Purification and Characterization of the RecombinantThermussp. Strain T2 α-Galactosidase Expressed inEscherichia coli

Abstract

The nucleotide sequence of the Thermus sp. strain T2 DNA coding for a thermostable ␣-galactosidase was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 474 amino acids (M r , 53,514). The observed homology between the deduced amino acid sequences of the enzyme and ␣-galactosidase from Thermus brockianus was over 70%. Thermus sp. strain T2 ␣-galactosidase was expressed in its active form in Escherichia coli and purified. Native polyacrylamide gel electrophoresis and gel filtration chromatography data suggest that the enzyme is octameric. The enzyme was most active at 75°C for p-nitrophenyl-␣-Dgalactopyranoside hydrolysis, and it retained 50% of its initial activity after 1 h of incubation at 70°C. The enzyme was extremely stable over a broad range of pH (pH 6 to 13) after treatment at 40°C for 1 h. The enzyme acted on the terminal ␣-galactosyl residue, not on the side chain residue, of the galactomanno-oligosaccharides as well as those of yeasts and Mortierella vinacea ␣-galactosidase I. The enzyme has only one Cys residue in the molecule. para-Chloromercuribenzoic acid completely inhibited the enzyme but did not affect the mutant enzyme which contained Ala instead of Cys, indicating that this Cys residue is not responsible for its catalytic function.␣-Galactosidases (␣-Gals) are known to occur widely in microorganisms, plants, and animals, and some of them have been purified and characterized (5). ␣-Gals catalyze the hydrolysis of 1,6-linked ␣-galactose residues from oligosaccharides and polymeric galactomannans (19,27,28). In the sugar beet industry, ␣-Gals have been used to increase the sucrose yield by eliminating raffinose, which prevents the crystallization of beet sugar (31). Raffinose and stachyose in beans are known to cause flatulence. ␣-Gal has the potential to alleviate these symptoms, for instance, in the treatment of soybean milk (6).We have studied the substrate specificity of ␣-Gals from eukaryotes by using galactomanno-oligosaccharides, such as 6 3 -mono-␣-D-galactopyranosyl-␤-1,4-mannotriose (Gal 3 Man 3 ) and 63 -mono-␣-D-galactopyranosyl-␤-1,4-mannotetraose (Gal 3 Man 4 ). The structures of these galactomanno-oligosaccharides are shown in Fig. 1. Mortierella vinacea ␣-Gal I (11) and yeast ␣-Gals (32) are specific for Gal 3 Man 3, having an ␣-galactosyl residue (designated the terminal ␣-galactosyl residue) attached to the O-to-6 position of the nonreducing end mannose of ␤-1,4-mannotriose. On the other hand, Aspergillus niger 5-16 ␣-Gal (12) and Penicillium purpurogenum ␣-Gal (27) show a preference for Gal 3 Man 4 , having an ␣-galactosyl residue (designated as the side chain ␣-galactosyl residue) attached to the O-to-6 position of the third mannose from the reducing end of ␤-1,4-mannotetraose. The M. vinacea ␣-Gal II (28) acts on both substrates to almost equal extents. These facts indicate that eukaryotic ␣-Gals were classified into three groups based on the substrate specificity of these galactomanno-oligosaccharides.Genes encoding ␣-Gals have been cloned ...

Cloning of a Novel Pyrethroid-Hydrolyzing Carboxylesterase Gene from Sphingobium sp. Strain JZ-1 and Characterization of the Gene Product

“…A similar result was reported for the ␣-galactosidase from Thermus sp. strain T2, which also had only one Cys residue (8).…”

The Tetramer Structure of the Glycoside Hydrolase Family 27 α-Galactosidase I fromUmbelopsis vinacea

“…[7][8][9] We have purified -galactosidases from several species and have determined the substrate specificities of each of them. [10][11][12][13] Umbelopsis vinacea (filamentous fungi, earlier we used the synonym Mortierella vinacea) possesses two types of -galactosidases, and one of which, -galactosidase I (UvGalI), has a molecular mass of 50-56 kDa and 240 kDa when analyzed by SDS-PAGE and gel filtration respectively. The broadness of the SDS-PAGE band suggests that UvGalI might be a heterogeneous glycoprotein.…”

Cloning and expression of the gene encoding Streptomyces coelicolor A3(2) α-galactosidase belonging to family 36

“…The sequence identities of S. coelicolor a-Gal with the enzymes from Thermus thermophilius (Fridjonsson et al 1999b), Thermus sp. strain T2 (Ishiguro et al 2001) and Thermotoga maritima (Liebl et al 1998) were 44, 43, and 41%, respectively (Figure 1). The amino acid sequence of S. coelicolor a-Gal is more homologous to those of thermophilic organisms than those of mesophile Lactobacillus plantarum and Pediococcus pentosaceus.…”