• Korean Journal of Food Science and Technology
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• v.29 no.2
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• pp.376-378
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• 1997

In an attempt to increase the conversion of lactose to galactoligosaccharides, two types of ${\beta}-galactosidases$ originated from Thermus caldophilus and Bacillus sp. A4442 reacted with 60% (w/w) lactose consecutively. Concentration of galactooligosaccharides reached up to 60% at the 85% conversion of the initial lactose maintaining transgalactosylation ratio ca. 90%.

• Journal of Microbiology and Biotechnology
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• v.13 no.1
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• pp.139-145
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• 2003

The gene encoding Thermus caldophilus GK24 polyphosphate kinase (Tca PPK) was cloned and sequenced. The gene contains an open reading frame encoding 608 amino acids with a calculated molecular mass of 69,850 Da. The deduced amino acid sequence of Tca PPK showed a 40% homology to Escherichia coli PPK, and $39\%$ to Klebsiella aerogenes PPK. The Tca ppk gene was expressed under the control of the T7lac promoter on pET-22b(+) in E. coli and its enzyme was purified about 70-fold with $36\%$ yield, following heating and HiTrap chelating HP column chromatography. The native enzyme was found to have an approximate molecular mass of 580,000 Da and consisted of eight subunits. The optimum pH and temperature of the enzyme were 5.5 and $70^{\circ}C$, respectively. A divalent cation was required for the enzyme activity, with $Mg^2+$ being the most effective.

• Journal of Microbiology and Biotechnology
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• v.13 no.4
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• pp.628-631
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• 2003

The recombinant alanine dehydrogenase (ADH) from E. coli containing Thermus caldophilus ADH was purified to homogeneity from a cell-free extract. The enzyme was purified 38-fold with a yield of 68% from the starting cell-free extract. The purified enzyme gave a single band in polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 45 kDa. The pH optimum was 8.0 for reductive amination of pyruvate and 12.0 for oxidative deamination of L-alanine. The enzyme was stable up to $70^{\circ}C$. The activity of the enzyme was inhibited by 1 mM $Zn^{2+}$, 20% hexane, and 20% $CHCl_3$. However, 10 mM $Mg^{2+}$ and 40% propanol had no effect on the enzyme activity. The Michaelis constants ($K_m$) for the substrates were $50\;\mu\textrm{M}$ for NADH, 0.2 mM for pyruvate, 39.4 mM for $NH_4+$, 2.6 mM for L-alanine, and 1.8 mM for $NAD^+$.

• BMB Reports
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• v.34 no.4
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• pp.347-354
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• 2001

$\alpha$-Glucosidase of an extreme thermophile, Thermus caldophilus GK24 (TcaAG), was purified 80-fold from cells to a homogeneous state and characterized. The enzyme exhibited optimum activity at pH 6.5 and $90^{\circ}C$, and was stable from pH 6.0 to 85 and up to $90^{\circ}C$. The enzyme had a half-life of 85 minutes at $90^{\circ}C$. An analysis of the substrate specificity showed that the enzyme hydrolyzed the non-reducing terminal unit of $\alpha$-1,6-glucosidic linkages of isomaltosaccharides and panose, $\alpha$-1,3-glycosidic bond of nigerose and turanose, and $\alpha$-1,2-glycosidic bond of sucrose. The gene encoding the TcaAG was cloned, sequenced, and sequenced in E. coli. The nucleotide sequence of the gene encoded a 530 amino acid polypeptide and had a G+C content of 68.4% with a strong bias for G or C in the third position of the codons (93.6%). A sequence analysis revealed that TcaAG belonged to the $\alpha$-amylase family. We suggest that this monomeric, thermostable, and broad-acting $\alpha$-glucosidase is a departure from previously exhibited specificities. It is, therefore, a novel $\alpha$-glucosidase.

• Food Industry And Nutrition
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• v.2 no.1
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• pp.7-9
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• 1997

내열설 미생물, Thermus caldophilus CK24에 대한 탄수화물 생합성을 연구하는 과정에서 다양한 탄수화물 관련효소를 탐색하고 그레 대한 생화학적 및 분자생물학적 연구를 수행하고 있다. 일차로 내열성 미생물내 1) 당핵산염 합성효소와 당전이 효소, 2) 탄수화물 대사효소. 3)탄수화물 분해 및 전환효소의 존재를 HPLC/Bio-LC분석을 통하여 확인하고 이들에 대한 연구를 진행하고 있다. 본 연구발표에서는 포도당을 과당으로 전환하는 이성화효소(xylose isomerase), 그리고 맥아당을 트레할로스로 전환하는 트레할로스 합성효소(trehalose synthase)를 소개하고저 한다. 이성화효소는 이미 산업적 과당 생산에서 대규모적으로 사용되고 있는 식품산업효소이다. 본 연구에서는 Thermus caldophilus GK24, Thermus thermophilus HB8, Thermus flavus AT62 3종의 내열성 미생물에 대한 이성화효소 유전자를 클로닝 하고, 각 재조합하고 이성화효소를 대량생산하였다. 이 내열성 이성화효소는 최적 반응 온도가 8$0^{\circ}C$이고, 포도당을 과당으로 전환하는 수유른 55%이었다. 이러한 과당전환률은 이미 산업적으로 사용되고 있는 이성화효소의 과당전환률(43%)보다 훨씬 높은 것으로 과당 생산공정의 단순화의 생산성 향상에 결정적인 요인이라 할 수 있다. 한편 본 이성화효소의 산업적 특성을 증대하기 위하여 구조-기능관계 연구를 착수하였다. 우선 내열성 이산화 효소의 입체 구조를 결정하였고, 구조조정에 따른 기능적 특성을 조사하기 위하여 특정 위치의 선택적 변이 연구를 진행하고 있다. 끝으로 포도당 전이 효소를 추적하던 과정에서 맥아당을 트레할로스로 전환하는 새로운 효소를 Thermus caldo-philus GK24에서 발견하였다. 그 트레할로스 합성효소는 분자량이 약 110kDa이고 최적 반응온도가 75$^{\circ}C$이면, 조효소없이 맥아당을 트레할로스로 80%이상 전환해 주는 가역효소이었다. 본 연구에서는 효소반응의 조건과 특성을 조사하였고, 효소 아미노-밀단의 서열결정정보를 통하여 효소의 유전자를 클로닝 하고 그 유전자의 구조와 발현연구를 진행하고 있다.

• Journal of Microbiology and Biotechnology
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• v.12 no.2
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• pp.301-305
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• 2002

The gene (ppaT) encoding Thermus caldophilus GK24 pyrophosphatase (Tca pyrophosphatase) was cloned and sequenced. The gene was found to contain an open reading frame encoding 175 amino acids with a calculated mass of 19,155 Da. The ppaT gene was expressed under the control of the tac promoter in Escherichia coli. The recombinant Tca pyrophosphatase was purified 21.4-fold with $56\%$ yield and specific activity of 25.7 U $mg^-1$, following a combination of heating (to denature the E. coli proteins) and one step of DEAE-Sephacel column chromatography. The native enzyme was found to have an approximate molecular mass of 110,000 Da and consisted of six subunits. The enzyme exhibited maximal activity at pH of 8.0-8.5 and was stable at $80-90^{\circ}C$. A divalent cation was absolutely required for the enzyme activity, with $Mg^2+$. being the most effective.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.638-642
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• 2000

A ${\beta}-glycosidase$ enzyme with $\beta$-D-fucosidase, ${\beta}-D-galactosidase$, and $\beta$-D-glucosidase activities has been purified from Thermus caldophilus GK24. The enzyme was monomeric with a molecular mass of 49 kDa, as evidenced by SDS-PAGE. The $K_m$ values for p-nitrophenyl ${\beta}-D-fucopyranoside$ (p-NPFuc), p-nitrophenyl ${\beta}-D-galactopyranoside$ (p-NPGal), and p-nitrophenyl ${\beta}-D-glucopyranoside$ (p-NPGlu) were 0.23 mM, 6.25 mM, and 0.28 mM, respectively. The enzyme showed optimal pH ranging between 5.5-6.5 and maximum temperature in the range of $85-90^{\circ}C$ for all the above mentioned activities. The half-life of the enzyme in sodium phosphate buffer (pH 6.0) at $80^{\circ}C$ was approximately 7 h. The p-NPGal hydrolyzing activity of Tca ${\beta}-glycosidase$ was strongly activated by L-histidine, while the p-NPFuc and p-NPGlu hydrolyzing activities of Tca ${\beta}-glycosidase$ were not affected at all by the amino acid. These results suggest differences in the conformation or in the reactive residues at the active site of Tca ${\beta}-glycosidase$.

• Genomics & Informatics
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• v.1 no.1
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• pp.50-54
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• 2003

Genome of an extreme thermophile, Thermus caldophilus GK24 has been analyzed to construct the genomic map. The genomic DNAs encapsulated in agarose gel were digested with SspI, EcoRI, SpeI, and HpaI restriction endonucleases, and then the resulting genomic DNA fragments were analyzed by pulsed-field gel electrophoresis. Its restriction map has been constructed by analyzing sizes of the restriction fragments obtained from both complete and partial digestions. The circular form of its genome was composed of about 1.98 Mbp and a megaplasmid. The genomic loci for the genes of xylose isomerase, thioredoxin, tRNA-16S rRNA, 23S rRNA, L5 ribosomal protein, ADP-glucose pyrophosphorylase, DNA-ligase, and Tca DNA polymerase were determined by both Southern hybridization and PCR.

• KSBB Journal
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• v.21 no.4
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• pp.298-301
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• 2006

A gene(GeneBank AF 135796) coding for a trehalose synthase from Thermus caldophilus GK24 was cloned into Escherichia coli K12 using five vector systems. The constitutive expression system(pHCETS) which shows the highest trehalose synthase activity from flask culture of recombinant E. coli was selected for the production of trehalose from maltose. For the shake flask culture, the final dry cell weight was 0.9 g/L and the trehalose synthase activity was 25 U/mL. Fed-batch culture of recombinant E. coli harboring plasmid pHCETS which uses the glycerolas a carbon source was performed in jar fermentor: the dry cell weight of 20 g/L and the trehalose synthase activity of 13.7 U/mL were attained in 48 h.

• Journal of Life Science
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• v.9 no.1
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• pp.26-34
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• 1999

A thermostable pullulanase has been isolated and purified from Thermus caldophilus GK-24 to a homogeneity by gel-filtration and ion-exchange chromatography. The specific activity of the purified enzyme was 431-fold increase from the crude culture broth with a recovery of 11.4%. The purified enzyme showed $M_{r}$ of 65 kDa on denaturated and natural conditions. The pI of the enzyme was 6.1 and Schiff staining was negative, suggesting that the enzyme is not a glycoprotein. The enzyme was most active at pH 5.5. The activity was maximal at $75^{\cire}C$ and stable up to $95^{\cire}C$ for 30 min at pH 5.5. The enzyme was stable to incubation from pH 3.5 to pH 8.0 at $4^{\cire}C$ for 24hr. The presence of pullulan protected the enzyme from heat inactivation, the extent depending upon the substrate concentration. The activity of the enzyme was simulated by $Mn^{2+}$ ion, }$Ni^{2+}$, $Ca^{2+}$, $Co^{2+}$ ions. The enzyme hydrolyzed the ${\alpha}$-1,6-linkages of amylopectin, glycogens, ${\alpha}$, ${\beta}$-limited dextrin, and pullulan. The enzyme caused the complete hydrolysis of pullulan to maltotriose and the activity was inhibited by $\alpha$, $\beta$, or $\gamma$-cyclodextrins. The $NH_{2}$-terminal amino acid sequence [(Ala-Pro-Gln-(Asp of Tyr)-Asn-Leu-Leu-Xaa-ILe-Gly-Ala(Ser)] was compared with known sequences of various sources and that was compared with known sequences of various sources and that was different from those of bacterial and plant enzymes, suggesting that the enzymes are structurally different.