PRODUCTION, PURIFICATION AND CHARACTERIZATION OF GLUCOSE-OXIDASE FROMA NEWLY ISOLATED STRAIN OF PENICILLIUM-PINOPHILUM

A number of nutritional factors influencing growth and glucose oxidase (EC 1.1.3.4) production by a newly isolated strain of Penicillium pin ophilum were investigated. The most important factors for glucose oxid ase production were the use of sucrose as the carbon sourer, and growt h of the fungus at non-optimal pH 6.5. The enzyme was purified to appa rent homogeneity with a yield of 74%, including an efficient extractio n step of the mycelium mass at pH 3.0, cation-exchange chromatography and gel filtration. The relative molecular mass (M-r) of native glucos e oxidase was determined to be 154 700 +/- 4970, and 77 700 for the de natured subunit. Electron-microscopic er;aminations revealed a sandwic h-shaped dimeric molecule with subunit dimensions of 5.0 x 8.0 nm, Glu cose oxidase is a glycoprotein that contains tightly bound FAD with an estimated stoichiometry of 1.76 mol/mol enzyme, The enzyme is specifi c for D-glucose, for which a K-m value of 6.2 mM was determined. The p H optimum was determined in the range pH 4.0-6.0. Glucose oxidase show ed high stability on storage in sodium citrate (pH 5.0) and in potassi um phosphate (pH 6.0), each 100 mM. The half-life of the activity was considerably more than 305 days at 4 degrees C and 30 degrees C, and 2 13 days at 40 degrees C. The enzyme was unstable at temperatures above 40 degrees C in the range pH 2.0-4.0 and at a pH above 7.0.