation

An extracellular beta-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 and Superose 12 gel filtration. The enzyme is monomeric with a M(r) 85,400, as estimated sodium dodecyl (SDS)-polyacrylamide electrophoresis, has pI 3.95, contains about 8.5% carbohydrate. N terminus appears to be blocked. catalyzes hydrolysis cellobiose p-nitrophenyl-beta-D-glucoside (PNPG). Km Vmax values substrate at pH 6.0 40 degrees C are 0.25 mM 27.1 mumol.min-1 x mg-1, respectively; PNPG substrate, corresponding 0.35 27.7 mg-1. Glucose (Ki = 8.75 mM, substrate) gluconolactone 1.68 10(-2) 2.57 substrates, respectively) competitive inhibitors. Optimal activity substrates 6.2 50 C. high against sophorose (beta-1,2-glucobiose) laminaribiose (beta-1,3-glucobiose) but no gentiobiose (beta-1,6-glucobiose). stimulated Mg2+, Mn2+, Co2+, Ni2+ inhibited Ag+, Fe2+, Cu2+, Hg2+, SDS, p-chloromercuribenzoate.