• Title/Summary/Keyword: dehydratase

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Effects of Na-alginate on the Toxicity Induced by Lead and Cadmium in Albino Rats (Na-alginate 투여가 白鼠의 鉛과 카드뮴 독성에 미치는 영향)

  • Lee, Sang Bong
    • Journal of Environmental Health Sciences
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    • v.12 no.2
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    • pp.17-26
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    • 1986
  • Earlier studies have shown that the administration of Na-alginate, a water-soluble, non absorbable acidic polysaccharide, effectively reduces the absorption of radioactive strontium, cadmium and other bivalent metals. In this study the effects of Na-alginate on the Toxicity induced by Lead and Cadmium were examined in rats and the following results were obtained. 1. The elevations of the activities of serum GOT, GPT by Pb and Cd administration in the experimental animals. However, the administration of Na-alginate significantly inhibited the elevation of serum GOT, GPT. 2. Generally serum TBA values were significantly elevated in Pb and Cd group in comparison with the control group. However, the administration of Na-alginate inhibited the elevation of serum TBA values. 3. The activity of Alkaline phosphatase was also significantly elevated in Cd group in comparison with the control group. However, the administration of Na-alginate significantly inhibited the elevation of serum alkaline phsphatase. 4. Furthermore, the activity of $\delta$-aminolevulinic acid dehydratase in red blood cells was also significantly inhibited by Pb and this inhibition was stimulated by the administration of Na-alginate. 5. The concentrations of Pb in liver and Cd in kidney were markedly increased by Pb and Cd. However, the administration of Na-alginate significantly inhibited the elevation of them.

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Premature Release of Polyketide Intermediates by Hybrid Polyketide Synthase in Amycolatopsis mediterranei S699

  • Hong, Jay-Sung-Joong;Choi, Cha-Yong;Yoo, Yeo-Joon
    • Journal of Microbiology and Biotechnology
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    • v.13 no.4
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    • pp.613-619
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    • 2003
  • The polyketide backbone of rifamycin B is assembled by the type I rifamycin polyketide synthase (PKS) encoded by the rifA-rifE genes. In order to produce novel analogs of rifamycin via engineering of the PKS genes, inactivation of the ${\beta}-ketoacyl:acyl$ carrier protein reductase (KR) domain in module 8 of rifD, by site-specific mutagenesis of the NADPH binding site, was attempted. Module 8 contains a nonfunctional dehydratase (DH) domain and a functional KR domain that is involved in the reduction of the ${\beta}-carbonyl$ group, resulting in the C-21 hydroxyl of rifamycin B. This mutant strain produced linear polyketides, from tetraketide to octaketide, which were also produced by a rifD-disruption mutant as a consequence of premature termination of the polyketide assembly. Another attempt to replace the DH domain of module 7, which has been considered nonfunctional, with a functional homolog derived from module 7 of rapamycin-producing PKS also resulted in the production of linear polyketides, including the heptaketide intermediate and its precursors. Premature release of the carbon chain assembly intermediates is an unusual property of the rifamycin PKS. that is not seen in other PKSs such as the erythromycin PKS.

Expression of Acid Stress-Induced Proteins of Streptococcus mutans Isolated from Korean Children with Caries (한국인 우식아동으로부터 분리한 Streptococcus mutans의 내산성 단백질의 발현)

  • Kang, Kyung-Hee;Nam, Jin-Sik;Jin, Ing-Nyol
    • Journal of the Korea Academia-Industrial cooperation Society
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    • v.10 no.7
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    • pp.1766-1772
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    • 2009
  • In this study, we are interested in comparing the protein profiles of acid-shocked and control cells of S. mutans isolated from Korean children with caries. The results of 2D gel electrophoresis showed that twelve proteins are up-regulated when the cells were grown under 20 mM lactic acid stress in the exponential phase. Up-proteins under acid stress were estimated a major key of the survival and proliferation of S. mutans in low pH environments. These proteins are estimated generally associated with three biochemical pathways: glycolysis, alternative acid production and branched-chain amino acid biosynthesis.

Shikimate Metabolic Pathway Engineering in Corynebacterium glutamicum

  • Park, Eunhwi;Kim, Hye-Jin;Seo, Seung-Yeul;Lee, Han-Na;Choi, Si-Sun;Lee, Sang Joung;Kim, Eung-Soo
    • Journal of Microbiology and Biotechnology
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    • v.31 no.9
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    • pp.1305-1310
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    • 2021
  • Shikimate is a key high-demand metabolite for synthesizing valuable antiviral drugs, such as the anti-influenza drug, oseltamivir (Tamiflu). Microbial-based strategies for shikimate production have been developed to overcome the unstable and expensive supply of shikimate derived from traditional plant extraction processes. In this study, a microbial cell factory using Corynebacterium glutamicum was designed to overproduce shikimate in a fed-batch culture system. First, the shikimate kinase gene (aroK) responsible for converting shikimate to the next step was disrupted to facilitate the accumulation of shikimate. Several genes encoding the shikimate bypass route, such as dehydroshikimate dehydratase (QsuB), pyruvate kinase (Pyk1), and quinate/shikimate dehydrogenase (QsuD), were disrupted sequentially. An artificial operon containing several shikimate pathway genes, including aroE, aroB, aroF, and aroG were overexpressed to maximize the glucose uptake and intermediate flux. The rationally designed shikimate-overproducing C. glutamicum strain grown in an optimized medium produced approximately 37.3 g/l of shikimate in 7-L fed-batch fermentation. Overall, rational cell factory design and culture process optimization for the microbial-based production of shikimate will play a key role in complementing traditional plant-derived shikimate production processes.

Crystal Structure of Mesaconyl-CoA Hydratase from Methylorubrum extorquens CM4

  • Jae-Woo Ahn;Jiyeon Hong;Kyung-Jin Kim
    • Journal of Microbiology and Biotechnology
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    • v.33 no.4
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    • pp.485-492
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    • 2023
  • Methylorubrum extorquens, a facultative methylotroph, assimilates C1 compounds and accumulates poly-β-hydroxylbutyrate (PHB) as carbon and energy sources. The ethylmalonyl pathway is central to the carbon metabolism of M. extorquens, and is linked with a serine cycle and a PHB biosynthesis pathway. Understanding the ethylmalonyl pathway is vital in utilizing methylotrophs to produce value-added chemicals. In this study, we determined the crystal structure of the mesaconyl-CoA hydratase from M. extorquens (MeMeaC) that catalyzes the reversible conversion of mesaconyl-CoA to β-methylmalyl-CoA. The crystal structure of MeMeaC revealed that the enzyme belongs to the MaoC-like dehydratase domain superfamily and functions as a trimer. In our current MeMeaC structure, malic acid occupied the substrate binding site, which reveals how MeMeaC recognizes the β-methylmalyl-moiety of its substrate. The active site of the enzyme was further speculated by comparing its structure with those of other MaoC-like hydratases.

Metabolic Engineering of Rational Screened Saccharopolyspora spinosa for the Enhancement of Spinosyns A and D Production

  • Jha, Amit Kumar;Pokhrel, Anaya Raj;Chaudhary, Amit Kumar;Park, Seong-Whan;Cho, Wan Je;Sohng, Jae Kyung
    • Molecules and Cells
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    • v.37 no.10
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    • pp.727-733
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    • 2014
  • Spinosyns A and D are potent ingredient for insect control with exceptional safety to non-target organisms. It consists of a 21-carbon tetracyclic lactone with forosamine and tri-Omethylated rhamnose which are derived from S-adenosyl-methionine. Although previous studies have revealed the involvement of metK1 (S-adenosylmethionine synthetase), rmbA (glucose-1-phosphate thymidylyltransferase), and rmbB (TDP-D-glucose-4, 6-dehydratase) in the biosynthesis of spinosad, expression of these genes into rational screened Saccharopolyspora spinosa (S. spinosa MUV) has not been elucidated till date. In the present study, S. spinosa MUV was developed to utilize for metabolic engineering. The yield of spinosyns A and D in S. spinosa MUV was $244mgL^{-1}$ and $129mgL^{-1}$, which was 4.88-fold and 4.77-fold higher than that in the wild-type ($50mgL^{-1}$ and $27mgL^{-1}$), respectively. To achieve the better production; positive regulator metK1-sp, rmbA and rmbB genes from Streptomyces peucetius, were expressed and co-expressed in S. spinosa MUV under the control of strong $ermE^*$ promoter, using an integration vector pSET152 and expression vector pIBR25, respectively. Here-with, the genetically engineered strain of S. spinosa MUV, produce spinosyns A and D up to $372/217mgL^{-1}$ that is 7.44/8.03-fold greater than that of wild type. This result demonstrates the use of metabolic engineering on rationally developed high producing natural variants for the production.

Effects of Ethanol on the Subacute Lead Poisoning in Rats (흰쥐에서 아급성 연중독에 미치는 에탄올의 영향에 관한 연구)

  • 이용욱;박성관;이선동
    • Journal of Environmental Health Sciences
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    • v.21 no.3
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    • pp.56-66
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    • 1995
  • This study was performed to investigate the effects of Ethanol on the lead poisoning in rats. For this experiment, 48 male Sprague-Dawley strain were used. The experimental groups were divided into six: a normal control(Control), 200 mg/kg b.w. lead(Pd), 5% ethanol(E5), 10% ethanol(E10), 200 mg/kg b.w. lead plus 5% ethanol(PE5) and 200 mg/kg b.w. lead plus 10% ethanol(PE10). Lead was dissolved in the distilled water and administered orally. Ethanol was given with drinking water ad libitum. The rats were allocated to each group by 8 and sacrificed for 5 weeks. The results were as follows: 1. The mean body weight of each group were increased constantly in all groups during experimental period, but the values of ethanol treatment groups were higher than that of control (Control), lead treatment group(Pb) (P<0.01). 2. Compared to Control and Pb, the relative weight of liver and brain were increased in all the ethanol fed groups. But the relative weight of organs were not observed significantly. 3. The lead concentration of organs were high in the group treated with lead(Pb, PES, PE10) (P<0.01), and PE5, PE10 were high compared with Pb in brain especially(P<0.01). However, no statistical significance were showed between PE5 and PE10. 4. The concentration of serum ALT was increased by lead plus ethanol (PE5, PE10) significantly (P<0.01). 5. The concentration of Hematocrit, hemoglobin, WBC and RBC were not observed difference significantly in all groups.

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Characterization of dihydroflavonol 4-reductase cDNA in tea [Camellia sinensis (L.) O. Kuntze]

  • Singh, Kashmir;Kumar, Sanjay;Yadav, Sudesh Kumar;Ahuja, Paramvir Singh
    • Plant Biotechnology Reports
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    • v.3 no.1
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    • pp.95-101
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    • 2009
  • Tea leaves are major source of catechins—antioxidant flavonoids. Dihydroflavonol 4-reductase (DFR, EC 1.1.1.219) is one of the important enzymes that catalyzes the reduction of dihydroflavonols to leucoanthocyanins, a key ''late'' step in the biosynthesis of catechins. This manuscript reports characterization of DFR from tea (CsDFR) that comprised 1,413 bp full-length cDNA with ORF of 1,044 bp (115-1,158) and encoding a protein of 347 amino acids. Sequence comparison of CsDFR with earlier reported DFR sequences in a database indicated conservation of 69-87% among amino acid residues. In silico analysis revealed CsDFR to be a membrane-localized protein with a domain (between 16 and 218 amino acids) resembling the NAD-dependent epimerase/dehydratase family. The theoretical molecular weight and isoelectric point of the deduced amino sequence of CsDFR were 38.67 kDa and 6.22, respectively. Upon expression of CsDFR in E. coli, recombinant protein was found to be functional and showed specific activity of 42.85 nmol $min^{-1}$ mg $protein^{-1}$. Expression of CsDFR was maximum in younger rather than older leaves. Expression was down-regulated in response to drought stress and abscisic acid, unaffected by gibberellic acid treatment, but up-regulated in response to wounding, with concomitant modulation of catechins content. This is the first report of functionality of recombinant CsDFR and its expression in tea.

Growth and Chlorophyll Biosynthesis of Vigna angularis under Lead Stress

  • Suh-Young Koo;Sun
    • Journal of Environmental Science International
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    • v.1 no.2
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    • pp.145-155
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    • 1992
  • The effect of various supplies of lead singly and in combination with aluminium on growth and chlorophyll biosynthesis was investigated in 7-day-old Vigna anguluris seedlings. Expose to 50 $\mu$N Pb or more drastically reduced root elongation rate. Significant depressions in root growth was observed within 1 day and no recovery of growth was seen over the duration of treatment period. Root elongation decreased depending on the Pb concentrations. Root growth inhibition was stronger than shoot growth inhibition. The initiation of lateral roots appeared to be more sensitive to Pb than the growth of main roots. Inhibition of root and shoot elongation by Pb was lessened by combined exposure of Pb and Al, suggesting that the presence of AA reverse the inhibitory effect of Pb alone. With the histochemical sodium rhodizonate method the rate of Pb uptake was dependent on the Pb concentration and exposure time of the roots to Pb salts. Pb was first deposited on the root surface and then translocated radially in the root cap cells. During a longer Pb administration (up to 72 h) Pb penetration was nonuniform, with accumulation within the cortex or endodermis. There was drastic reduction in chlorophyll content by Pb. The Pb inhibition of chlorophyll synthesis was concentration dependent. 5-Aminolevulinic acid dehydratase (ALAD) activity exhibited distinct inhibition from control. Reduction in chlorophyll content was accompanied by proportional changes in ALAD activity. Chlorophyll content and ALAD activity were less affected by combined exposure of Pb and Al, suggesting that Al has a protective effect against the inhibiting action of Pb on photosynthetic activity.

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Identification of Genes for Growth with Oxygen in Escherichia coli by Operon Fusion and Southern Blot Techniques

  • Kim, Il-Man;Lee, Yong-Chan;Won, Jae-Seon;Choe, Mu-Hyeon
    • Journal of Microbiology and Biotechnology
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    • v.13 no.6
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    • pp.976-983
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    • 2003
  • Seven Escherichia coli cells defective with aerobic growth were isolated by the insertion of ${\lambda}placMu53$, a hybrid bacteriophage of ${\lambda}$ and Mu, which created a transcriptional fusion to lacZY. These insertion mutant cells were tested on an XG ($5-bromo-4-chloro-3-indolyl-{\beta}-D-galactopyranoside$) medium for anaerobic expression of lacZ by fusion to a promoter. The chromosomal DNA from these strains were digested by EcoRI, and the EcoRI fragments that contained the fused gene and lacZ sequence were identified by Southern hybridization, using lacZ containing plasmid as a probe. The EcoRI fragment from each strain was cloned and sequenced. The sequence data were compared with the GenBank database. The mutated gene of three strains, CYT4, CYT5, and OS11, was found to be identical, and it was nrdAB that encoded ribonucleoside diphosphate reductase. The gene nrdAB was at min 50.5 on the Escherichia coli linkage map and 2,348,084 on the physical map, and is involved in hemAe-related reduction-oxidation reaction. OS6 and OS14 mutant strains had insertion at min 8.3 and the mutated gene was hemB. The hemB encodes 5-aminolevulinate dehydratase or porphobilinogen synthase. The OS3 mutant had insertion in cydB at min 16.6. The cydAB encodes cytochrome d oxidase. In the case of OS1, the fusion was made with sucA, the E1 component of ${\alpha}-ketoglutarate$ dehydrogenase.