• Journal of Microbiology
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• 제35권4호
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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.

• 한국식품과학회지
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• 제29권6호
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• pp.1304-1308
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• 1997

충치 예방물질의 지속적인 연구로 Jack Fruit 잎으로부터 새로운 procyanidin류를 분리하였고 thiolysis 및 기기 분석에 의해 구조를 결정하였다. 신규화합물의 화학 구조는 flavan-3-ol 화합물의 삼량체인 $(-)-epiafzele-chin-(4{\beta}{\rightaroow}8)-afzelechin-(4{\alpha}{\rightaroow}8)-catechin$의 구조로, 분자량이 $833\;[M-H]^-$ 이었다. 화학 구조가 결정된 신규화합물의 glucosyltransferase저해 실험 결과 1.0 mM에서 완벽한 저해 효과가 관찰되었고 저해기작은 비경쟁적 저해제임을 알았다.

• 한국식품과학회지
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• 제27권1호
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• pp.92-96
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• 1995

Theobroma cacao L. bean husk에서 새로운 flavan-3-ol 화합물을 분리하여 이 화합물의 화학구조를 thiolysis, 탈유황반응 및 기기분석에 의해 결정하였다. 이 화합물은 FAB-MS에서 [1153] 으로 epicatechin 4분자가 결합된 cinnamtannin A-2였다. GTase 저해는 0.03 mM에서 완벽한 저해효과를 보였고 비경쟁적 저해제로 이 화합물의 수산기가 GTase를 저해하는 중요한 요인으로 추정된다.

• Bulletin of the Korean Chemical Society
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• 제28권12호
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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.