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

• A novel gene encoding an α-galactosidase was cloned from deep sea bacterium.
• Detailed enzymatic characteristics of the enzyme were studied.
• Directed evolution of the enzyme was carried out by site-directed mutagenesis.
• Two mutants with improved catalytic efficiency were obtained.

A novel gene (BmelA) (1323 bp) encoding an α-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 α-galactosidase Mel4A from Bacillus halodurans among the characterized α-galactosidases. The recombinant BmelA displayed its maximum activity at 35 °C and pH 8.5–9.0 in 50 mM Tris–HCl buffer, and could hydrolyze different substrates with the Km values against p-nitrophenyl-α-d-galactopyranoside (pNP-α-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 (kcat/Km) against pNP-α-Gal, respectively, while mutant I38F showed a 3.5-fold decrease against pNP-α-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.