• Microbiology and Biotechnology Letters
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• v.16 no.5
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• pp.393-397
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• 1988

The adenylate kinase was purified from an extreme thermophile by adenosine-pentaphospho-adenosine elution from phosphocellulose column. The molecular weight was estimated to be 22,000 by SDS-PAGE and gel filtration. The optimum temperature of the enzyme activity was 8$0^{\circ}C$ and the activation energy was given as 22.4 kcal/mole. The enzyme even showed full activity after incubation at 9$0^{\circ}C$ or in 6M guanidine-HCI at 3$0^{\circ}C$ and retained 75% of its original activity even after 1 hour at 10$0^{\circ}C$. The Michaelis constants of the enzymes for AMP, ADP, and ATP were 0.01mM, 0.017mM and 0.067mM, respectively.

• Journal of Microbiology and Biotechnology
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• v.18 no.2
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• pp.287-294
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• 2008

Three amino acid residues (His119, Glu164, and Glu338) in the active site of Thermus caldophilus GK24 ${\beta}$-glycosidase (Tca ${\beta}$-glycosidase), a family 1 glycosyl hydrolase, were mutated by site-directed mutagenesis. To verify the key catalytic residues, Glu164 and Glu338 were changed to Gly and Gln, respectively. The E164G mutation resulted in drastic reductions of both ${\beta}$-galactosidase and ${\beta}$-glucosidase activities, and the E338Q mutation caused complete loss of activity, confirming that the two residues are essential for the reaction process of glycosidic linkage hydrolysis. To investigate the role of His119 in substrate binding and enzyme activity, the residue was substituted with Gly. The H119G mutant showed 53-fold reduced activity on 5mM p-nitrophenyl ${\beta}$-D-galactopyranoside, when compared with the wild type; however, both the wild-type and mutant enzymes showed similar activity on 5mM p-nitrophenyl ${\beta}$-D-glucopyranoside at $75^{\circ}C$. Kinetic analysis with p-nitrophenyl ${\beta}$-D-galactopyranoside revealed that the $k_{cat}$ value of the H119G mutant was 76.3-fold lower than that of the wild type, but the $K_m$ of the mutant was 15.3-fold higher than that of the wild type owing to the much lower affinity of the mutant. Thus, the catalytic efficiency $(k_{cat}/K_m)$ of the mutant decreased to 0.08% to that of the wild type. The $k_{cat}$ value of the H119G mutant for p-nitrophenyl ${\beta}$-D-glucopyranoside was 5.l-fold higher than that of the wild type, but the catalytic efficiency of the mutant was 2.5% of that of the wild type. The H119G mutation gave rise to changes in optima pH (from 5.5-6.5 to 5.5) and temperature (from $90^{\circ}C\;to\;80-85^{\circ}C$). This difference of temperature optima originated in the decrease of H119G's thermostability. These results indicate that His119 is a crucial residue in ${\beta}$-galactosidase and ${\beta}$-glucosidase activities and also influences the enzyme's substrate binding affinity and thermostability.

• The Korea Genome Conference
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• 2003.09a
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• pp.43.1-43.1
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• 2003

• KSBB Journal
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• v.18 no.1
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• pp.8-13
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• 2003

Recombinant trehalose synthase from Thermus caldophilus GK24 showed an ability to produce trehalose from maltose. The activity of the partially purified enzyme was not influenced by most metal ions at 1 mM but was inhibited by 10 mM $Co^{2+}$, $Mn^{2+}$, and $Fe^{2+}$. Enzyme activity varied during prolonged reaction due to changes in the environmental conditions. Thus, the reaction was carried out for an extended time with optimized conditions of $45^{\circ}C$ and pH 7.0. An yield of 32.9% was reached at $60^{\circ}C$ after reaction for 22 h, and, maximum trehalose conversion (69.2%) was attained at $25^{\circ}C$. The yields obtained using enzyme dosages of 10, 25, and 50 U/g were 62.3, 62.3 and 59.0 %, respectively, though the initial conversion rate was higher when the higher dose was used. Similar profiles of trehalose production yields were observed with reaction working volumes of 10 ml to 2,000 ml.

• Microbiology and Biotechnology Letters
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• v.36 no.3
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• pp.182-188
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• 2008

A hyperthermophilic bacteria (strain HJ6) was isolated from a hot springs located in the Arima-cho, Hyogo, Japan. The cells were long-rod type ($2-4{\mu}m$), about $0.4{\mu}m$ in diameter. The pH and temperature for optimal growth were 6.5 and $80^{\circ}C$, respectively. Phylogenetic analysis based on the 16S rDNA sequence and biochemical studies indicated that HJ6 belonged to the genus Thermus thermophilus (Tt). The gene encoding the Trehalose synthase (TS) was cloned and sequenced. The open reading frame (ORF) of the TtTS gene was composed of 2,898 nucleotides and encoded a protein (975 amino acids) with a predicted molecular weight of 110.56 kDa. The deduced amino acid sequence of TtTS showed 99% and 83% identities to the Thermus caldophilus TS and Meiothermus ruber TS, respectively. TtTS gene was expressed in Escherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for Trehalose synthase activity were found to be $80^{\circ}C$ and 7.5, respectively. The half-life of heat inactivation was about 40 min at $90^{\circ}C$. The maximum trehalose conversion rate of maltose into trehalose by the enzyme increased as the substrate concentration increased, and reached 55.7% at the maltose concentration of 500 mM, implying that the enzyme conversion was dependent of the substrate concentration.

• The Korea Genome Conference
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• 2002.08a
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• pp.49.1-49.1
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• 2002

• Proceedings of the PSK Conference
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• 2002.04a
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• pp.94-95
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• 2002

• KSBB Journal
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• v.22 no.5
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• pp.345-350
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• 2007

Phosphosugars are found in all living organisms and are commercially valuable compounds with possible applications in the development of a wide range of specialty chemicals and medicines. In carbohydrate metabolism, fructose 6-phosphate (F6P) is an essential intermediate formed by phosphorylation of 6' position of fructose in glycolysis, gluconeogenesis, pentose phosphate pathway and Calvin cycle. In glycolysis, F6P lies within the glycolysis metabolic pathway and is produced by isomerisation of glucose 6-phosphate. For large-scale production, F6P could be produced from starch using many enzymes such as pullulanase, starch phosphorylase, isomerase and mutase. In enzymatic reactions carried out at high temperatures, the solubility of starch is increased and microbial contamination is minimized. Thus, thermophile-derived enzymes are preferred over mesophile-derived enzymes for industrial applications using starch. Recently, we reported the production of glucose 1-phosphate (G1P) from starch by Thermus caldophilus GK24 enzymes. Here we report the production of F6P from starch through three steps; from starch to glucose 1-phosphate (glucan phosphorylase, GP), then glucose 6-phosphate (phosphoglucomutase, GM) and then F6P (phosphoglucoisomerase, GI). Using 200 L of 1.2% soluble starch solution in potassium phosphate buffer, 1,253 g of G1P were produced. Then, 30% yields of F6P were attained at the optimum reaction conditions of GM : G1 (1 : 2.3), 63.5$^{\circ}C$, and pH 6.85. The optimum conditions were found by response surface methodology and the theoretical values were confirmed by the experiments. The optimum starch concentrations were 20 g/L under the given conditions.