The Sulfolobus tokodaii gene ST1704 codes highly thermostable glucose dehydrogenase

NAD(P)-dependent glucose-1-dehydrogenase (GDH) has been used for glucose determination and NAD(P)H production in bioreactors. Thermostable glucose dehydrogenase exhibits potential advantage for its application in biological processes. The function of the putative GDH gene (ST1704, 360-encoding amino acids) annotated from the total genome analysis of a thermoacidophilic archeaon Sulfolobus tokodaii strain 7 was investigated to develop more effective application of GDH. The gene encoding S. tokodaii GDH was cloned and the activity was expressed in Escherichia coli, which did not originally possess GDH. This shows that the gene (ST1704) codes the sequence of GDH. The enzyme was effectively purified from the recombinant E. coli with three steps containing a heat treatment and two successive chromatographies. The native enzyme (molecular mass: 160 kDa) is composed of a tetrameric structure with a type of subunit (41 kDa). The enzyme utilized both NAD and NADP as the coenzyme. The maximum activity for glucose oxidation in the presence of NAD was observed around pH 9 and 75 degreesC in the presence of 20 mM Mg2+. The enzyme showed broad substrate specificity: several monosaccarides such as 6-deoxy-D-glucose, 2-amino-2-deoxy-D-glucose and D-Xylose were oxidized as well as D-glucose as the electron donor. D-Mannose, D-ribose and glucose-6-phosphate were inert as the donor. The enzyme showed high thermostability: remarkable loss of activity was not observed up to 80 degreesC by incubation for 15 min at pH 8.0. In addition, the enzyme was stable in a wide pH range of 5.0-10.5 by incubation at 37 degreesC. From the steady-state kinetic analysis, the enzyme reaction of D-glucose oxidation proceeds via a sequential ordered Bi-Bi mechanism: NAD and D-glucose bind to the enzyme in this order and then D-glucono-1,5-lactone and NADH are released from the enzyme in this order. The amino acid sequence alignment showed that S. tokodaii GDH exhibited high homology with the Sulfolobus solfataricus hypothetical glucose dehydrogenase and a Thermoplasma acidophilum one. (C) 2003 Elsevier B.V. All rights reserved.