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‣ Exigências de valina para frangos de corte; Requirements of valine for broilers

Ferreira, Nayara Tavares
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 15/12/2011 Português
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O presente estudo teve como objetivo avaliar níveis ótimos de valina digestível. Foram conduzidos três ensaios com frangos de corte da linhagem Cobb nas fases inicial (1-14 dias), crescimento (14-28 dias) e terminação (28-42 dias), com ensaios realizados separadamente para cada fase, tendo como base o método dose-resposta. Para os ensaios de 1 a 14dias e de 14 a 28 dias, 672 amimais foram distribuídos em delineamento inteiramente ao acaso, em oito tratamentos (7 níveis de valina e 1 controle), com 7 repetições, sendo cada unidade experimental composta por 12 aves , porém no ensaio de 28 a 42 dias foram utilizados 560 frangos, igualmente distribuídos como nos ensaios anteriores em 8 tratamentos com 7 repetições com 10 aves cada. Foram formuladas dietas basais pela técnica da diluição, deficientes em valina, contendo níveis de energia, minerais e vitaminas conforme recomendações de Rostagno et al. (2005), para cada fase. As recomendações dos níveis de valina digestível para cada fase, obtidas no presente estudo, foram realizadas com base nas respostas de CA. As recomendações obtidas pelo método dose resposta são de 0,917; 0,905 e 0,783% de valina digestível para frangos de 1 a 14 dias, 14 a 28 e 28 a 42 dias de idade...

‣ Effect of branched-chain amino acids, valine, isoleucine and leucine on the biosythesis of bitespiramycin 4"-O-acylspiramycins

Li,Zhen-Lin; Wang,Yong-Hong; Chu,Ju; Zhuang,Ying-Ping; Zhang,Si-Liang
Fonte: Sociedade Brasileira de Microbiologia Publicador: Sociedade Brasileira de Microbiologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/12/2009 Português
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Bitespiramycin, a group of 4"-O-acylated spiramycins with 4"-O-isovalerylspiramycins as the major components, was produced by recombinantspiramycin-producing strain Streptomyces spiramyceticus harboring a 4"-O-acyltransferase gene. The experiment was initially performed in synthetic medium with 0.5 g l-1 Valine, Isoleucine or Leucine feeding at 36 h cultivation. When valine was fed, the biological titer of bitespiramycin was 45.3% higher than that of the control group, but the relative content of total isovalerylspiramycin components decreased by 22.5%. In the case of ilecine, the biological titer of bitespiramycin and the total isovalerylspiramycins alone were 85% and 72.1% of the control group, respectively. In contrast, the relative content of other acylated spiramycins increased by 54.41%. However, leucine feeding increased the relative content of total isovalerylspiramycins by 41.9% while the biological titer of bitespiramycin was nearly equal to that of the control group. The improvement effect of leucine on the biosynthesis of isovalerylspiramycins was further confirmed by feeding of 2.0 g l-1 leucine to the culture with complex medium. After batch feeding with a total amount of 2.0 g l-1 leucine to the culture from 70 h to 90 h...

‣ Global Expression Profiling and Physiological Characterization of Corynebacterium glutamicum Grown in the Presence of l-Valine

Lange, C.; Rittmann, D.; Wendisch, V. F.; Bott, M.; Sahm, H.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/2003 Português
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Addition of l-valine (50 to 200 mM) to glucose minimal medium had no effect on the growth of wild-type Corynebacterium glutamicum ATCC 13032 but inhibited the growth of the derived valine production strain VAL1 [13032 ΔilvA ΔpanBC(pJC1ilvBNCD)] in a concentration-dependent manner. In order to explore this strain-specific valine effect, genomewide expression profiling was performed using DNA microarrays, which showed that valine caused an increased ilvBN mRNA level in VAL1 but not in the wild type. This unexpected result was confirmed by an increased cellular level of the ilvB protein product, i.e., the large subunit of acetohydroxyacid synthase (AHAS), and by an increased AHAS activity of valine-treated VAL1 cells. The conclusion that valine caused the limitation of another branched-chain amino acid was confirmed by showing that high concentrations of l-isoleucine could relieve the valine effect on VAL1 whereas l-leucine had the same effect as valine. The valine-caused isoleucine limitation was supported by the finding that the inhibitory valine effect was linked to the ilvA deletion that results in isoleucine auxotrophy. Taken together, these results implied that the valine effect is caused by competition for uptake of isoleucine by the carrier BrnQ...

‣ Incorporation of [3H]Leucine and [3H]Valine into Protein of Freshwater Bacteria: Uptake Kinetics and Intracellular Isotope Dilution

Jørgensen, Niels O. G.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1992 Português
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Incorporation of [3H]leucine and [3H]valine into proteins of freshwater bacteria was studied in two eutrophic lakes. Incorporation of both amino acids had a saturation level of about 50 nM external concentration. Only a fraction of the two amino acids taken up was used in protein synthesis. At 100 nM, the bacteria respired 91 and 78% of leucine and valine taken up, respectively. Respiration of 3H and 14C isotopes of leucine gave similar results. Most of the nonrespired leucine was recovered in bacterial proteins, while only up to one-half of the nonrespired valine occurred in proteins. In intracellular pools of the bacteria, [3H]leucine reached an isotope saturation of 88 to 100% at concentrations of >40 nM. For [3H]valine, an isotope equilibrium of about 90% was obtained at concentrations of >80 nM. Within an incubation period of typically 1 h, tritiated leucine and valine incorporated into proteins of the bacteria reached an isotope saturation of 2 to 6%. In a 99-h batch experiment, bacterial protein synthesis calculated from incorporation of leucine and valine corresponded to 31 and 51% (10 nM) and 89 and 97% (100 nM), respectively, of the chemically determined protein production. Measured conversion factors of 100 nM leucine and valine were 6.4 × 1016 and 6.6 × 1016 cells per mol...

‣ Incorporation of [3H]Leucine and [3H]Valine into Protein of Freshwater Bacteria: Field Applications

Jørgensen, Niels O. G.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1992 Português
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Incorporation of leucine and valine into proteins of freshwater bacteria as a measure of bacterial production was tested in two eutrophic Danish lakes and was related to bacterial production measured by thymidine incorporation. In a depth profile (0 to 8 m) in Frederiksborg Castle Lake, incorporation of 100 nM leucine and valine gave similar rates of protein production. In terms of carbon, this production was about 50% lower than incorporation of 10 nM thymidine. In another depth profile in the same lake, incorporations of 10 nM valine and 100 nM leucine were identical, but differed from incorporations of 10 nM leucine and 100 nM valine. Bacterial carbon production calculated from incorporations of 10 nM thymidine and 10 nM leucine was similar, whereas 10 nM valine and 100 nM leucine and valine indicated an up to 2.4-fold-higher rate of carbon production. In a diel study in Lake Bagsvaerd, incorporation of 100 nM leucine and valine indicated a similar protein production, but the calculated carbon production was about 1.9-fold higher than the production based on uptake of 10 nM thymidine. Different diel changes in incorporation of the two amino acids and in incorporation of thymidine were observed. In both lakes, concentrations of naturally occurring leucine and valine were <5 nM in most samples. This means that the specific activity of a 3H isotope added at a concentration of 100 nM usually was diluted a maximum of 5%. Net assimilation of natural free amino acids in the lakes sustained 8 to 69% of the net bacterial carbon requirement...

‣ Regulation of the Pool Size of Valine in Escherichia coli K-12

Felice, Maurilio De; Guardiola, John; Malorni, Maria C.; Klopotowski, Tadeusz; Iaccarino, Maurizio
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1974 Português
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Three mutations (ilvH611, ilvH612, and ilvH613) are described which make Escherichia coli K-12 resistant to valine inhibition and are located near leu. The expression of the ilv genes appears to be normal in these mutants since the isoleucine-valine biosynthetic enzymes are not derepressed relative to the wild type. The intracellular concentration of valine is, however, higher in the mutants than in the isogenic ilvH+ strain. These mutants also excrete valine, probably because of the high intracellular concentration of this amino acid. The pool size of valine is regulated independently from that of isoleucine and leucine. The increased intracellular concentration of valine is due to a decreased feedback inhibition that valine exerts on its own biosynthetic pathway. In fact, acetolactate synthase activity assayed in extracts of ilvH612 and ilvH613 mutants is more resistant to valine inhibition than the activity assayed in the ilvH+ isogenic strain. Two forms of acetolactate synthase activity can be separated from these extracts by adsorption and elution on hydroxylapatite. One of them is as sensitive to valine inhibition as that of the wild type, the other is more resistant to valine inhibition.

‣ Effect of a leu-linked mutation on the valine sensitivity of acetohydroxy acid synthase activity in Escherichia coli.

Kline, E L; Brown, C S; Umbarger, H E
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1975 Português
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A spontaneous leu-linked mutation (ilvH2015) in Escherichia coli K-12 made the strain resistant to 1 mM valine and l mM glycylvaline (Val-r) and caused the isoleucine and valine biosynthetic enzyme, acetohydroxy acid synthase, to be less sensitive to feedback inhibition by valine than the wild-type enzyme. Transfer of the ilvDAC deletion into a strain carrying ilvH2015 abolished the effect of the marker on the acetohydroxy acid synthase and rendered it as sensitive to valine as the enzyme in the isogenic control strain without the Val-r marker under both repressing and limiting conditions. In contrast, auxotrophy caused by transfer of an ilvC lesion into the Val-r strain did not interfere with the effect of ilvH2015 on valine sensitivity of acetohydroxy acid synthase. In addition, the presence of the Val-r marker produced minor but significant pleiotropic effects on several other isoleucine and valine biosynthetic enzymes but did not cause derepression of the ilv gene cluster. These studies suggest some type of interaction between a product produced by a gene close to leu and the isoleucine and valine biosynthetic enzymes.

‣ Multivalent Repression and Genetic Derepression of Isoleucine-Valine Biosynthetic Enzymes in Serratia marcescens

Kisumi, Masahiko; Komatsubara, Saburo; Chibata, Ichiro
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1971 Português
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The regulation of the formation of isoleucine-valine biosynthetic enzymes was examined to elucidate the mechanism of isoleucine-valine accumulation by α-aminobutyric acid-resistant (abu-r) mutants of Serratia marcescens. In the isoleucine-valine auxotroph, l-threonine dehydratase, acetohydroxy acid synthetase, and transaminase B were repressed when isoleucine, valine, and leucine were simultaneously added to minimal medium. These enzymes were derepressed at the limitation of any single branched-chain amino acid. Pantothenate, which stimulated growth of this auxotroph, had no effect on the enzyme levels. It became evident from these results that in S. marcescens isoleucine-valine biosynthetic enzymes are subject to multivalent repression by three branched-chain amino acids. The abu-r mutants had high enzyme levels in minimal medium, with or without three branched-chain amino acids. Therefore, in abu-r mutants, isoleucine-valine biosynthetic enzymes are genetically derepressed. This derepression was considered to be the primary cause for valine accumulation and increased isoleucine accumulation.

‣ Regulation of Valine Catabolism in Pseudomonas putida

Marshall, Vincent dePaul; Sokatch, John R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1972 Português
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The activities of six enzymes which take part in the oxidation of valine by Pseudomonas putida were measured under various conditions of growth. The formation of four of the six enzymes was induced by growth on d- or l-valine: d-amino acid dehydrogenase, branched-chain keto acid dehydrogenase, 3-hydroxyisobutyrate dehydrogenase, and methylmalonate semialdehyde dehydrogenase. Branched-chain amino acid transaminase and isobutyryl-CoA dehydrogenase were synthesized constitutively. d-Amino acid dehydrogenase and branched-chain keto acid dehydrogenase were induced during growth on valine, leucine, and isoleucine, and these enzymes were assumed to be common to the metabolism of all three branched-chain amino acids. The segment of the pathway required for oxidation of isobutyrate was induced by growth on isobutyrate or 3-hydroxyisobutyrate without formation of the preceding enzymes. d-Amino acid dehydrogenase was induced by growth on l-alanine without formation of other enzymes required for the catabolism of valine. d-Valine was a more effective inducer of d-amino acid dehydrogenase than was l-valine. Therefore, the valine catabolic pathway was induced in three separate segments: (i) d-amino acid dehydrogenase, (ii) branched-chain keto acid dehydrogenase...

‣ Alanine and Aspartate Formation During Growth on Valine-C14 by Pseudomonas aeruginosa

Sokatch, J. R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1966 Português
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Sokatch, J. R. (University of Oklahoma School of Medicine, Oklahoma City). Alanine and aspartate formation during growth on valine-C14 by Pseudomonas aeruginosa. J. Bacteriol. 92:72–75. 1966.—Pseudomonas aeruginosa grown with dl-valine-4,4′-C14 synthesized alanine labeled mainly in carbons 1 and 3, indicating that the isopropyl carbons of valine were the precursors of pyruvate for alanine formation by a pathway which did not involve randomization of isotope. Alanine from cells grown on valine-1-C14 contained isotope only in the carboxyl carbon, suggesting another route to pyruvate from valine by carbon dioxide fixation. Oxidation of valine to propionyl-coenzyme A (CoA), as it occurs in animal tissues, followed by the oxidation of propionyl-CoA to acrylyl-CoA, lactyl-CoA, and pyruvate, would account for the isotope data. Cells grown on valine oxidized valine, isobutyrate, and propionate immediately, whereas cells grown on acetate did not oxidize valine or isobutyrate and required an induction period before propionate was oxidized. P. aeruginosa grown with propionate-1-C14 or propionate-2-C14 formed alanine-1-C14 and alanine-2-C14, respectively, which agrees with the contention that at least part of the propionate is oxidized via the acrylate pathway. Aspartate formed from valine-1-C14 was labeled only in the carboxyl carbons...

‣ Induced Phenotypic Resistance to Valine in Mycobacterium pellegrino

Horvath, Istvan; Szentirmai, A.; Zsadanyi, J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1967 Português
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Valine coordinately increases the levels of three of the enzymes participating in the biosynthesis of isoleucine and valine in Mycobacterium pellegrino. The amount of valine required for end-product induction depends on the condition of the cells. Isoleucine inhibits the effect of valine. Acetohydroxy acid synthetase, the enzyme catalyzing the first common step in the biosynthesis of valine and isoleucine, is inhibited by valine. The induction effect of valine appears to be due to its ability to inhibit the activity of this enzyme, thus causing isoleucine deficiency, which in turn leads to derepression. This conclusion is supported by the fact that valine, under certain conditions, inhibits growth.

‣ REGULATORY MECHANISMS IN THE BIOSYNTHESIS OF ISOLEUCINE AND VALINE I. : Genetic Derepression of Enzyme Formation

Ramakrishnan, T.; Adelberg, Edward A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1964 Português
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Ramakrishnan, T. (Yale University, New Haven, Conn.), and Edward A. Adelberg. Regulatory mechanisms in the biosynthesis of isoleucine and valine. I. Genetic derepression of enzyme formation. J. Bacteriol. 87:566–573. 1964.—A total of 60 mutants of Escherichia coli K-12 resistant to 10−2m valine were isolated from the valine-sensitive F′ strain AB1206. Conjugation experiments showed that in five of these mutants the valine-resistance locus is closely linked to the structural genes governing isoleucine-valine biosynthesis. In these five valine-resistant mutants, three enzymes of the isoleucine-valine pathway were found to be coordinately derepressed: l-threonine deaminase, dihydroxy acid dehydrase, and transaminase B. Two other enzymes of this pathway, the condensing enzyme and the reductoisomerase, were unaffected. The mutation from valine-sensitivity to valine-resistance appears to have altered an operator locus, because the derepressed state is dominant over the repressed state in diploids heterozygous for the valine-resistance locus. The valine-resistant mutants excrete isoleucine into the medium. The significance of these findings with respect to the valine-sensitivity of E. coli K-12 and the regulation of the biosynthesis of isoleucine and valine by this organism are discussed.

‣ VALINE-ISOLEUCINE METABOLISM IN ACETOBACTER SUBOXYDANS AND THE INHIBITION OF GROWTH BY VALINE1

Kerwar, Suresh S.; Cheldelin, Vernon H.; Parks, L. W.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1964 Português
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Kerwar, Suresh S. (Oregon State University, Corvallis), Vernon, H. Cheldelin, and L. W. Parks. Valine-isoleucine metabolism in Acetobacter suboxydans and the inhibition of growth by valine. J. Bacteriol. 88:179–186. 1964.—Extracts of Acetobacter suboxydans can synthesize valine and isoleucine via acetolactate and acetohydroxybutyrate, respectively. The amounts of these amino acids synthesized from different intermediates were determined. The pathways appear to be identical to those described for yeast, Neurospora, and Escherichia coli. When exogenous valine was added to a synthetic growth medium inoculated with A. suboxydans, no growth of the culture was observed. The inhibitory effect of valine was reversed by the addition of isoleucine. The site and mechanism of valine inhibition were investigated. Threonine deaminase was inhibited by valine and isoleucine but not by leucine. Repression of the deaminase by isoleucine but not by valine was indicated. The data reported in this paper suggest that valine prevented growth of the organism through false feedback inhibition of threonine deaminase, thereby limiting isoleucine biosynthesis.

‣ ISOLEUCINE AND VALINE METABOLISM IN ESCHERICHIA COLI XI. K-12: Valine Inhibition of the Growth of Escherichia coli Strain1

Leavitt, Richard I.; Umbarger, H. E.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1962 Português
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Leavitt, Richard I. (Harvard Medical School, Boston, Mass.) and H. E. Umbarger. Isoleucine and valine metabolism in Escherichia coli. XI. Valine inhibition of the growth of Escherichia coli strain K-12. J. Bacteriol. 83:624–630. 1962.—The inhibition of the growth of Escherichia coli strain K-12 by valine was shown to be due to the sensitivity of the acetohydroxybutyrate-forming system to valine. It was demonstrated that both E. coli strain W, a strain whose growth is unaffected by valine, and a valine-resistant mutant of strain K-12 have acetolactate- and acetohydroxybutyrate-forming systems which are less sensitive to valine than that of strain K-12. It was further shown that α-aminobutyrate accumulates in the culture fluid of the valine-sensitive strain when incubated in the presence of valine. The levels of valine in the “free amino acid pool” were examined and found to be related to the differences in valine sensitivity of the acetolactate-forming systems of the three strains.

‣ Valine-Resistance, a Potential Marker in Plant Cell Genetics. I. Distinction between Two Types of Valine-Resistant Tobacco Mutants Isolated from Protoplast-Derived Cells

Bourgin, J. P.; Goujaud, J.; Missonier, C.; Pethe, C.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1985 Português
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In previous experiments, seven lines of valine-resistant plants were regenerated from protoplast-derived haploid tobacco mesophyll cells which had been UV mutagenized and submitted to selection by toxic concentrations of valine. In this study we described the transmission of valine-resistance to progeny and a preliminary phenotypical and biochemical characterization of the resistant plants.—Two types were thus distinguished among the seven mutant lines. Valine-resistance of the mutants of the first type (three lines) was transmitted as a single Mendelian dominant character (Vr1), whereas valine-resistance of the second type (four lines) was transmitted as a digenic recessive character (vr2 and vr3). Allelism tests revealed that the four recessive mutant lines yielded resistant progeny when intercrossed and, therefore, bear recessive mutant alleles at the same two unlinked loci.—When cultured at a density of 100 cell/ml, protoplast-derived cells of mutants of the first type had a low level of resistance to valine, whereas protoplast-derived cells of mutants of the second type displayed a high level of resistance to valine and to other amino acids.—According to the results of 14C-labelled amino acid uptake experiments, the amino acid resistance of mutants of the second type...

‣ Valine-Resistance, a Potential Marker in Plant Cell Genetics. II. Optimization of Uv Mutagenesis and Selection of Valine-Resistant Colonies Derived from Tobacco Mesophyll Protoplasts

Grandbastien, M. A.; Bourgin, J. P.; Caboche, M.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1985 Português
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The induction and selection of valine-resistant mutants from haploid tobacco (Nicotiana tabacum L.) mesophyll protoplast-derived cells have been studied. Using cells from an original mutant plant obtained previously, we performed reconstruction experiments in order to determine the best conditions for the recovery of resistant cells among a population of sensitive cells. Optimal selective conditions were shown to depend on various factors including cell density, time of addition of valine and seasonal variations affecting the mother plants.—Using cell densities of approximately 10 4 cells/ml, we defined efficient selective conditions: more than 25% of the putative mutant clones selected from UV-mutagenized protoplasts were reproducibly confirmed to be valine resistant. Further characterization of some regenerated mutant plants indicated that valine-resistance was associated with an uptake deficiency, as in the case of the original mutant plant of the Valr-2 line used for reconstruction experiments. Spontaneous mutation rates for valine-resistance were below accurately detectable levels, i.e., less than 10-6 per cell per generation. Induced mutation frequency varied nonlinearily with UV dose from 10-5 to 5 x 10-4 resistant clones per surviving colony. Two independent loci (vr2 and vr3) were previously shown to be involved in valine-resistance due to amino acid uptake deficiency. Haploid tobacco plants were produced through anther culture from an F1 double-heterozygous plant obtained from a cross between the original mutant plant and a wild-type plant. Study of the level of resistance to valine of protoplast-derived cells allowed the classification of these haploid plants in four types: sensitive...

‣ The role of valine in the biosynthesis of penicillin N and cephalosporin C by a Cephalosporium sp

Warren, S. C.; Newton, G. G. F.; Abraham, E. P.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1967 Português
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1. The production of penicillin N, but not that of cephalosporin C, was inhibited by the addition of d-valine to suspensions in water of washed mycelium of Cephalosporium sp. 8650. The production of cephalosporin C was selectively inhibited by γ-hydroxyvaline. 2. l-[14C]Valine was taken up rapidly and virtually completely by suspensions of washed mycelium but d-[14C]valine and α-oxo[14C]-isovalerate were taken up relatively slowly. 3. Part of the l-valine was rapidly degraded in the mycelium and part was incorporated into protein. Turnover of the valine in the amino acid pool was estimated to occur in 10–17min. 4. No detectable amount of l-[14C]valine was converted into the d-isomer in the mycelium. α-Oxo[14C]isovalerate was rapidly converted into l-[14C]valine in mycelium and mycelial extracts. 5. d-[14C]Valine was partially converted into the l-isomer in the mycelium and 14C from d-valine was incorporated into protein. 6. The labelling of penicillin N and cephalosporin C by 14C from l-[14C]valine was consistent with the view that l-valine is a direct precursor of C5 fragments of both antibiotics and that any intermediates involved are present in relatively small pools in rapid turnover. 7. Labelling of the antibiotics with 14C from d-[1-14C]valine appeared to occur after the latter had been converted into the l-isomer. Unlabelled d-valine did not decrease the efficiency of incorporation of 14C from l-[1-14C]valine. 8. Intracellular peptide material which contained...

‣ Metabolic engineering of Escherichia coli for the production of l-valine based on transcriptome analysis and in silico gene knockout simulation

Park, Jin Hwan; Lee, Kwang Ho; Kim, Tae Yong; Lee, Sang Yup
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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The l-valine production strain of Escherichia coli was constructed by rational metabolic engineering and stepwise improvement based on transcriptome analysis and gene knockout simulation of the in silico genome-scale metabolic network. Feedback inhibition of acetohydroxy acid synthase isoenzyme III by l-valine was removed by site-directed mutagenesis, and the native promoter containing the transcriptional attenuator leader regions of the ilvGMEDA and ilvBN operon was replaced with the tac promoter. The ilvA, leuA, and panB genes were deleted to make more precursors available for l-valine biosynthesis. This engineered Val strain harboring a plasmid overexpressing the ilvBN genes produced 1.31 g/liter l-valine. Comparative transcriptome profiling was performed during batch fermentation of the engineered and control strains. Among the down-regulated genes, the lrp and ygaZH genes, which encode a global regulator Lrp and l-valine exporter, respectively, were overexpressed. Amplification of the lrp, ygaZH, and lrp-ygaZH genes led to the enhanced production of l-valine by 21.6%, 47.1%, and 113%, respectively. Further improvement was achieved by using in silico gene knockout simulation, which identified the aceF, mdh, and pfkA genes as knockout targets. The VAMF strain (Val ΔaceF Δmdh ΔpfkA) overexpressing the ilvBN...

‣ Improvement of the Redox Balance Increases l-Valine Production by Corynebacterium glutamicum under Oxygen Deprivation Conditions

Hasegawa, Satoshi; Uematsu, Kimio; Natsuma, Yumi; Suda, Masako; Hiraga, Kazumi; Jojima, Toru; Inui, Masayuki; Yukawa, Hideaki
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /02/2012 Português
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Production of l-valine under oxygen deprivation conditions by Corynebacterium glutamicum lacking the lactate dehydrogenase gene ldhA and overexpressing the l-valine biosynthesis genes ilvBNCDE was repressed. This was attributed to imbalanced cofactor production and consumption in the overall l-valine synthesis pathway: two moles of NADH was generated and two moles of NADPH was consumed per mole of l-valine produced from one mole of glucose. In order to solve this cofactor imbalance, the coenzyme requirement for l-valine synthesis was converted from NADPH to NADH via modification of acetohydroxy acid isomeroreductase encoded by ilvC and introduction of Lysinibacillus sphaericus leucine dehydrogenase in place of endogenous transaminase B, encoded by ilvE. The intracellular NADH/NAD+ ratio significantly decreased, and glucose consumption and l-valine production drastically improved. Moreover, l-valine yield increased and succinate formation decreased concomitantly with the decreased intracellular redox state. These observations suggest that the intracellular NADH/NAD+ ratio, i.e., reoxidation of NADH, is the primary rate-limiting factor for l-valine production under oxygen deprivation conditions. The l-valine productivity and yield were even better and by-products derived from pyruvate further decreased as a result of a feedback resistance-inducing mutation in the acetohydroxy acid synthase encoded by ilvBN. The resultant strain produced 1...

‣ Valine substituted winter flounder 'antifreeze': preservation of ice growth hysteresis

Haymet, A D J; Ward, Leanne G; Harding, Margaret; Knight, Charles A
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
Português
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Three mutant polypeptides of the type 1 37-residue winter flounder 'antifreeze' protein have been synthesized. All four threonine residues in the native peptide were been mutated to serine, valine and glycine respectively and two additional salt bridges were incorporated into the sequences in order to improve aqueous solubility. The peptides were analyzed by nanoliter osmometry, the 'ice hemisphere' test, the 'crystal habit' test, measurement of ice growth hysteresis and CD spectroscopy. While the valine and serine mutants retain the α-helical structure, only the valine mutant retains 'antifreeze' activity similar to that of the native protein. These data show that the threonine hydroxyl groups do not play a crucial role in the accumulation of the native 'antifreeze' protein at the ice/water interface and the inhibition of ice growth below the equilibrium melting temperature.