Properties of an α-galactosidase, and Structure of its Gene galA, within an α- and β-galactoside Utilization Gene Cluster of the Hyperthermophilic Bacterium Thermotoga maritima

Thermotoga maritima represents one of the few hyperthermophilic bacteria currently known. The chromosomal alpha-galactosidase gene of T. maritima strain MSB8 has been cloned and its nucleotide sequence was determined. The gene, designated galA, has coding capacity for a 552 residue polypeptide with a calculated molecular mass of 63,653 Da. GalA was found to be flanked by other genes probably involved in galactoside breakdown and utilization. The previously sequenced beta-galactosidase gene, lacZ, is localized immediately upstream of galA while two open reading frames that putatively encode enzymes of galactose catabolism, i.e. galactose-1-phosphate uridylytransferase (galT) and galactokinase (galK), were found downstream of galA. The identified genes are extremely close together or even overlap and have the same orientation, so they could all be part of one galactoside utilization operon of T. maritima MSB8. GalA displayed low-level amino acid sequence similarity with alpha-galactoside of glycosyl hydrolase family 36. However, GalA is smaller than the other members of this enzyme family. The galA gene was expressed in Escherichia coli and the recombinant alpha-galactosidase was purified and characterized. The molecular mass of the recombinant enzyme was estimated at about 62 kDa by denaturting gel electrophoresis. Maximal hydrolysis of the chromogenic substrate p-nitrophenyl-alpha-D-galactopyranoside was measured at pH 5.0-5.5 and 90-95 degrees C (5 min assay). Divalent cations were not required for activity. The enzyme released galactose from raffinose, melibiose and the synthetic substrates p-nitrophenyl-and omicron-nitrophenyl-alpha-D-galactopyranoside. The T. maritima alpha-galactosidase thus was highly specific for the galactose moiety and the alpha-anomeric configuration of the glycosidic linkage. Its extreme thermal stability (t 1/2 = 6.5 h at 85 degrees C) makes this enzyme an interesting candidate for biotechnological applications.