Purification and Characterization of the Quinoproteind-Glucose Dehydrogenase Apoenzyme fromEscherichia coli

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Abstract
Purification and characterization of the quinoprotein d-glucose dehydrogenase (EC 1.1.99.17) from Escherichia coli K-12 were carried out, and the purified enzyme was obtained as the apo-form. The enzyme was purified 1,400-fold with an overall yield of 28% from the membrane fraction of the organism by a procedure involving solubilization of the enzyme with Triton X-100, ion exchange chromatographies and gel filtration. The homogeneity of the enzyme was confirmed by SDS-polyacrylamide gel electrophoresis and analytical ultracentrifugation, and its molecular weight was estimated to be 88,000. The purified enzyme itself had no enzyme activity, but it was markedly activated on the addition of pyrroloquinoline quinone in the presence of magnesium ions. The enzyme showed a broad substrate specificity for various monosaccharides including d-glucose, d-fucose, d-galactose, d-mannose and d-xylose. Oxidation of substrates proceeded rapidly at fairly acidic pH with ubiquinone, which may be the intrinsic electron acceptor of the organism. EDTA, p-benzoquinone, citrate and semicarbazide showed inhibitory effects on the enzyme activity. The stability as to pH and heating was increased when the enzyme was converted to the holo-enzyme.

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