• KSBB Journal
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• v.28 no.5
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• pp.332-337
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• 2013

The gene encoding for xylose isomerase from the thermophilic bacterium Thermotoga maritima was cloned and recombinantly expressed in E. coli cells. Optimal activity was shown at $90^{\circ}C$ and pH 8.0. Treatment of saponin by recombinant xylose isomerase increased the growth inhibitory effect against human gastric cancer (AGS) cells and human colon cancer (HT-29) cells. On the other hand, treatment of fucoidan by the enzyme could not change the growth inhibitory effect against the same cancer cells. One ${\mu}g/ml$ of enzyme-treated saponin exhibited the same or higher growth inhibitory effect against both cancer cells compared with 100 ${\mu}g/ml$ of enzymeuntreated saponin. These results would be useful in the development of functional food or drug.

• Journal of Microbiology and Biotechnology
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• v.22 no.12
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• pp.1724-1730
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• 2012

An ${\alpha}$-L-arabinofuranosidase (TmAFase) from Thermotoga maritima MSB8 is a highly thermostable exo-acting hemicellulase that exhibits a relatively higher activity towards arabinan and arabinoxylan, compared with other glycoside hydrolase 51 family enzymes. In the present study, we carried out the enzymatic characterization and structural analysis of TmAFase. Tight domain associations found in TmAFase, such as an inter-domain disulfide bond (Cys306 and Cys476) in each monomer, a novel extended arm (amino acids 374-385) at the dimer interface, and total 12 salt bridges in the hexamer, may account for the thermostability of the enzyme. One of the xylan binding determinants (Trp96) was identified in the active site, and a region of amino acids (374-385) protrudes out forming an obvious wall at the substrate-binding groove to generate a cavity. The altered cavity shape with a strong negative electrostatic distribution is likely related to the unique substrate preference of TmAFase towards branched polymeric substrates.

• Journal of Life Science
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• v.16 no.7 s.80
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• pp.1148-1157
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• 2006

A hyperthermophilic bacterium Thernotoga maritima produced thermostable ${\beta}-glucosidase$. The gene encoding ${\beta}-glucosidase$ from T. maritima MSB8 was cloned and expressed in Escherichia coli. The en-zyme (BgIB) hydrolyzed ${\beta}-glucosidase$ linkages between glucose and alkyl, aryl of saccharide groups such as salicin, arbutin, and $_pNPG$. The insert DNA contained ORF with 2,166 bp encodes a 721 amino acids (calculated molecular mass of 80,964 and pl of 4.93). The amino a.id sequence of BglB showed the similarity to family 3 glycosyl hydrolases. The molecular weight of the enzyme was estimated to be approximately 81kDa by MUG-nondenaturing PAGE (4-methylumbelliferyl 13-D-glucoside-nondenaturing polyacrylamide gel electophoresis) and SDS-PACE. The ${\beta}-glucosidase$ exhibited maximal activity at pH 7.0 and $80^{\circ}C$. By exchanging two possible residues (Glu-232 and Asp-242) to Ala by site-directed mutagenesis method, it was found that these were essential for enzymatic activity.

• Journal of Life Science
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• v.21 no.2
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• pp.221-226
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• 2011

The gene encoding 4-$\alpha$-glucanotransferase (mgtA) from Thermotoga neapolitana was cloned and expressed in Escherichia coli in order to investigate whether this enzyme was capable of producing cycloamylose for industrial applications. MgtA was purified to homogeneity by HiTrap Q HP and Sephacryl S-200 HR column chromatographies. The size of the enzyme as determined by SDS-PAGE was about 52 kDa, which was in good agreement with its deduced molecular mass of 51.9 kDa. The optimal temperature and pH for the activity of the 4-$\alpha$-glucanotransferase was found to be $85^{\circ}C$ and 6.5, respectively. The enzyme hydrolyzed the 1,4-$\alpha$-glucosidic bonds in oligomeric 1,4-$\alpha$-glucans and transferred oligosaccharides (maltotriose being the shortest one) to acceptor maltodextrins. However, the enzymes had no activity against pullulan, glycogen, and other di- or trioligosaccharides with rare types of $\alpha$-bond. MgtA is distinguished from 4-$\alpha$-glucanotransferase from Thermotoga maritima in that it can convert maltotriose into maltooligosaccharides. The treatment of glucoamylase after the reaction of MgtA with maltotriose, maltotetraose, maltopentaose, or maltohexaose as sole substrate revealed that MgtA yielded linear maltooligosaccharides instead of cycloamylose.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.587-587
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• 2003

• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.477-480
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• 2002

A thermostable xylanase from Thermotoga maritima (Xyn B) cleaves several pNP-glycosides of monosaccharides. We found that the initial product of the cleavage of pNP-xyloside (pNP-Xy1) was a disaccharide, not xylose, indicating that xylosyl unit of pNP-Xyl was transglycosylated to another pNP-Xyl. We determined that the disaccharide was xylobiose which has the linkage of the ${\beta}$ 1-4, and described the reaction mechanism of the Xyn B. Also, we produced the several pNP-glycosides and propyl-disaccharides from the transglycosylation of Xyn B with varial glycosides and/or 1-propanol. All reaction products were purified by column chromatography (Toyo-pearl HW-40C, 45 cm${\times}$2.5 cm or 45 cm ${\times}$ 2.5 cm${\times}$ 2). The isolated products were analyzed by means of 1D and 2D NMR.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.429-429
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• 2005

• Proceedings of the Korean Society of Crop Science Conference
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• 2018.10a
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• pp.204-204
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• 2018

• Food Science and Biotechnology
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• v.17 no.5
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• pp.990-995
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• 2008

A thermostable $\alpha$-L-arabinofuranosidases ($\alpha$-L-AFase) is an industrially important enzyme for recovery of L-arabinose from hemicellulose. The recombinant $\alpha$-L-AFase from Thermotoga maritima was expressed in Escherichia coli by using a constitutive pHCE or an inducible pRSET vectors. In batch fermentation, the constitutive expression system resulted in slightly faster growth rate (0.78 vs. 0.74/hr) but lower enzyme activity (2,553 vs. 3,723 units/L) than those of the induction system. When fed-batch fermentation was performed, biomass and enzyme activity reached the highest levels of 36 g/L and 9,152 units/L, respectively. The fed batch cultures performed superior results than batch culture in terms of biomass yield (4.62-5.42 folds) and enzyme synthesis (3.39-4.00 folds). In addition, the fed-batch induction strategy at high cell density resulted in the best productivity in cell growth as well as enzyme activity rather than the induction method at low cell density or the constitutive expression.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2004.06a
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• pp.273-277
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• 2004

$\alpha$-L-Arabinofuranosidase ($\alpha$-L-AFase, EC 3.2.1.55) was isolated from hyperthermophilic microorganism, Thermotoga maritima. The open reading frame (ORF) of $\alpha$-L-AFase gene is 1,455 bp long and encodes 484 amino acid residues with a molecular weight of 55,265 Da. The ORF of $\alpha$-L-AFase gene was introduced into the E. coli expression vector, $_p/RSET-B, and overexpressed in E. coli BL21. The purified recombinant $\alpha$-L-AFase showed the highest activity at 10$0^{\circ}C$ and pH 5.5. The purified enzyme appeared to have no metal cofactor requirement. The Km and specific activity values of the recombinant enzyme were 0.99 mM and 1,200 U/mg on p-nitrophenyl-$\alpha$-L-arabinofuranoside. It released only L-arabinose from sugar beet arabinan, sugar beet debranched arabinan and oat spelts arabinoxylan but had no activity onarabinogalactan and gum arabic. This result suggests that L-arabinose could be produced from natural polysaccharides using this enzyme. Mutant enzymes which Glu26, Glu172 and Glu281 residues were replaced to alanine, aspartic acid or glutamine caused Kcat to decrease by a factor of between 10$^3$ and 10$^4$. Glu172 and Glu281 residues of $\alpha$-L-AFase are seemed to be the acid/base and nucleophile in catalytic reaction, respectively, and Glu26 is supposed to playa key role in substrate binding.ng.