title: Characterization and site-directed mutagenesis of an α-galactosidase from the deep-sea bacterium Bacillus megaterium.
abstract: A novel gene (BmelA) (1323bp) 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 (k(cat)/K(m)) 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.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Haibo
authors.initials: H
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Yongjun
authors.initials: Y
authors.lastname: Qin
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Zongqing
authors.initials: Z
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China. Electronic address: lzd@mail.hzau.edu.cn.
authors.forename: Ziduo
authors.initials: Z
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D001410:Bacillus megaterium / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D016680:Genome, Bacterial; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D000519:alpha-Galactosidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D009243:NAD; D000519:alpha-Galactosidase
keywords: Glycoside hydrolase family 4; NAD(+) binding; Site-directed mutagenesis; α-Galactosidase