• 한국미생물·생명공학회지
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• 제21권6호
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• pp.577-581
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• 1993

The amino acid threonine was produced from glycine and ethanol in a reaction mixture using cell free extract of the methylotrophic bacterium isolated from soil and identified as mellthylo-bacterium sp. KJ29. Although the isolate could grow on carbon source other than methanol, only the cell free extract from the cells grown on methanol produced threonine. Methanol dehydrogenase (MDH) activity was present only in the cells grown on methanol when compared to the cells grown on heterotrophic substrates.

• 한국식품영양학회지
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• 제15권3호
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• pp.235-240
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• 2002

랫트에 있어서 상이한 단백질과 threonine급여수준에 의한 간 TDG의 활성을 조사하였다. 식이 중의 단백질 급여수준과 TDG 활성과의 연계성을 조사하였을때, 12.0% CP까지는 TDG의 총활성이 단계적으로 상승하고, 더 높은 CP 함량 (18.0%∼24,0% CP)에 있어서는 경향적으로 threonine 분해의 쇠퇴가 나타났다. 전반적으로 단백질 함량의 증가에 의해 간 TDC를 통한 threonine의 산화가 증가하지 않았음을 나타내었다. 또 한식이 중의 단백질 및 threonine 급여수준과 TDG활성과의 연계성을 조사하였을 때, 단계적으로 상승시킨 threonine 급여수준이 (0.28∼0.72% Thr) 12.0% CP에 있어서 단계적으로 TDC활성을 상승시켰다. 또한 유사한 현상이 18.0%CP에 있어서도 관찰되었으며 약간 더 높은 TDG 활성을 나타내었다.

• 약학회지
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• 제34권6호
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• pp.447-456
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• 1990

Various methods are available for the production of L-threonine. The microbial production of L-threonine has been achieved by breeding L-threonine analog-resistant auxotrophic mutants of various bacteria. The enzymatic production of L-threonine has been demonstrated by use of threonine metabolic enzymes such as threonine deaminase, threonine aldolase, or threonine dehydrogenase complex. Threonine synthesis from glycine and ethanol seems to be catalyzed by the enzymes Methanol dehydrogenase(MDH) and Serine hydroxymethyltransferase(SHMT), which was also found to catalyze the aldol condensation of glycine with acetaldehyde. The improved production of L-threonine has been achieved by amplifying the genes for the L-threonine biosynthetic enzymes using recombinant DNA techniques.

• Asian-Australasian Journal of Animal Sciences
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• 제24권10호
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• pp.1417-1424
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• 2011

This study was conducted to assess the relation between threonine (Thr) oxidation rate and threonine efficiency on rat and chicken fed with graded levels of protein and threonine. The increase in threonine content from 0.28 to 0.72% in a diet containing 12.0% crude protein (CP) caused a gradual increase in threonine dehydrogenase (TDG) activity in rat liver. Similar, but more pronounced results were observed after 18.0% CP in the diet. Both protein levels in combination with the highest level of threonine supplementation increased liver TDG activity significantly, indicating enhanced threonine catabolism. Parameters of efficiency of threonine utilization calculated from parallel nitrogen balance studies decreased significantly and indicated threonine oversupply after a maximum of threonine supplementation. At the lower levels of threonine addition the efficiency of threonine utilization was not significantly changed. In the chicken liver up to 0.60% true digestible threonine (dThr) in the 18.5% CP diet produced no effect on the TDG activity. However, TDG activity in the liver was elevated by the diet containing 22.5% CP (0.60% dThr) and the efficiency of threonine utilization decreased, indicating the end of threonine limiting range. In conclusion, the in vitro TDG activity in the liver of rat and growing chicken has an indicator function for the dietary supply of threonine.

• Journal of Microbiology and Biotechnology
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• 제24권6호
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• pp.748-755
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• 2014

In the genome annotation of Escherichia coli MG1655, the orf382 (1,149 bp) is designated as a gene encoding an alcohol dehydrogenase that may be Fe-dependent. In this study, the gene was amplified from the genome by PCR and overexpressed in Escherichia coli BL21(DE3). The recombinant $6{\times}$His-tag protein was then purified and characterized. In an enzymatic assay using different hydroxyl-containing substrates (n-butanol, $\small{L}$-threonine, ethanol, isopropanol, glucose, glycerol, $\small{L}$-serine, lactic acid, citric acid, methanol, or $\small{D}$-threonine), the enzyme showed the highest activity on $\small{L}$-threonine. Characterization of the mutant constructed using gene knockout of the orf382 also implied the function of the enzyme in the metabolism of $\small{L}$-threonine into glycine. Considering the presence of tested substrates in living E. coli cel ls and previous literature, we believed that the suitable nomenclature for the enzyme should be an $\small{L}$-threonine dehydrogenase (LTDH). When using $\small{L}$-threonine as the substrate, the enzyme exhibited the best catalytic performance at $39^{\circ}C$ and pH 9.8 with $NAD^+$ as the cofactor. The determination of the Km values towards $\small{L}$-threonine (Km = $11.29{\mu}M$), ethanol ($222.5{\mu}M$), and n-butanol ($8.02{\mu}M$) also confirmed the enzyme as an LTDH. Furthermore, the LTDH was shown to be an ion-containing protein based on inductively coupled plasma-atomic emission spectrometry with an isoelectronic point of pH 5.4. Moreover, a circular dichroism analysis revealed that the metal ion was structurally and enzymatically essential, as its deprivation remarkably changed the ${\alpha}$-helix percentage (from 12.6% to 6.3%).

• KSBB Journal
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• 제22권1호
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• pp.62-65
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• 2007

대사 공학을 이용한 생산 균주 개발의 핵심 기술은 원하는 대사산물을 과량으로 얻기 위하여 기존의 대사회로에서 제거, 증폭, 변경을 시켜야 할 유전자를 선정하는 것이다. 대사조절 분석 기법은 대사 흐름이 특정 효소의 활성에 따라 어떻게 변하는지를 예측하는 기술이다. 본 논문에서는 대장균의 threonine 생합성 효소 반응 kinetic model과 대사조절 분석 기법을 이용하여 threonine 생합성 flux를 가장 효과적으로 증가시키기 위하여 활성 증가가 필요한 효소가 aspartate semialdehyde dehyogenase라는 것을 밝혔다. 이러한 결과를 확인하기 위하여 asd가 과발현된 vector와 threonine 생합성 경로의 다른 효소인 aspartate kinase를 coding하는 thrA를 과발현 시키는 vector를 제작하여 threonine 생산 균주인 TF5015에 형질전환하여 threonine 농도를 측정하였다. Flask 배양결과 대사조절 분석 기법으로 확인된 유전자 asd를 과발현시킬 경우가 생합성 경로의 다른 유전자를 과발현시킨 경우보다 더 높은 threonine 농도의 증가를 보였다. 이러한 연구 결과들은 효소 반응 kinetic model과 대사조절 분석 기법을 이용하여 원하는 product를 효율적으로 생산할 수 있는 생산 균주를 제작할 수 있게 할 것이다.

• Journal of Plant Biology
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• 제39권1호
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• pp.41-48
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• 1996

발아 후 8일된 해녀콩 자엽에서 성질이 서로 다른 두 개의 homoserine dehydrogenase를 분리하였다. 자엽에서 얻은 조효소액을 열처리, 황산 암모늄 침전, DEAE-Sephacel 및 Sephacryl S-300 겔 크로마토그래피와 Procion red dye, Cibacron blue dye 및 Resource Q 컬럼 크로마토그래피로 정제하였다. 겔 크로마토그래피에서 얻은 2개의 활성분획 중 T-형(트레오닌 감수성)과 K-형(트레오닌 비감수성)의 분자량은 각각 230 kD과 135 kD이었다. 10mM 트레오닌 첨가로 T-형 효소는 70% 이상의 활성저해를 받았으나 K-형 효소는 전혀 억제를 받지 않았다. Homoserine에 대한 Km은 T-형이 1.6mM, K-형이 0.3mM이었고, NAD에 대한 Km은 T-형이 2.34mM, K-형이 0.03mM이었으며 NADP에 대한 Km은 두 효소에서 동일하게 0.01mM로 산출되었다. 400mM KCl에서 T-형은 4.9배, K-형은 2.8배의 활성증가를 보였다. 부분정체(Sephacryl S-300 겔 크로마토그래피)된 상태의 T-형과 K-형은 조건에 따라 쉽게 상호전환되었다.

• Asian-Australasian Journal of Animal Sciences
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• 제27권1호
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• pp.69-76
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• 2014

This study was carried out to evaluate the relationship between threonine (Thr) efficiency and Thr dehydrogenase (TDG) activity as an indicator of Thr oxidation on chicks fed with levels of diets (CP [17.5% and 21.5%] and Thr [3.8 and 4.7 g/100 g CP]; glycine [Gly][0.64% and 0.98%] and true digestible Thr [dThr] [0.45% and 0.60%]). Calculation of the Thr efficiency was based on N-balance data and an exponential N-utilization model, and TDG activity was determined as accumulation of aminoacetone and Gly during incubation of hepatic mitochondria. This study found that in the liver of chicks who received a diet containing up to 0.79% Thr (4.7 g Thr/100 g of CP) in the 17.5% CP diet, no significant (p>0.05) effect on TDG activity was observed. However, significantly (p = 0.014) increased TDG activity was observed with a diet containing 21.5% CP (4.7 g Thr/100 g of CP) and the efficiency of Thr utilization showed a significant (p = 0.001) decrease, indicating the end of the Thr limiting range. No significant (p>0.05) effect on the total TDG activity and accumulation of Gly was observed with addition of Gly to a diet containing 0.45% dThr. In addition, addition of Gly to a diet containing 0.60% dThr also did not result in a change in accumulation of Gly. Due to an increase in accumulation of aminoacetone, an elevated effect on total TDG activity was also observed. No significant (p>0.05) reduction in the efficiency of Thr utilization was observed after addition of Gly at the level of 0.45% dThr. However, significantly (p<0.001) reduced efficiency of Thr utilization was observed after addition of Gly at the level of 0.60% dThr. Collectively, we found that TDG was stimulated not only by addition of Thr and protein to the diet, but also by addition of Gly, and efficiency of Thr utilization was favorably affected by addition of Gly at the level near to the optimal Thr concentration. In addition, no metabolic requirement of Gly through the TDG pathway was observed with almost the same accumulation of Gly and a slight increase in TDG activity by addition of Gly. Thus, our findings suggest that determination of TDG activity and parameter of efficiency of Thr utilization may be useful for evaluation of dietary Thr level.

• BMB Reports
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• 제35권4호
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• pp.437-441
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• 2002

Dihydrolipoamide dehydrogenase (E3) is a member of the pyridine nucleotide-disulfide oxidoreductase family. Thr residues are highly conserved. They are at the active site disulfide-bond regions of most E3s and other oxidoreductases,. The crystal structure of Azotobacter vinelandii E3 suggests that the hydroxyl group of Thr that are involved in the FAD binding interact with the adenosine phosphate of FAD. However, several prokaryotic E3s have Val instead of Thr. To investigate the meaning and importance of the Thr conservation in many E3s, the corresponding residue, Thr-44, in human E3 was substituted to Val by site-directed mutagenesis. The mutant’s E3 activity showed about a 2.2-fold decrease. Its UV-visible and fluorescence spectra indicated that the mutant might have a slightly different microenvironment at the FAD-binding region.

• 한국식품위생안전성학회지
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• 제18권3호
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• pp.146-152
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• 2003

당 대사에 관여하는 Pyruvate dehydrogenase phosphatase (PDP)는 해당과정에서의 대사 산물인 pyruvate 를 acetyl CoA로 만들어 구연산 회로로 진입시켜주는 효소인 Pyruvate dehydrogenase complex(PDP)의 활성을 조절하는 중요한 효소이다. PDP의 catalytic subunit는 PDP의 dihydrolipoamide acetyltransferase(E2), PDP regulatory subunit (PDPr), 그리고 칼슘 결합 도메인 등으로 구성되어 있는 것으로 추측되어지고 있다. 본 연구에서는 PDP 단백질을 분리정제하고 결정화 하고자하였다. PDP는 catalytic subunit(PDPc, Mr 52,600 Da)과, regulatory subunit (PDPr, 95,600 Da)으로 구성되어 있으며 칼슘 존재하에 PDPc는 dihydrolipoamide acetyltransferase(E2) component와 결합하여 기질인 인산 E1 component의 탈인산화율을 증가시킨다. PDPc는 intrinsic 칼슘 결합부위를 가지며 두 번째 칼슘 부위는 E2 존재 하에 형성된다. 이러한 특이한 상호반응을 이용한 GSH-Sepharose-GST-L2 matrix를 이용하여 약 1000 U/mg의 specific activity를 갖는 순수 PDPc를 약 80%의 yield로 얻어 결정화에 사용하였다.