• 한국미생물·생명공학회지
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• 제20권1호
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• pp.25-33
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• 1992

Serratia marcescens ATCC 25419를 질소원이 풍부한 BHI 배지에서 황산 암모늄 분별 침전을 시킨 후 DAEA-Sephacel chromatography, Phenyl-Sepharose hydrophobic chromatography, Sephacryl S-400 gel filtration, native gel elution을 거쳐 ALS isozyme Rf 0.83을 분리하였다. 분리한 ALS isozyme Rf 0.83의 native 형태는 gel filtration을 이용하여 분자량을 측정한 결과, 531,400이었고, SDS-PAGE를 수행한 결과 55,000의 large subunit와 38,900의 small subunit로 구성된 multimer임을 알 수 있었다.

• Bulletin of the Korean Chemical Society
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• 제28권7호
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• pp.1109-1113
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• 2007

Acetohydroxyacid synthase (AHAS, EC 2. 2. 1. 6) is the enzyme that catalyses the first step in the common pathway of the biosynthesis of the branched chain amino acids, valine, leucine and isoleucine. For the first time, the AHAS gene from Bacillus anthracis was cloned into the expression vector pET28a(+), and was expressed in the E. coli strain BL21(DE3). The purified enzyme was checked on 12% SDS-PAGE to be a single band with molecular weight of 65 kDa. The optimum pH and temperature for B. anthracis AHAS was at pH 7.5 and 37 oC, respectively. Kinetic parameters of B. anthracis were as follows: Km for pyruvate, K0.5 for ThDP and Mg2+ was 4.8, 0.28 and 1.16 mM respectively. AHAS from B. anthracis showed strong resistance to three classes of herbicides, Londax (a sulfonylurea), Cadre (an imidazolinone), and TP (a triazolopyrimidine). These results indicated that these herbicides could be used in the search for new anti-bacterial drugs.

• Bulletin of the Korean Chemical Society
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• 제26권6호
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• pp.916-920
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• 2005

Acetohydroxy acid synthase catalyzes the first common step in the biosynthesis of branched chain amino acids. AHAS plays two distinct metabolic roles, and is designated as anabolic AHAS and catabolic AHAS, depending on its function. Anabolic AHAS is FAD-dependent, while its catabolic counterpart is not. In this work, a conserved motif was identified in the $\beta$-domain of anabolic AHASs, but not in catabolic AHAS ($_{372}RFDDR_{376}$). In order to determine the functions of this motif, we replaced the motif with the corresponding sequence in FAD-independent AHAS, SPVEY. None of these three mutants (SPV, SPVE, and SPVEY) was detected with bound FAD. However, two of these mutants (SPVE and SPVEY) were active at a low level of specific activity. Although they exhibited pyruvate- and ThDP- dependent characteristics, the activity of the two active mutants appears to be FAD-independent. The SPVEY mutant was completely insensitive to the three tested herbicides, even at extremely high concentrations and is also somewhat more thermolabile than the wild type enzyme. The data provided in this work suggest that the RFDDR motif is a possible determinant of the FAD-dependent and herbicide-resistant properties of tobacco AHAS. The SPVEY mutant appears to exhibit catabolic AHAS-like activity.

• 대한유전성대사질환학회지
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• 제18권2호
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• pp.43-49
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• 2018

단풍시럽뇨병은 드문 상염색체 열성 대사 질환으로 측쇄 알파 케토산 탈수소효소 돌연변이에 의해 발생되는 질환이다. 측쇄 아미노산인 류신, 이소류신, 발린의 분해가 되지 않아 몸에 축적이 되고 식욕 저하, 구토, 기면, 이상행동, 발작 및 심한 경우 죽음을 초래한다. 세계적으로 단풍시럽뇨병의 유병율은 185,000명당 한명 이지만 한국에서는 극히 드물어 1,148,413명당 한명으로 보고된다. 이 증례의 여아는 만삭에 2.54 kg로 주산기 특이 병력 없이 출생한 첫째 아이로 특이 가족력 없었던 환아이다. 생후 10일경 식욕저하, 구토, 기면으로 타병원에서 대증치료 중에 선천성 대사이상 선별 검사 결과 상류신이 높아 전원 되었다. 환아는 기면, 혼수를 보였고 중추 수면무호흡으로 인공호흡기를 적용하였으며 소변에서 단풍시럽 냄새를 보였다. 뇌 초음파, 뇌 자기공명영상에서 뇌부종 소견을 보였으며 혈중아미노산 검사상 류신, 이소류신, 발린의 수치가 정상치보다 매우 높았다. 측쇄 아미노산 없는 특수분유를 시작했고 뇌부종으로 만니톨과 이뇨제를 사용했으며 고암모니아혈증으로 벤조산을 투약하였다. 경련 예방을 위해 페노바비탈을 투약하였으며 측쇄 알파 케토산 탈수소효소의 조효소인 타이아민 보충을 시작하였다. 치료함에 따라 혈중 측쇄 아미노산 수치가 감소하였고 뇌부종의 감소가 확인되었다. 29일 간의 신생아중환자실 처지 후 환아는 호흡 및 구강 식이 진행이 안정적이었고 병동으로 전동 후 퇴원하였다. 환아는 현재 5개월로 페노바비탈을 점차 줄여가고 있으며 측쇄 아미노산 수치도 안정적으로 유지되고 있다. 고개 가누기는 완벽하지 않지만 옹알이 및 뒤집기 가능하며 따라잡기 성장 및 발달을 보이고 있다. 유전학적 검사에서는 두 개의 BCKDHB 이형접합 돌연변이 p.Ala32Phefs*48와 p.Val130Phe를 확인 하였으며, 가족 검사를 통하여 부모 모두 보인자임을 확인하였다. 이 두 변이는 모두 이전에 한국에서 보고되지 않은 변이이다. 단풍시럽뇨병의 빠른 진단과 즉각적이고 적절한 치료가 환아의 생명을 유지하고 예후를 호전시킴을 본 증례를 통하여 다시 한 번 확인할 수 있다.

• 한국식품과학회지
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• 제39권1호
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• pp.50-54
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• 2007

단백질을 식품의 원료로서 여러 가지 식품에 응용하려고 할때 제일 먼저 문제되는 것이 영양가이지만 동시에 물리화학적 특성도 식품 원료로서의 적합성을 결정하는데 중요한 요인이 되고 있다. 그런 면에서 유청(cheese whey)의 성분에서 단백질 함량을 90% 이상으로 농축 분리시킨 whey protein isolate(WPI)는 단백질 보충제로서 뿐만 아니라 다양한 물리화학적 특성을 가지고 있기 때문에 여러 가지의 가공식품에 이용할 수 있다고 사료된다. 따라서 본 실험에서는 WPI의 아미노산 함량, 용해성, 유화성, 기포성을 조사하여 식품의 기능성 원료 및 대체물로서의 이용 가능성을 알아보고자 하였다. WPI의 총 아미노산 함량은 89.5%였고 그 중에서 필수 아미노산 함량이 44.6%를 차지하였다. 필수 아미노산 중에서는 leucine, isoleucine, valine 등의 BCAA(branched chain amino acid) 함량이 높았다. pH에 따른 WPI의 용해성은 82-88%의 범위로 pH의 영향을 받지 않았고 유화용량은 302.7mL/g으로 난황의 187.0mL/g보다 높았으며 시간이 경과함에 따라 유화액의 이장량으로 측정한 유화안정성도 65-97%로 나타나 난황의 60-89%보다 안정함을 보였다. 기포형성력은 323.3%로 난백의 186.6%보다 약 2배 정도 높았고 시간이 경과함에 따라 남은 기포의 부피로 측정한 기포안정성은 85.9-97.7%로 난백의 84.8-95.3%와 유사하였다. 이상의 결과에서 WPI는 우수한 단백질 보충제로서 뿐만 아니라 용해성, 기포성, 유화성도 우수하게 나타났으므로 각종 가공식품의 품질향상에 영향을 주는 기능성 원료 및 대체물로서의 활용도가 매우 높을 것으로 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• 제14권4호
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• pp.479-484
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• 2001

The effect of dietary supplementation of sodium salt of isobutyric acid in low protein (10% CP) wheat straw based diet on nutrient utilization and rumen fermentation was studied in ruminally fistulated male crossbred cattle. The study included a 7 day metabolism and a 3 day rumen fermentation trials. The cattle were distributed into two equal groups of 4 each. The animals of control group were fed a basal diet consisting of wheat straw, concentrate mixture and green maize fodder in 40:40:20 proportion whereas branched chain volatile fatty acid (BCFA) supplemented group received a basal diet + isobutyric acid at 0.75 percent of basal diet. The duration of study was 36 days. The feed intake between experimental groups did not differ significantly and the average total DMI (% BW) was 2.01 and $2.28kg\;day^{-1}$ in control and BCFA supplemented diets. The dietary supplementation of BCFA improved (p<0.05) the DM, OM, NDF and cellulose digestibility by 4.46, 6.63, 10.57 and 11.31 per cent over those fed control diet. The total N retention on BCFA supplementation was improved (p<0.01) due to decreased (p<0.05) urinary N excretion. The concentrations of ruminal total N was 37.07 and $34.77mg\;100ml^{-1}$ in control and BCFA fed groups, respectively. Dietary supplementation BCFA significantly (p<0.01) reduced the ruminal ammonia N concentration as compared to control and the mean values ($mg\;100ml^{-1}$) were 13.18 and 9.42 in control and BCFA fed groups. The total VFA concentration was higher (p<0.01) in BCFA supplemented group (101.14 mM) than the control (93.05 mM). Among the VFAs, the molar proportion of acetate was higher (p<0.01) in BCFA supplemented group (71.07 mM) as compared to control (64.98 mM). However, the concentration of propionate and butyrate remained unchanged. Amino acids composition of bacterial hydrolysates was similar in both the groups. Ruminal outflow rate of liquid digesta was higher (p<0.01) in BCFA fed group ($67.56l\;day^{-1}$) than control ($52.73l\;day^{-1}$). It is concluded that the dietary supplementation of Na-salt of isobutyric acid in low protein diet improved the nutrient utilization and ruminal fermentation characteristics.

• Journal of Microbiology and Biotechnology
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• 제28권11호
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• pp.1916-1927
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• 2018

Corynebacterium glutamicum is an excellent platform for the production of amino acids, and is widely used in the fermentation industry. Most industrial strains are traditionally obtained by repeated processes of random mutation and selection, but the genotype of these strains is often unclear owing to the absence of genomic information. As such, it is difficult to improve the growth and amino acid production of these strains via metabolic engineering. In this study, we generated a complete genome map of an industrial L-valine-producing strain, C. glutamicum XV. In order to establish the relationship between genotypes and physiological characteristics, a comparative genomic analysis was performed to explore the core genome, structural variations, and gene mutations referring to an industrial L-leucine-producing strain, C. glutamicum CP, and the widely used C. glutamicum ATCC 13032. The results indicate that a 36,349 bp repeat sequence in the CP genome contained an additional copy each of lrp and brnFE genes, which benefited the export of L-leucine. However, in XV, the kgd and panB genes were disrupted by nucleotide insertion, which increase the availability of precursors to synthesize L-valine. Moreover, the specific amino acid substitutions in key enzymes increased their activities. Additionally, a novel strategy is proposed to remodel central carbon metabolism and reduce pyruvate consumption without having a negative impact on cell growth by introducing the CP-derived mutant $H^+$/citrate symporter. These results further our understanding regarding the metabolic networks in these strains and help to elucidate the influence of different genotypes on these processes.

• 한국축산식품학회지
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• 제37권6호
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• pp.940-947
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• 2017

Goat milk has a protein composition similar to that of breast milk and contains abundant nutrients, but its use in functional foods is rather limited in comparison to milk from other sources. The aim of this study was to prepare a goat A2 ${\beta}$-casein fraction with improved digestibility and hypoallergenic properties. We investigated the optimal conditions for the separation of A2 ${\beta}$-casein fraction from goat milk by pH adjustment to pH 4.4 and treating the casein suspension with calcium chloride (0.05 M for 1 h at $25^{\circ}C$). Selective reduction of ${\beta}$- lactoglobulin and ${\alpha}_s$-casein was confirmed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and reverse-phase high-performance liquid chromatography. The hypoallergenic property of A2 ${\beta}$-casein fraction was examined by measuring the release of histamine and tumor necrosis factor alpha from HMC-1 human mast cells exposed to different proteins, including A2 ${\beta}$-casein fraction. There was no significant difference in levels of both indicators between A2 ${\beta}$-casein treatment and the control (no protein treatment). The A2 ${\beta}$-casein fraction is abundant in essential amino acids, especially, branched-chain amino acids (leucine, valine, and isoleucine). The physicochemical properties of A2 ${\beta}$-casein fraction, including protein solubility and viscosity, are similar to those of bovine whole casein which is widely used as a protein source in various foods. Therefore, the goat A2 ${\beta}$-casein fraction may be useful as a food material with good digestibility and hypoallergenic properties for infants, the elderly, and people with metabolic disorders.

• Animal cells and systems
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• 제1권2호
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• pp.323-328
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• 1997

We have previously shown that chick muscle extracts contained at least 10 different ubiquitin C-terminal hydrolases (UCHs). In the present studies, one of the enzymes, called UCH-9, was purified by conventional chromatographic procedures using $^{125}l$-labeled ubiquitin-${\alpha}$NH-MHISPPEPESEEEEE HYC (Ub-PESTc) as a substrate. The purified enzyme behaved as a 27-kDa protein under both denaturing and nondenaturing conditions, suggesting that it consists of a single polypeptide chain. It was maximally active at pHs between 7 and 8.5, but showed little or no activity at pH below 6 and above 10. Lice other UCHs, its activity was strongly inhibited by sulfhydryl blocking reagents, such as iodoacetamide, and by Ub-aldehyde. In addition to Ub-PESTc, UCH-9 hydrolyzed Ub-aNH-protein extensions, including Ub-${\alpha}NH$-carboxyl extension protein of 80 amino acids and Ubo-${\alpha}NH$-dihydrofolate reductase. However, this enzyme was not capable of generating free Ub from mono-Ub-${\varepsilon}NH$-protein conjugates and from branched poly-Ub chains that are ligated to proteins through ${\varepsilon}NH$-isopeptide bonds. This enzyme neither could hydrolyze poly-His-tagged di-Ub. These results suggest that UCH-9 may play an important role in production of free Ub and ribosomal proteins from their conjugates.

• BMB Reports
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• 제33권3호
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• pp.195-201
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• 2000

A human gene has been reported that may encode the enzyme acetohydroxyacid synthase. Previously this enzyme was thought to be absent from animals although it is present in plants and many microorganisms. In plants, this enzyme is the target of a number of commercial herbicides and the use of these compounds may need to be reassessed if the human enzyme exists and proves to be susceptible to inhibition. Here we report the construction of several plasmid vectors containing the cDNA sequence for this protein, and their expression in Escherichia coli. High levels of expression were observed, but most of the protein proved to be insoluble. The small amounts of soluble protein contained little or no acetohydroxyacid synthase activity. Attempts to refold the insoluble protein were successful insofar as the protein became soluble. However, the refolded protein did not gain any acetohydroxyacid synthase activity. In vivo complementation tests of an E. coli mutant produced no evidence that the protein is active. Incorrect folding, or the lack of another subunit, may explain the data but we favor the interpretation that this gene does not encode an acetohydroxyacid synthase.