• BMB Reports
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• v.32 no.1
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• pp.20-24
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• 1999

The first step of the branch pathway in tryptophan biosynthesis is catalyzed by anthranilate synthase, which is subjected to feedback inhibition by the end product of the pathway. The $trpE^{FBR}$ gene from a mutant Escherichia coli strain coding for anthranilate synthase that was insensitive to feedback inhibition by tryptophan has been cloned. To identify the amino acid changes involved in the feedback regulation of anthranilate synthase, the nucleotide sequence of the mutant $trpE^{FBR}$ gene was determined. Sequence analysis of the $trpE^{FBR}$ gene revealed that four bases were changed in the structural gene while alteration was not found in the 5' control region. Among these base changes, only two base substitutions caused the alterations in amino acid sequences. From the results of restriction fragment exchange mapping, the 61st nucleotide, C to A substitution, that changed $Pro^{21}{\rightarrow}Ser$ was identified as the cause of the desensitization to feedback inhibition by tryptophan. Additional feedback-resistant enzymes of the E. coli anthranilate synthases were constructed by site-directed mutagenesis to examine the effect of the $Ser^{40}\;{\rightarrow}\;Arg^{40}$ change found in the $trpE^{FBR}$ gene of Brevibacterium lactofermentum. From the feedback inhibition analysis, the $Pro^{21}{\rightarrow}Ser$ and $Ser^{40}{\rightarrow}Arg$ mutants maintained about 50% and 90% of their maximal activities, respectively, even at the extreme concentration of 10 mM tryptophan. From these results, we suggest that the $Pro^{21}$ and $Ser^{40}$ residues are involved in the tryptophan binding in the E. coli enzyme.

• Bulletin of the Korean Chemical Society
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• v.7 no.1
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• pp.78-81
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• 1986

The photosensitized lysis of egg lecithin lipid membranes (liposomes) have been performed to UV-B light (270-320 nm) by L-tryptophan(L-Trp) and its peptide such as N-acetylphenylalanyl-L-tryptophan(NAPT) incorporated in the liposomes(ca. 0.1% by weight) or in the external buffer (0.1-0.3 mM). Requirement of oxygenation suggests that the lysis of liposomes is caused by the photosensitized oxidation of lipids. There was significant protection against lysis photosensitized by Trp in the external buffer by low concentration of ferricyanide (0.8 mM), but there was no effect on the lytic efficiency by $N_3^-$ which is singlet oxygen($^1O_2$) quencher, indicative of an electron transfer mechanism involved in the photosensitization. The small change of the lytic efficiency with increasing pH from 4 to 9 was interpreted by large target theory and subsequently indicates that superoxide($O_2^-$) may be an active intermediate for the oxidation. The efficiency of photosensitization of Trp was higher than that of NAPT under the same experimental condition. The weak lytic efficiency of liposomes photosensitized by NAPT was enhanced by incorporating NAPT in liposomes, but it was again quenched by ${\beta}$-carotene incorporated in the bilayer of liposomes. These results indicate that a portion of liposome lysis may be due to $^1O_2$ formation from the excited NAPT.

• Journal of Plant Biology
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• v.39 no.4
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• pp.243-247
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• 1996

Phosphoribosylanthranilate transferase (PAT) catalyzes the second step of the tryptophan biosynthetic pathway and is encoded by a single-copy gene that complements all the visible phenotypes of the tryptophan mutant (trp1-100) of Arabidopsis. The trp1-100 is blue fluorescent under UV light becuase it accumulates anthranilate. To obtain a plant with reduced PAT activity, PAT1 genes with several internal deletions in different promoter regions (pHS 101, pHS102, pHS104, pHS105, and pHS107) were induced into trp1-100 via Agrobacterium. Then, homozygous T3 plants were isolated and examined for blue fluorescence. Introduction of the PAT1 gene fusants results in the reversion of fluorescence phenotype except in the case of pHS105. These results prompted us to perform a parallel analysis of anthranilate synthase and PAT interms of the genetic complementation. A plant line carrying pHS105 gene fusant does not completely complement the blue fluorescence but it accumulates less anthranilate than trp1-100. The activity of PAT was reduced in the transgenic mutant as well. The plant carrying these constructs will add to the growing collection of molecular tools for the study of the indolic secondary metabolism.

• Journal of the Korean Dietetic Association
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• v.3 no.2
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• pp.105-111
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• 1997

The inhibitory effect of rice(Oryza sartiva L., illpumbyeo) against mutagenicity induced by tryptophan pyrolysates were investigated using Salmonella typhimurium reversion assay. Both methanol extracts of obtained from brown and white rice were found to possess strong activites of inhibiting the mutagenicities of 3-amino-1,4-dimethyl-5H-pyriod[4,3-b]indol(Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indol(Trp-P-2) on Salmonella typhimurium reversion assay. As the concentration of methanol extract increased, inhibitory effect on mutagenicity increased but reached at steady state as inhibition rate of 90% when the concentration was above 10mg/plate. There was no significant difference(p>0.05) in inhibitory effect of methanol extracts between brown and white rice against tryptophan pyrolysates.

• KSBB Journal
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• v.7 no.4
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• pp.284-289
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• 1992

The optimum conditions for the production of tryptophan using a recombinant Klebsiella pnuemoniae phe A tyr A trp R/pSC 101-$trp^{+}$ and its plasmid stability during tryptophan production were studied. The optimum temperature was $37^{\circ}C$ and the specific growth rate was 1.05$h^{-1}$ at $37^{\circ}C$. Tryptophan production was increased by glucose fed-batch culture, and tryptophan was accumulated to 0.175 g/l after 36 hrs. This amount was about 1.2 and 1.6 times greater than that obtained from batch culture and flask culture, respectively. The stability of the strain in fed-batch cu1ture was greatly different from that in repeated flask culture. After 6 generation, 95% of total cells was stable in repeated flash culture, but in fed-batch culture only 50% was stable.

• Korean Journal of Agricultural Science
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• v.51 no.1
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• pp.1-8
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• 2024

We conducted this research to examine the reducing level of lysine : tryptophan ratios in the diet affected the performance and meat quality of finishing pigs. At the end of the experiment, 144 crossbred finishing pigs (Duroc × [Yorkshire × Landrace]) having an average body weight of 70.6 ± 3.9 kg were randomly assigned to four dietary treatments (9 replications, 4 pigs per pen). The pigs in the 4 treatments were fed diets with different lysine : tryptophan ratios, such as 1 : 0.175, 1 : 0.160, 1 : 0.145, and 1 : 0.130. In considering average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR), the ratio of tryptophan and lysine (Lys : Trp) did not show any significant effect (p > 0.05). Moreover, nutrient digestibility had no significant impact (p > 0.05). However, the decreasing level of tryptophan linearly decreased the back-fat thickness at overall period (p = 0.038) and reduced at week 5 (p = 0.007). Additionally, the lean meat percentage (LMP) showed a tendency to increase at initial (linear effect, p = 0.097) and increased at overall period (linear effect, p = 0.045). Therefore, we suggest that Lys : Trp ratio of 0.130 could enhance the meat quality in finishing pigs.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.2
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• pp.247-253
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• 2002

Two experiments were conducted to investigate the impact of high temperature and dietary tryptophan (Trp) on performance, selected organ weights and plasma free amino acid (AA) concentrations in broiler chickens. In Experiment 1, exposure to $27-33^{\circ}C$ of chickens for 2 weeks from 2 weeks of age did not affect growth and plasma free AA concentration except for a decrease in the concentration of plasma tyrosine (Tyr). In Experiment 2, 2-week-old birds were allocated to one of three temperature treatments; $24^{\circ}C$ (control), $36^{\circ}C$ (heat stress, HS) and $24^{\circ}C$ pair-fed (24PF) for 2 weeks and fed on diets containing 50, 100 and 300% of NRC requirement for Trp. Heat stress caused a reduction of weight gain and feed intake irrespective of dietary Trp levels compared with control counterparts, while feeding of 300% Trp diet did not attenuate the reduced performance by HS exposure. In groups fed the 100% Trp diets, plasma aromatic AA (AAA) and Tyr concentrations were decreased in the HS birds compared with the 24PF group. Plasma concentrations in most of AA groups were increased by HS in chickens fed the 50% Trp diet, while those were not changed by HS in chickens fed the 300% Trp diet, compared with 24PF counterparts. The plasma Trp/LNAA (LNAA=large neutral AAs, which are comprised of BCAA, AAA and Trp) ratio was increased by HS in chickens fed the 100% Trp diet, while it was decreased in chickens fed on 50% Trp diet as compared with 24PF group. From these results, it is suggested that performance and plasma amino acid profile deranged by heat stress are modulated, at least, to be relieved from the heat stress by feeding 50% Trp diet but not at all by feeding 300% Trp diet. The involvement of altered plasma AA profiles, in particular plasma Tyr concentrations and Trp/LNAA ratio, is discussed in association with the performance characteristics of HS chickens.

• Journal of Plant Biotechnology
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• v.2 no.2
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• pp.55-60
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• 2000

Experiments initiated 30 years ago to obtain selectable markers have led to a series of studies of Trp biosynthesis and anthranilate synthase (AS) the control enzyme using largely plant tissue cultures since they have experimental properties that can be readily exploited. Enzymological and compound feeding studies provided evidence that AS is the control point in the Trp biosynthesis branch and that altering the AS feedback control by the selection of mutants resistant to the Trp analog 5-methyl-tryptophan (5MT) can lead to the overproduction of this important amino acid. Plants regenerated from these Trp overproducing lines of most species also had high free Trp levels but Nicotiana tabaum (tobacco) plants expressed the feedback altered AS only in cultured cells and not in the regenerated plants. further tests by transient and stable expression of the cloned promoter for the naturally occurring tobacco feedback-insensitive AS, denoted ASA2, confirmed the tissue culture specific nature of the expression control. The 5MT caused by the expression of a feedback-insensitive AS from tobacco has been used to select protoplast fusion hybrids with several species since the resistance is expressed dominantly. Recently the ASA2 gene has been used successfully as a selectable marker to select transformed Astragalus sinicus and Glycine max hairy roots induced by Agrobactetium rhizogenes. These results show that the ASA2y-subunit can interact with the y-subunit of another species to form active feedback-insensitive enzyme that may be useful for selecting transformed cells. Plastid DNA transformation of tobacco has also effectively expressed ASA2 in the compartment in which Trp biosynthesis is localized in the cell.

• Membrane Journal
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• v.16 no.2
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• pp.133-143
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• 2006

A hollow fiber membrane containing BSA as ligand was Prepared by radiation-induced grafting GMA onto a porous polyethylene hollow fiber and subsequent reacting with DEA and TEA. The density of the DEA and TEA of the membrane were 3.4 mmol/g, 1.7r mmol/g, respectively. The DEA membrane exhibited a higher amount of than the TEA membrane. BSA was immobilized by the graft chains during the permeation of BSA solution throught the DEA and TEA membrane. The BSA was adsorbed in multilayer binding of 8 onto the DEA membrane whereas adsorption onto the TEA membrane remained constant. A two-stage stepwise BTC was observed due to independent chiral recognition for L, D-Trp solution by DEA-BSA membrane.

• Journal of Plant Biotechnology
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• v.42 no.3
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• pp.186-195
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• 2015

Anthranilate synthase (AS) is a key enzyme in the biosynthesis of tryptophan (Trp), which is the precursor of bioactive metabolites like indole-3-acetic acid and other indole alkaloids. Alpha anthranilate synthase 2 (OsASA2) plays a critical role in the feedback inhibition of tryptophan biosynthesis. In this study, two vectors with single (F124V) and double (S126F/L530D) point mutations of the OsASA2 gene for feedback-insensitive ${\alpha}$ subunit of rice anthranilate synthase were constructed and transformed into wildtype Dongjinbyeo by Agrobacterium-mediated transformation. Transgenic single and double mutant lines were selected as a single copy using TaqMan PCR utilized nos gene probe. To select intergenic lines, the flanking sequence of RB or LB was digested with a BfaI enzyme. Four intergenic lines were selected using a flanking sequence tagged (FST) analysis. Expression in rice (Oryza sativa L.) of the transgenes resulted in the accumulation of tryptophan (Trp), indole-3-acetonitrile (IAN), and indole-3-acetic acid (IAA) in leaves and tryptophan content as a free amino acid in seeds also increased up to 30 times relative to the wildtype. Two homozygous event lines, S-TG1 and D-TG1, were selected for characterization of agronomic traits and metabolite profiling of seeds. Differentially expressed genes (DEGs), related to ion transfer and nutrient supply, were upregulated and DEGs related to co-enzymes that work as functional genes were down regulated. These results suggest that two homozygous event lines may prove effective for the breeding of crops with an increased level of free tryptophan content.