Characterization and site-directed mutagenesis of an α-galactosidase from the deep-sea bacterium Bacillus megaterium

A novel gene (BmelA) (1323 bp) encoding an alpha-galactosidase of 440 amino acids was cloned from the deep-sea bacterium Bacillus megaterium and the protein was expressed in Escherichia coli BL21 (DE3) with an estimated molecular mass of about 45 kDa by SDS-PAGE. The enzyme belongs to glycoside hydrolase family 4, with the highest identity (74%) to alpha-galactosiclase Mel4A from Bacillus halodurans among the characterized alpha-galactosidases. The recombinant BmelA displayed its maximum activity at 35 C and pH 8.5-9.0 in 50 mM Tris-HCI buffer, and could hydrolyze different substrates with the K values against p-n, nitrophenyl-alpha-D-galactopyranoside (pNP-alpha-Gal), raffinose and stachyose being 1.02 +/- 0.02, 2.24 +/- 0.11 and 3.42 +/- 0.17 mM, respectively. Besides, 4 mutants (I38 V, I38A, I38F and Q84A) were obtained by site-directed mutagenesis based on molecular modeling and sequence alignment. The kinetic analysis indicated that mutants I38 V and I38A exhibited a 1.7- and 1.4-fold increase over the wild type enzyme in catalytic efficiency (k(cat)/K-m,) against pNP-alpha-Gal, respectively, while mutant I38F showed a 3.5-fold decrease against pNP-alpha-Gal and mutant Q84A almost completely lost its activity. All the results suggest that I38 and Q84 sites play a vital role in enzyme activity probably due to their steric and polar effects on the predicted tunnel structure and NAD(+) binding to the enzyme. (c) 2014 Elsevier Inc. All rights reserved.