International Conference and Workshop on Basic and Applied Sciences ISBN: 978-979-16649-0-5 UNAIR-RuG-KNAW-UTM 2007 An NAD + , Mn 2+ and DTT-dependent α-galactosidase from Bacillus halodurans Andian Ari Anggraeni 1 , Makiko Sakka 2 , Tetsuya Kimura 2 , Motomitsu Kitaoka 3 , and Kazuo Sakka 2 ( 1 State University of Yogyakarta, 2 Mie University Japan , 3 National Food Research Institute Japan ) Author for correspondence, email: sakka@bio.mie-u.ac.jp 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 37 o C in E. coli. In order to reduce the expression level, cultivation temperature was decreased to 20 o C 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 2+ for activity. Co 2+ and Ni 2+ also activated Mel4A, albeit less efficiently than Mn 2+ . 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 37 o C 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. INTRODUCTION α-Galactosidases (α-Gal; α-D-galactoside galactohydrolase; melibiase; EC 3.2.1.22) are enzymes that catalyze hydrolysis of the α-1,6- galactosidic linkages from the non-reducing end in galactose-containing oligosaccharides, lipo saccha- rides and/or polysaccharides. α-Gals widely occur in bacteria (5, 6), filamentous fungals (3), yeasts (11), plants (2), animals (7) and human (1). Some of them have been purified and characterized. Based on the amino acid sequence similarity, α-Gals have been classified under the families 4, 27, 36 and 57 of glycoside hydrolases (4). Almost all of the eukaryotic α-Gals showed a significant degree of amino acid sequence homology and they have been assigned to family 27. To date, some bacterial α-Gals have been characterized and grouped mainly into families 4, 36 and 57. Galactose is the constituent of oligosaccharides (melibiose, raffinose, and stachyose) and polysaccharides such as galactomannan. These oligosaccharides are present in beans and mushrooms. Because monogastric animal and human body lack of α-galactosidase, these oligosaccharides end up in the large intestine undigested and cause flatulence. The use of microbial α-galactosidase treatment prior consumption has been proposed to prevent flatulence. α-Galactosidase have been used in soymilk production to reduce the content of undigested oligosaccharides. In the sugar beet industry, α- galactosidases have been used to increase the sucrose yield by eliminating raffinose, which prevents normal crystallization of beet sugar. Bacillus halodurans C-125 was isolated in 1977 and reported as a β-galactosidase and xylanase producer. B. halodurans is a haloalkaliphilic bacteria which grow well in the pH range of 6.8 – 10.8. It is the strain most thoroughly characterized, physiologically, biochemically and genetically along with B. subtilis. This bacterium produces extracellular enzymes of industrial interest: protease, pectinase, amylase, xylanase. Extracellular enzymes produced by B. halodurans show relatively high optimum temperatures (50-70 o C) and alkaline pH optima (8). The complete genome sequence of B. halodurans has been determined (9) and it revealed the presence of three putative α-galactosidase genes; BH2228 belonging to glycoside hydrolases family 4, BH1870 belonging to family 27 and BH2223 173