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Purification and preliminary characterisation of β-glucosidase from Alcaligenes faecalis (ATCC 21400) Day, Anthony George


A β-glucosidase was isolated from A. faecalis and purified 880 fold by a combination of classical and medium pressure chromatographic techniques to a specific activity of 31.6 units/mg. The protein was homogeneous by the criteria of SDS-PAGE and gel chromatography. The sub-unit molecular weight was determined to be 51,000 by SDS-PAGE. The apparent oligomeric molecular weight was determined to be 75,000 by Superose gel chromatography and 98,000 by Waters 1-250 gel chromatography, suggesting that the enzyme is a dimer. The enzyme was shown to be a retaining β-glucosidase with exo-glucanase activity only. The kinetic parameters of a number of substrates and inhibitors were determined allowing deductions to be made about the nature of the active site and catalytic mechanism. The Km's determined for cellobiose and PNPG were low for a bacterial β-glucosidase, 0.70 mM and 0.083 mM respectively. In the cellodextrin series, cellobiose to cellopentaose, the enzyme was most efficient (as defined by Vm/Km) with cellotriose as substrate. In common with other cellulolytic β-glucosidases, the glycone site showed a high specificity for glucose (although it would tolerate some modifications) and poor specificity at the aglycone site. Catalytic activity was (unusually) observed with p-nitrophenyl-β-D-mannopyranoside as substrate. Activation energies were determined by means of Arrhenius plots.

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