Raffinose, Sucrose and Inulin Hydrolysis by Enzymes of the Hyperthermophilic Bacterium Thermotoga maritima
Wolfgang LIEBL*
Department of Microbiology, Technical University Muenchen, Arcisstr. 21, D-80290 Muenchen, Germany
More than 50 different species of hyperthermophilic procaryotes (those with an optimal growth temperature of at least 80℃) have been described (1). While most of the currently known hyperthermophiles are Archaea, only a few of these organisms belong to the domain of Bacteria. Thermotoga maritima, a heterotroph with a maximum growth temperature of 90℃, in the last few years has become a model organism for the study of hyperthermophilic bacteria. T. maritima, strain MSB8 (DSM 3109), is able to utilize various oligo- and polysaccharides, and some of the enzymes involved in their breakdown have been characterized (2-4). Recently we observed that T. maritima has two enzymes that hydrolyse raffinose [α-D- galActopyranosyl-( 1→6)-α-D-glucopyranosyl-( 1→2)- β-D-fructofuranose], a trisaccharide widely found in nature, i.e. anα-galactosidase and aβ-fructosidase. Presently very little information is available concerning the occurrence, properties, and primary structures of these types of enzymes of hyperthermophiles, even though they may have considerable biotechnological potential. We have now cloned the genes for anα-galactosidase and aβ-fructosidase of T. maritima. The genes were expressed in E. coli and the extremely thermostable enzymes were purified and characterised. The β-fructosidase gene, bfrA, codes for a 432 residue, about 50 kDa polypeptide. The enzyme released fructose from sucrose and raffinose. Interestingly, the fructose polymer inulin was hydrolysed quantitatively in an exo-type fashion. BfrA displayed similar catalytic efficiencies for the hydrolysis of sucrose and inulin. BfrA is the most thermostableβ-fructosidase and also the most thermostable inulinase described to date. The T. maritima MSB8 gene forα-galactosidase, designated galA, has coding capacity for a 552 residue polypeptide with a molecular mass of 63 kDa. The gala gene is flanked by other genes probably involved in galactoside breakdown and utilization. The identified genes could all be part of one galactoside utilisation operon. The recombinantα-galactosidase released galactose from raffinose, melibiose, and the synthetic substrates pNP- and oNP-α-D-galactoside. The enzyme thus was highly specific for the galactose moiety and theα-anomeric configuration of the glycosidic linkage. Both BfrA and GalA were extremely insensitive to thermoinactivation. The most interesting results of the sequence analyses and of the biochemical and kinetic characterisation of the hydrolytic enzymes will be presented.
1. Stetter, K. O. (1996) FEMS Microbiol. Rev., 18, 149-158.
2. Winterhalter, C., Heinrich, P., Candussio, A., Wich, G., and Liebl, W. (1995) Mol. Microbiol., 15, 431-444.
3. Liebl, W., Stemplinger, I., and Ruile, P. (1997) J. Bacteriol., 179, 941-948.
4. Ruile, P., Winterhalter, C., and Liebl, W. (1997) Mol. Microbiol., 23, 267-279.
