A gene having high sequence homology (45-49%) with the glycerol-1-phosphate dehydrogenase gene from Methanobacterium thermoautotrophicum was cloned from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820). This gene expressed in Escherichia coli with the pET vector system consists of 1113 nucleotides with an ATG initiation codon and a TAG termination codon. The molecular mass of the purified enzyme was estimated to be 38 kDa by SDS/PAGE and 72.4 kDa by gel column chromatography, indicating presence as a dimer. The optimum reaction temperature of this enzyme was observed to be 94-96°C at near neutral pH. This enzyme was subjected to two-substrate kinetic analysis. The enzyme showed substrate specificity for NAD(P)Hdependent dihydroxyacetone phosphate reduction and NAD + -dependent glycerol-1-phosphate (Gro1P) oxidation. NADP + -dependent Gro1P oxidation was not observed with this enzyme. For the production of Gro1P in A. pernix cells, NADPH is the preferred coenzyme rather than NADH. Gro1P acted as a noncompetitive inhibitor against dihydroxyacetone phosphate and NAD(P)H. However, NAD(P) + acted as a competitive inhibitor against NAD(P)H and as a noncompetitive inhibitor against dihydroxyacetone phosphate. This kinetic data indicates that the catalytic reaction by glycerol-1-phosphate dehydrogenase from A. pernix follows a ordered bi-bi mechanism.

Tofu containing both low and high viscosity chitosan was prepared and changes in the microbiological, physicochemical and sensory properties of the tofu during storage were investigated. The colony forming units of mesophilic and psychrotrophic microorganisms in tofu containing high viscosity chitosan were markedly lower during storage than those in the control tofu as well as the tofu containing low viscosity chitosan. The pH of the tofu samples increased during storage. The L and a values of the tofu, especially the control tofu, increased during storage. In the sensory test, the roasted nutty and beany aromas of the tofu decreased during storage. Instrumental analysis of hardness and chewiness of the tofu decreased during storage. Overall preference for the tofu gradually decreased during storage, but overall preference for the tofu containing high viscosity chitosan scored higher than the other tofu samples.

The enzyme sn-glycerol-1-phosphate dehydrogenase (Gro1PDH, EC 1.1.1.261) is key to the formation of the enantiomeric configuration of the glycerophosphate backbone (sn-glycerol-1-phosphate) of archaeal ether lipids. This enzyme catalyzes the reversible conversion between dihydroxyacetone phosphate and glycerol-1-phosphate. To date, no information about the active site and catalytic mechanism of this enzyme has been reported. Using the sequence and structural information for glycerol dehydrogenase, we constructed six mutants (D144N, D144A, D191N, H271A, H287A and D191N/H271A) of Gro1PDH from Aeropyrum pernix K1 and examined their characteristics to clarify the active site of this enzyme. The enzyme was found to be a zinc-dependent metalloenzyme, containing one zinc ion for every monomer protein that was essential for activity. Site-directed mutagenesis of D144 increased the activity of the enzyme. Mutants D144N and D144A exhibited low affinity for the substrates and higher activity than the wild type, but their affinity for the zinc ion was the same as that of the wild type. Mutants D191N, H271A and H287A had a low affinity for the zinc ion and a low activity compared with the wild type. The double mutation, D191N/H271A, had no enzyme activity and bound no zinc. From these results, it was clarified that residues D191, H271 and H287 participate in the catalytic activity of the enzyme by binding the zinc ion, and that D144 has an effect on substrate binding. The structure of the active site of Gro1PDH from A. pernix K1 seems to be similar to that of glycerol dehydrogenase, despite the differences in substrate specificity and biological role.

Chitinouspolymers have been experimented with for thepulpose of shelf-life extension of foods due to their antimicrobial activity. Food additives, however, may impair the taste, color or texture of foods. Therefore, it is necessary to evaluate the f l e e t of a food additive on foods before it is used. In this study, we investigated how the physicochemical properties, microstmcture, textural properties and sensory characteristics of tofu are aflected by the addition of chitooligosaccharide during its preparation. The addition of chitooligosaccharide to tofu did not signi3cantly aflect its physicochemical properties including moisture content, yield, turbidity and color. m e chitooligosaccharide tofu, however, had lower hardness and smaller protein aggregates than the control tofu. The chitooligosaccharide did not influence most sensory attributes of tofu except for imparting a bitter taste.

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