• Journal of Microbiology
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• v.35 no.4
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• pp.360-364
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• 1997

A ${\beta}$-ketothiolase was purified 180-fold from the cell extracts of Alcaligenes sp. SH-69 by a series of chromatography on DEAE-Dephadex A-50, Sephacryl S-200, and hydrozyapatitie columns, The optimum pH values of the partially purified enzyme were 7.5 for condensation reaction and 8.3 for thiolysis reaction were estimated to be 0.12mM and $18.7\;{\mu}M$, respectively. The $K_m$ valued for acetoacetyl-CoA and free CoASH in the thiolusis in the condensation reaction was 0.70mM. The condensation reaction of the ${\beta}$-ketothiolase was inhibited even by low concentrations of free CoASH($K_i=30.4{\mu}M$). Pretreatment of the enzyme with NADH and NADPH markedly inhibited the thiolysis reaction of the enzyme. The potent inhibition of the enzyme by sulfhydryl reagents suggests the involvement of cystein residue in the active site.

• Korean Journal of Food Science and Technology
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• v.29 no.6
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• pp.1304-1308
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• 1997

In the course of studies for anti-plaque agents, novel procyanidin structure isolated from Artocarpus heterophyllus folium was established by thiolysis and spectroscopic analysis. The chemical structure was identified for $(-)-epiafzelecin-(4{\beta}{\rightarrow}8)-afzelecin-(4{\alpha}-8)-catechin$ containing the trimeric flavan-3-ols and molecular weight was 833[M-H] by FAB-MS negative ion method. The inhibitory effect on the glucosyltransferase activity was investigated, novel compound showed complete inhibition at 1.0 mM and inhibited on the glucosyltransferase noncompetitively.

• Korean Journal of Food Science and Technology
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• v.27 no.1
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• pp.92-96
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• 1995

In the course of our studies, novel flavan-3-ol structure isolated from Theobroma cacao L. husk was established by the thiolysis, desulfurization and spectroscopic method. The structure was identified for cinnamtannin A-2 containing the tetrameric epicatechin and molecular weight was [1153] by FAB-MS negative ion. The inhibitory effect on the glucosyltransferase activity was investigated. Cinnamtannin A-2 showed complete inhibition at 0.03 mM and inhibited on the glucosyltransferase noncompetitively. The hydroxyl group of flavan-3-ol was supposed to be the essential element for inhibition on the glucosyltransferase.

• Bulletin of the Korean Chemical Society
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• v.28 no.12
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• pp.2303-2309
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• 2007

Expressed protein ligation (EPL) technique, joining recombinantly expressed proteins to polypeptides, has been widely adopted for addressing various biological questions and for drug discovery. However, joining two recombinant proteins together is sometimes difficult when proteins are expressed insoluble and unrefoldable, because ligation-active proteins via intein-fusion are obtainable when they are folded correctly. We overcame this limitation coexpressing target protein with additional methionine aminopeptidase (MAP) which enhances removal of the initiation methionine of recombinantly expressed protein. Our approach demonstrated that two domains of 46 kDa 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, a target of herbicide glyphosate, were successfully joined by native chemical ligation, although its C-terminal domain was expressed as an inclusion body. The intein-fused N-terminal fragment of EPSP synthase (EPSPSN, residues 1-237) was expressed and the ligation-active thioester tagged N-terminal fragment (EPSPSN-thioester) was purified using a chitin affinity chromatography and mercapto-ethanesulphonate (MESNA) as intein thiolysis reagent. Its Cterminal fragment (EPSPSC, residues Met237-238CYS-427), expressed as an inclusion body, was prepared from an additional MAP-expressing strain. Protein ligation was initiated by mixing ~1 mM of EPSPSN-thioester with ~2 mM of EPSPSCCYS (residues 238CYS-427). Also we found that addition of 2% thiophenol increased the ligation efficiency via thiol exchange. The ligation efficiency was ~85%. The ligated full-length EPSP synthase was dissolved in 6 M GdHCl and refolded. Circular dichroism (CD) and enzyme activity assay of the purified protein showed that the ligated enzyme has distinct secondary structure and ~115% specific activity compared to those of wild-type EPSP synthase. This work demonstrates rare example of EPL between two recombinantly expressed proteins and also provides hands-on protein engineering protocol for large proteins.