Analysis of the gene for beta-fructosidase (invertase, inulinase) of the hyperthermophilic bacterium Thermotoga maritima, and characterisation of the enzyme expressed in Escherichia coli.
This is the first report describing the gene structure and the enzymatic properties of a beta-fructosidase of a hyperthermophilic organism. The bfrA gene of the ancestral bacterium Thermotoga maritima MSB8 codes for a 432-residue, polypeptide of about 50 kDa, with significant sequence similarity to other beta-fructosidases. On the basis of its primary structure, BfrA can be assigned to glycosyl hydrolase family 32. The bfrA gene was expressed in Escherichia coli and the recombinant enzyme was purified and characterised. BfrA was specific for the fructose moiety and the beta-anomeric configuration of the glycosidic linkages of its substrates. The enzyme released fructose from sucrose and raffinose, and the fructose polymer inulin was hydrolysed quantitatively in an exo-type fashion. BfrA displayed similar catalytic efficiencies for the hydrolysis of sucrose and inulin with Kcat/K(m) values (at 75 degrees C, pH 5.5) of about 4.1 x 10(4) M-1S-1 and 3.1 x 10(4) M-1S-1 respectively. BfrA had an optimum temperature of 90-95 degrees C (10-min assay) and was extremely insensitive to thermo-inactivation. During 5 h at temperatures up to 80 degrees C at pH 7, the enzyme retained at least 85% of its initial activity. Thus, BfrA is the most thermostable beta-fructosidase and also the most thermostable inulinase described to date. In conclusion, the T. maritima enzyme can be classified as an exo-beta-D-fructofuranosidase (EC 3.2.1.26) with invertase and inulinase activity. Its catalytic properties along with the extreme thermostability recommend it for use in biotechnology.
Liebl
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Institut für Mikrobiologie und Genetik, Georg-August-Universität, Göttingen, Germany.
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Gotschlich
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D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000032:analysis; D001483:Base Sequence; D015139:Blotting, Southern; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007444:Inulin / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013395:Sucrose / Q000737:chemistry; D043324:beta-Fructofuranosidase
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D013395:Sucrose; D007444:Inulin; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase