Molecular and biochemical characterization of a recombinant glycosyl hydrolase family 3 β-glucosidase overexpressed in Escherichia. coli; bioprospecting metagenomes for cellulolytic processing function

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Molecular Catalysis


Enzyme activity on a synthetic substrate (p-nitrophenyl-β-D-glucopyranoside (pNPG)) ranked by specificity (kcat/Km), placed Bgl-3 (a recombinant P. carotovorum subsp. carotovorum-ß-glucosidase expressed in Escherichia coli) in a group of ß-glucosidases, whereas ranking according to turnover number (kcat) placed Bgl-3 at the top of the group, implying its potential for high activity in biomass processing. The role of Lys211, His212, Arg172, and Asp114, in substrate recognition and stabilization of a glucose unit in catalysis is proposed here due to interactions with the substrate C1-C6 hydroxyls through hydrogen bonding, as well as the role of two methionines, Met255, Met322, in hydrophobic stabilization. However, enzyme inactivation due to ion pair dissociation, at pH range >8 and <5, of the enzyme nucleophile and the acid-base, can influence enzyme-intermediate complex formation, suggesting the latter as rate limiting in Bgl3 catalysis. Asp290 and Glu517 in the substrate binding clefts, are the nucleophile and the catalytic acid/base suggested, respectively. The close proximity of His212 and His522 stabilizes the enzyme glycosyl-intermediate through hydrogen bonding.



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