An NAD + , Mn 2+ and DTT-dependent α-galactosidase from Bacillus halodurans

TLDR: The α-galactosidase Mel4A gene of Bacillus halodurans was recombinantly expressed in Escherichia coli, purified and characterized, and it was found that this enzyme preferred small saccharides to highly polymerized galactomannans.

Abstract: The α-galactosidase mel4A (previously called melA) gene of Bacillus halodurans was recombinantly expressed in Escherichia coli, purified and characterized. The mel4A gene consists of 1305 nucleotides encoding a protein of 434 amino acids with a predicted molecular weight of 49,761. According to its primary structure as deduced from the nucleotide sequence of the gene, Mel4A was assigned to family 4 of glycoside hydrolases. Almost all of the enzyme was produced as inclusion bodies at 37C in E. coli. In order to reduce the expression level, cultivation temperature was decreased to 20C so that the enzyme could be collected from soluble fraction. Recombinant α-galactosidase Mel4A was purified to homogeneity in a single step using His-binding metal affinity chromatography. B. halodurans Mel4A has the unusual property, i.e., absolutely depending on NAD and Mn for activity. Co and Ni also activated Mel4A, albeit less efficiently than Mn. In addition, Mel4A activity required reducing condition which met by the addition of dithiothreitol (DTT). In the presence of all cofactors, optimum activity was achieved at 37C and pH 7.4. The enzyme hydrolyzed p-nitrophenyl-α-D-galactopyranoside, melibiose, raffinose, and stachyose but not guar gum, indicating that this enzyme preferred small saccharides to highly polymerized galactomannans. Western immunoblots of intracellular and extracellularproteins of B. halodurans revealed that raffinose induced the expression of intracellular Mel4A of B. halodurans. This bacterium was also able to utilize guar gum as the carbon source, but Western blot analysis indicated that the production of Mel4A was not enhanced by the addition of guar gum.