Purification, characterization and mechanistic study of beta-glucosidase from Flavobacterium meningosepticum (ATCC 13253)

Abstract

A beta-glucosidase (EC 3.2.1.21) from Flavobacterium meningosepticum has been purified and characterized. Purity was enhanced at least 530-fold from crude cell extract with 16.6% yield. The estimated molecular mass of the purified enzyme is 150 kDa by gel filtration and 78 kDa by SDS-PAGE. This dimeric enzyme has a pI=9.0 and an optimal activity at pH 5.0 and temperature of 50 degrees C. Divalent metal ions (Hg2+ CU2+, Ca2+, Mg2+) and EDTA have negligible effect on the enzyme activity. The enzyme exhibited a high specificity on the glycon portion of aryl-P-D-glycosides. NMR spectroscopy revealed the enzyme catalyzed hydrolysis of p-nitrophenyl-beta-D-glucopyranoside with the retention of anomeric configuration, indicating that a double displacement mechanism was involved. A preliminary study of the Bronsted relationship showed a concave-downward plot, which is consistent with the two-step mechanism.