• Parasites, Hosts and Diseases
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• 제21권1호
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• pp.49-57
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• 1983

The distribution and Properties of branched chain amino acid aminotransferase (EC 2.6. 1.42) was investigated in adult Fasciola hepatica. Fascicla hepatica was fractionated by differential centrifugation into nuclear, mitochondrial and cytosolic fractions. The activity of branched chain amino acid aminotransferase was measured by the method of Ichihara and Koyama (1966) . Isozyme patterns of this enzyule was also examined by DEAE-cellulose column chromatography. The results obtained were as follows; 1. The activity in homogenate was found to be 12.69 units/g wet tissue. The activity of this enzyme was relatively high compared with those in rat tissues. 2. The distribution of branched chain amino acid aminotransferase in the subcellular organelles showed that 87.8% of the activity was in cytosolic, 10.9% in mitochondrial and 1.3% was in nuclear fraction. 3. Cytosolic fraction of Fasciola hepatica contained Enzyme I, but not Enzyme II and III, of branched chain amino acid aminotransferase. Ensyme I was eluted by 50mM phosphate buffier from DEAE-cellulose column and catalyzed the transamination of all three branched chain amino acids. 4. The Enzyme I was purified about 22-folds increase in specific activity after chromatography on DEAE-cellulose. 5. The best substrate among three amino acids (leucine, isoleucine and valise) was L-isoleucine. 6. The optimal temperature of Enzyme I was $45^{\circ}C$ and the optimal pH was 8.2. 7. The Km value for leucine of Enzyme I was 4.17 mM. 8. The Km values for a-ketoglutarate and pyridoxal phosphate of Enzyme I were 0.41mM and $4.76{\times}10^{-3}{\;}mM$, respectively.

• 미생물학회지
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• 제35권2호
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• pp.133-138
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• 1999

Bacillus subtilis ED 213의 cytidine deaminase 의 활성부위에 존재하는 필수 아미노산잔기를 화학수식 방법으로 측정하였다. 본 효소는 1mM o-phenanthroline 에 의하여 효소활성이 43% 저해되어 효소활성 발현에 Fe\sup 2+\가 요구된다고 추정되며, 1mM ethylenediaminetetraacetic acid 에 의해서는 효소활성이 오히려 28% 정도 촉진되었다. 본 효소는 1mM N-bromosuccinimide, 1mM chloramine-T 와 1mM $\rho$-chloromercuribenzoic acid에 의하여 100% 저해되었으며, 그의 저해 양상은 경쟁적 저해 양상을 나타내었다. 본 효소의 효소활성은 1mM pyridoxal-5-phosphate 에 의항 36% 저해되었으며, 1mM 1ethyl-3-carbodiamide 와 1mM glycine methylester에 의해 저해된 효소활성이 5mM cysteine에 의해 완전히 회복되었다. 이상의 결과로부터 Bacillus subtilis ED 213 cytidine deaminase의 활성부위에는 tyrosine, methionine, cysteine 과 serine 잔기가 관여할 뿐만 아니라 lysine 과 glycine 도 효소활성에 관여하는 것으로 추정된다.

• Journal of Microbiology and Biotechnology
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• 제18권6호
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• pp.1136-1140
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• 2008

The Rhodopseudomonas palustris KUGB306 hemA gene codes for 5-aminolevulinic acid (ALA) synthase. This enzyme catalyzes the condensation of glycine and succinyl-CoA to yield ALA in the presence of the cofactor pyridoxal 5'-phosphate. The R. palustris KUGB306 hemA gene in the pGEX-KG vector system was transformed into Escherichia coli BL21. The effects of physiological factors on the extracellular production of ALA by the recombinant E. coli were studied. Terrific Broth (TB) medium resulted in significantly higher cell growth and ALA production than did Luria-Bertani (LB) medium. ALA production was significantly enhanced by the addition of succinate together with glycine in the medium. Maximal ALA production (2.5 g/l) was observed upon the addition of D-glucose as an ALA dehydratase inhibitor in the late-log culture phase. Based on the results obtained from the shake-flask cultures, fermentation was carried out using the recombinant E. coli in TB medium, with the initial addition of 90 mM glycine and 120 mM succinate, and the addition of 45 mM D-glucose in the late-log phase. The extracellular production of ALA was also influenced by the pH of the culture broth. We maintained a pH of 6.5 in the fermenter throughout the culture process, achieving the maximal levels of extracellular ALA production (5.15 g/l, 39.3 mM).

• 한국미생물·생명공학회지
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• 제19권1호
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• pp.14-20
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• 1991

Pseudomonas syringae의 intact cell에서 에틸렌 생성을 극대화 하기 위한 전환조건은 $30^{\circ}C$. pH7.5로 조사되었고, 다양한 기질의 전환효과를 검초한 결과, Asn>Gln>Asp>Glu>$\alpha$-KG>citrate>oxalacetate의 순으로 많은양의 에틸렌을 생성하였다. 또한, arginine과 histidine을 상기 유기산과 함께 넣었을 때 에틸렌 생성에 현저한 상승효과를 나타냈다. Cell-free system 에서는 $\alpha$-KG>Glu>citrate>Gln>Ser순으로 0.5mM $\alpha$-KG에서 310.8(nl.mg $protein^[-1}.h^[-1}$)로 가장 많은 에틸렌을 생성하였고, aminotransferse 억제제인 AOA를 사용해 본 결과, Glu는 glutamate dehydrogenase에 의하여 $\alpha$-KG를 거쳐서 에틸렌으로 전환된 것이라 생각된다.

• Journal of Microbiology and Biotechnology
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• 제27권7호
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• pp.1216-1222
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• 2017

To develop starters for the production of functional foods or materials, lactic acid bacteria producing ${\gamma}-aminobutyric$ acid (GABA) were screened from jeotgals, Korean fermented seafoods. One isolate producing a high amount of GABA from monosodium $\text\tiny{L}$-glutamate (MSG) was identified as Enterococcus avium by 16S rRNA gene sequencing. E. avium M5 produced $18.47{\pm}1.26mg/ml$ GABA when incubated for 48 h at $37^{\circ}C$ in MRS broth with MSG (3% (w/v)). A gadB gene encoding glutamate decarboxylase (GAD) was cloned and overexpressed in E. coli BL21 (DE3) using the pET26b (+) expression vector. Recombinant GAD was purified through a Ni-NTA column and the size was estimated to be 53 kDa by SDS-PAGE. Maximum GAD activity was observed at pH 4.5 and $55^{\circ}C$and the activity was dependent on pyridoxal 5'-phosphate. The $K_m$ and $V_{max}$ values of GAD were $3.26{\pm}0.21mM$ and $0.0120{\pm}0.0001mM/min$, respectively, when MSG was used as a substrate. Enterococcus avium M5 secretes a lot of GABA when grown on MRS with MSG, and the strain is useful for the production of fermented foods containing a high amount of GABA.

• BMB Reports
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• 제32권5호
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• pp.480-485
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• 1999

Tyrosine phenol-lyase of thermophilic Symbiobacterium sp. SC-1, which is obligately and symbiotically dependent on thermophilic Bacillus sp. SK-1, was purified and characterized. The enzyme is composed of four identical subunits and contains approximately 1 mol of pyridoxal 5'-phosphate (PLP) per mol subunit as a cofactor. The enzyme showed absorption maxima at 330 and 420 nm, and lost this absorption profile by treatment with phenylhydrazine. The apparent dissociation constsnt, $K'_D$, for PLP was determined with the apoenzyme to be about $1.2\;{\mu}M$. The isoelectric point was 4.9. The optimal temperature and pH for the $\alpha,\beta$-elimination of L-tyrosine were found to be $80^{\circ}C$ and pH 8.0, respectively. The substrate specificity of the enzyme was very broad: L-amino acids including L-tyrosine, 3,4-dihydroxyphenyl-L-alanine (L-DOPA), L-cysteine, L-serine, S-methyl-L-cysteine, $\beta$-chloro-L-alanine, and S-(o-nitrophenyl)-L-cysteine all served as substrates. D-Tyrosine and D-serine were also decomposed into pyruvic acid and ammonia at rates of 7% and 31% relative to their corresponding L-enantiomers, respectively. D-Alanine, which was inert as a substrate in a, $\beta$-elimination, was the only D-amino acid racemized by the enzyme. The $K_m$ values for L-tyrosine, L-DOPA, S-(o-nitrophenyl)-L-cysteine, $\beta$-chloro-L-alanine, and S-methyl-L-cysteine were 0.19, 9.9, 0.36, 12, and 5.5 mM, respectively.

• Journal of Microbiology and Biotechnology
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• 제18권1호
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• pp.55-58
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• 2008

Alanine racemase, a bacterial enzyme belonging to the fold-type III group of pyridoxal 5'-phosphate (PLP)-dependent enzymes, has been shown to catalyze the interconversion between L- and D-alanine. The alanine racemase from the pathogenic bacterium Enterococcus faecalis v583 has been overexpressed in E. coli and was shown to crystallize an enzyme at 295 K, using polyethylene glycol (PEG) 8000 as a precipitant. X-ray diffraction data to $2.5{\AA}$ has been collected using synchrotron radiation. The crystal is a member of the orthorhombic space group, $C222_1$ with unit cell parameter of a=94.634, b=156.516, $c=147.878{\AA},\;and\;{\alpha}={\beta}={\gamma}=90{\AA}$. Two or three monomers are likely to be present in the asymmetric unit, with a corresponding $V_m\; of\;3.38{\AA}^3\;Da^{-1}\;and\;2.26{\AA}^3\;Da^{-1}$ and a solvent content of 63.7% and 45.5%, respectively.

• Journal of Applied Biological Chemistry
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• 제54권4호
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• pp.231-237
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• 2011

A gene encoding a putative alanine racemase in Xanthomonas. oryzae pv. oryzae was cloned, expressed and characterized. Expression of the cloned gene was performed in Escherichia coli BL21(DE3)pLys using a pET-21(a) vector harbouring $6{\times}histidine$ tag. Purification of the recombinant alanine racemase by affinity chromatography resulted in major one band by sodium dodecyl sulfate polyacryl amide gel electrophoresis analysis, showing about 45 kDa of molecular weight. The alanine racemase gene, cloned in this experiment, appears to be constitutively expressed in X. oryzae, as analyzed by reverse transcriptase polymerase chain reaction. The enzyme was the most active toward L-alanine and secondly D-alanine, showing a racemic reaction, thus the enzyme is considered as an alanine racemase. The enzyme was considerably activated by addition of pyridoxal-5-phosphate (PLP), showing that 75% increase in activity was observed at 0.3 mM, compared with control. D-Cysteine as well as L-cysteine significantly inhibited the enzyme activity. The inhibitions by cysteines were more prominent in the absence of PLP, showing 9 and 5% of control activity at 2 mM of addition, respectively. The enzyme was the most active at pH 8.0 and more stable at alkaline pHs than acidic pH condition.

• Journal of Microbiology and Biotechnology
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• 제25권7호
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• pp.1108-1113
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• 2015

Cadaverine (1,5-diaminopentane) is an important industrial chemical with a wide range of applications. Although there have been many efforts to produce cadaverine through fermentation, there are not many reports of the direct cadaverine production from lysine using biotransformation. Whole-cell reactions were examined using a recombinant Escherichia coli strain overexpressing the E. coli MG1655 cadA gene, and various parameters were investigated for the whole-cell bioconversion of lysine to cadaverine. A high concentration of lysine resulted in the synthesis of pyridoxal-5'-phosphate (PLP) and it was found to be a critical control factor for the biotransformation of lysine to cadaverine. When 0.025 mM PLP and 1.75 M lysine in 500 mM sodium acetate buffer (pH6) were used, consumption of 91% lysine and conversion of about 80% lysine to cadaverine were successfully achieved.

• Nutritional Sciences
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• 제8권3호
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• pp.169-174
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• 2005

This study investigated the effect of allium vegetable intake on the storage and utilization of energy substrates before, during, and after exercise in tissues of rats. Ninety mts were fed either a control diet or a diet with added allium sativum (AS), allium cepa (AC), allium fistulosum (AF), or allium tuberosum (AT) for 4 weeks and then subdivided into 3 groups: before-exercise (BE) during-exercise (DE) after-exercise (AE). The DE group exercised on treadmill for 1 hour just before being sacrificed at the end of the 4th week of the dietary treatment Rats in the AE group were allowed to recuperate for 2 horns after being exercised like the DE group. Pyriooxal 5'-phospwe (PLP) levels were compared in plasma, liver and skeletal muscle of rats. There was no difference between AS animals and control animals in plasma PLP levels regardless of exercise. The plasma PLP levels of AC animals were higher than those of control animals before exercise but this PLP was decreased with exercise and lower than that of control animals after exercise. The plasma PLP levels of AF animals were higher than those of control animals during exercise but there was no difference before and after exercise. The plasma PLP levels of AT animals were higher than those of control animals regardless of exercise. Compared to those of control mts, the PLP levels of liver and muscle were significantly lower in AS, AC, AF and AT mts before exercise. The levels of liver PLP were significantly decreased in control mts while not changed in AS, AC, AF and AT mts during exercise. The levels of liver PLP tended to decrease in AS, AC and AF mts after exercise. The levels of muscle PLP were significantly decreased in control rats, while not changed in AS, AC and AF mts during exercise. The levels of muscle PLP were decreased in control mts but not changed in AS, AC and AF mts after exercise. Thus, it is suggested that the changes of PLP concentrations in plasma and tissues induced by exercise are affected by allium vegetable diet and demonstrated that allium vegetable intake induced an alteration in the redistribution of PLP among tissues.