Isolation and properties of an extracellular beta-glucosidase from the polycentric rumen fungus Orpinomyces sp. strain PC-2.
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 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-beta-D-glucoside (PNPG). The Km and Vmax values with cellobiose as the substrate at pH 6.0 and 40 degrees C are 0.25 mM and 27.1 mumol.min-1 x mg-1, respectively; with PNPG as the substrate, the corresponding values are of 0.35 mM and 27.7 mumol.min-1 x mg-1. Glucose (Ki = 8.75 mM, with PNPG as the substrate) and gluconolactone (Ki = 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 degrees C. The enzyme has high activity against sophorose (beta-1,2-glucobiose) and laminaribiose (beta-1,3-glucobiose) but has no activity against gentiobiose (beta-1,6-glucobiose). The activity of the beta-glucosidase is stimulated by Mg2+, Mn2+, Co2+, and Ni2+ and inhibited by Ag+, Fe2+, Cu2+, Hg2+, SDS, and p-chloromercuribenzoate.
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Center for Biological Resource Recovery, University of Georgia, Athens 30602.
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D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002417:Cattle; D002475:Cellobiose / Q000378:metabolism; D004187:Disaccharides / Q000378:metabolism; D005110:Extracellular Space / Q000201:enzymology; D005658:Fungi / Q000201:enzymology*; D005936:Glucans / Q000378:metabolism; D005960:Glucosides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D012417:Rumen / Q000382:microbiology; D013379:Substrate Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D002241:Carbohydrates; D004187:Disaccharides; D005936:Glucans; D005960:Glucosides; D008670:Metals; C014637:laminaribiose; D002475:Cellobiose; C025193:4-nitrophenyl beta-D-glucoside; D001617:beta-Glucosidase; C003328:sophorose