Tryptophan Synthetase Activity Research Articles

Improving the Activity of Tryptophan Synthetase via a Nucleic Acid Scaffold.

Tryptophan synthetase (TSase), which functions as a tetramer, is a typical enzyme with a substrate channel effect, and shows excellent performance in the production of non-standard amino acids, histamine, and other biological derivatives. Based on previous work, we fused a mutant CE protein (colistin of E. coli, a polypeptide with antibacterial activity) sequence with the sequence of TSase to explore whether its catalytic activity could be enhanced, and we also analyzed whether the addition of a DNA scaffold was a feasible strategy. Here, dCE (CE protein without DNase activity) protein tags were constructed and fused to the TrapA and TrapB subunits of TSase, and the whole cell was used for the catalytic reaction. The results showed that after the dCE protein tag was fused to the TrapB subunit, its whole cell catalytic activity increased by 50%. Next, the two subunits were expressed separately, and the proteins were bound in vitro to ensure equimolar combination between the two subunits. After the dCE label was fused to TrapB, the activity of TSase assembled with TrapA also improved. A series of experiments revealed that the enzyme fused with dCE9 showed higher activity than the wild-type protein. In general, the activity of assembly TSase was optimal when the temperature was 50 °C and the pH was about 9.0. After a long temperature treatment, the enzyme maintained good activity. With the addition of exogenous nucleic acid, the activity of the enzyme increased. The maximum yield was 0.58 g/L, which was almost three times that of the wild-type TSase (0.21 g/L). The recombinant TSase constructed in this study with dCE fusion had the advantages of higher heat resistance and higher activity, and confirmed the feasibility of adding a nucleic acid scaffold, providing a new idea for the improvement of structurally similar enzymes.

Tryptophanase and tryptophan synthetase in Escherichia Coli

The general objective of this study was to evaluate the effects of glucose and tryptophan in the induction or repression of the enzymes tryptophanase and tryptophan synthetase in Escherichia coli under different incubation time. In general, the tryptophanase activity expressed in the amount of indole produced was significantly higher in tubes with added L-tryptophan during the assay. It was also observed that tryptophanase activity has increased as the time of incubation increases. Cells B (grown in basal medium + DL-tryptophan) had the highest tryptophanse activity across time of incubation as compared with Cells A (basal medium only), Cells C (basal medium + glucose) and Cells D (basal medium + tryptophan + glucose). Cells C had the lowest tryptophanase activity followed by Cells D. The enzyme tryptophanase, which degrades tryptophan to indole, pyruvic acid and ammonia, is repressed by glucose. The inhibition by glucose and other carbohydrates of induced enzyme formation has been attributed to repression by intermediates of carbohydrate metabolism. It appeared that tubes without DL-serine had higher tryptophan synthetase activity or lower unutilized indole as compared with tubes with DL-serine. It was expected that higher synthetase activity should be present in tubes with DL-serine because together with indole, they served as reactants in the activity of the enzyme synthetase. There was also observed increase in tryptophan synthethase activity as the time of incubation was prolonged. Cells C had the highest tryptophan synthetase activity as compared to Cells A, Cells B and Cells D.

Light quality regulation of growth and endogenous IAA metabolism of Ganoderma lucidum mycelium

To study the effect and mechanism of the light quality acting on Ganoderma lucidum, and provide a theoretical basis for G. lucidum mycelium cultivation, we focused on growth and endogenous IAA metabolism of G. lucidum mycelium under different light-emitting diode (LED) condition. The growth index, endogenous levels of IAA and Enzymes related to IAA metabolism and Polysaccharides content were investigated in different growth periods. Results showed that blue light irradiation was the best from the viewpoint of steady growth and polysaccharides accumulation, red light irradiation improved endogenous IAA level and promoted growth of mycelium in early stage of cultivation, green light irradiation decreased growth rate and fresh weight of mycelium, but increased drying rate. Enzymes related to IAA metabolism also significantly influenced by light quality. The activity of indole acetic acid oxidase (IAAO), peroxidase (POD) and tryptophan synthetase with blue light irradiation were showed high level in early time, but decreased later, and the IAA content was consistently at lower level than that in other treatments, while mycelium irradiated with yellow light showed the highest activity of both IAAO and tryptophan synthetase, and medium level of IAA content. In conclusion, the light quality affects growth and regulation of the level of endogenous IAA of G. lucidum mycelium.

Effect of phytohormones on fiber initiation of cotton ovule

In order to study the effect of phytohormones on cotton fiber initiation, contents of four endogenous phytohormones and activities of four related enzymes in ovules (in vivo) of a fuzzless–lintless mutant (fl) and its wild-type (FL) line were measured from 4 days before anthesis (day −4) to 4 days after anthesis (day 4). The results showed that contents of indole-3-acetic acid, gibberellic acid (GA), and zeatin riboside in fl ovules were lower than those in FL ovules. Therefore, indole-3-acetic acid, GA, and zeatin riboside were thought to be the promoters of fiber initiation. Although abscisic acid (ABA) content in fl ovule was slightly higher than that in FL ovule on day 0, which might imply that ABA inhibited fiber initiation. Fiber initiation could also be influenced by enzyme through regulating synthesis and degradation of related phytohormones since fl ovules were significantly higher in activities of indole-3-acetic acid oxidase, cytokinin oxidase and peroxidase, but lower in activity of tryptophan synthetase than those in FL ovules. To test the above hypothesis, exogenous plant growth regulators were also applied for the culture of ovules from fl and FL in vitro. When no regulators were added, no fiber was induced on fl ovule, but a few fibers were induced in FL ovule. Higher total fiber units (TFU) were observed when indole-3-acetic acid and gibberellic acid (GA3) were applied either separately or in combination to media. TFU did not increased with zeatin riboside alone, but the highest TFU was achieved when zeatin riboside was applied together with indole-3 acetic acid and GA3, which implied that fiber initiation could be promoted by them as additive.

The tryptophan synthetase gene TRP1 of Nodulisporium sp.: molecular characterization and its relation to nodulisporic acid A production

Nodulisporic acid A (NAA), an insecticidal indole diterpene, is produced by the fungus Nodulisporium sp. Since indole-3-glycerolphosphate is the precursor of the indole moiety of NAA, it is suggested that the activity of tryptophan synthetase may play a role in NAA biosynthesis. To investigate this hypothesis, the tryptophan synthetase gene TRP1 of Nodulisporium sp. was cloned and characterized. The gene consists of three introns of 146, 68, and 57 bp. The four exons encode a protein of 712 amino acids, the sequence of which is highly homologous to that of other fungal tryptophan synthetase proteins. The transcription initiation site was mapped 66 bp upstream to the ATG, and the polyA tail attachment site is 169 bp downstream to the translation stop codon. Replacement of the N-terminal half of the gene with a hygromycin selection marker yielded mutants with the tryptophan auxotroph/hygromycin-resistance (trp(-)/hyr) phenotype. The TRP1 mutants required a high concentration of tryptophan supplement in solid medium (10 mM) to sustain minimal growth and failed to produce NAA in the production medium (FFL-CAM) supplemented with high concentrations of tryptophan.

Prolonged production of tryptophan using immobilized bacteria

Tryptophan production of a genetically modified E. coli strain possessing elevated tryptophan synthetase activity but sensitive to indole could be prolonged by the immobilization of cells in calcium alginate gel beads with a calculated half-life time of 12.2 days.

Synthesis of the essential amino acid tryptophan in the pea aphid ( Acyrthosiphon pisum) symbiosis

Pea aphids ( Acyrthosiphon pisum) containing their symbiotic bacteria do not require a dietary supply of the essential amino acid tryptophan over two generations on chemically-defined diets; but aphids, whose bacteria are disrupted by chlortetracycline, die as first-generation larvae on tryptophan-free diet. Consistent with the interpretation that the symbiotic bacteria in the aphids synthesize tryptophan, tryptophan synthetase activity was detected in both symbiont-bearing aphids and isolated bacteria, but not in chlortetracycline-treated aphids. Aphids reared on tryptophan-free diet produced honeydew containing 0.32 mM tryptophan, from which it can be calculated that each bacterial cell in the aphid releases at least 0.04 fmol tryptophan d −1.

Cloning and expression of tryptophan genes from [formula omitted] in [formula omitted

A gene bank from the amino acid producer Brevibacterium lactofermentum has been prepared in Escherichia coli using pBR322 as vector. Four clones containing genetic information needed to complement mutations in A,B,C and D genes from E. coli have been isolated. The cloned fragments range between 4.3 kb (pULT61) and 7.9 kb (pULT62). All the four clones contain genetic information that complements trp B gene from E. coli . The cloned trp B gene is very stable and is maintained extrachromosomally in E. coli . It is expressed very efficiently showing high levels of tryptophan synthetase activity.

Continuous production of L‐tryptophan from indole and L‐serine by immobilized Escherichia coli cells

Escherichia coli B 10, which has high activity of tryptophan synthetase, was grown in a 50-L batch culture in order to determine in which growth phase the cells have the highest specific tryptophan productivity. Accordingly, whole cells of the stationary phase were used for immobilization in polyacrylamide beads. After immobilization, these immobilized cells had 56% activity of tryptophan synthetase compared with that of free cells. First, the properties of immobilized cells were investigated. Next, discontinuous productions of L-tryptophan were carried out by using immobilized cells. In discontinuous production of L-tryptophan by the batch, the activity remaining of immobilized cells was 76-79% after 30 times batchwise use. In continuous production of L-tryptophan with a continuous stirred tank reactor (CSTR), the activity remaining of the immobilized cells was 80% after continuous use for 50 days. The maximum productivity of L-tryptophan in this CSTR system was 0.12 g tryptophan L(-1) h(-1).

Production L‐tryptophan by Escherichia coli cells

Whole cells of Escherichia coli B 10 having high tryptophan synthetase activity were used directly as an enzyme source to produce L-tryptophan from indole and L- or D,L-serine. This strain is tryptophan auxotrophic, which is tryptophanase negative and, in addition, L- and D-serine deaminase negative under production conditions. To avoid inhibition of tryptophan synthetase by a high concentration of indole, nonaqueous organic solvents, Amberlite XAD-2 adsorbent, and nonionic detergents were used as reservoirs of indole in the reaction mixture for the production of L-tryptophan. As a result, different effects were observed on the production of L-tryptophan. Particularly, among the nonionic detergents, Triton X-100 was very efficient. Using Triton X-100 for production of L-tryptophan from indole and L- or D,L-serine by whole cells of Escherichia coli B 10, 14.14 g/100 mL and 14.2 g/100 mL of L-tryptophan were produced at 37 degrees C for 60 h.

Control of expression of a cloned yeast (Saccharomyces cerevisiae) gene (trp5) by a bacterial insertion element (IS2).

A hybrid ColE1 plasmid [pYe(trp5)1], containing a yeast DNA segment that complements auxotrophic point mutations and deletions in the Escherichia coli tryptophan synthetase gene (trpAB), has been isolated. Expression of the yeast tryptophan synthetase activity from the cloned yeast gene (trp5) is relatively inefficient in E. coli, as measured by growth rates of trpAB/pYe(trp5)1 strains on minimal media lacking tryptophan and by enzyme assays. Faster growing variants occur spontaneously at a frequency of one in 10(4)--10(5) cells plated and produce higher levels of the yeast enzyme. Plasmid DNA [pYe(trp5)2] from one of these variants was shown to contain a DNA insertion (1.3 kilobase pairs) in the cloned yeast DNA segment in relatively close proximity to the trp5 gene. This DNA insert was identified as a bacterial IS2 element, which carries a promoter for RNA transcription when inserted in the proper orientation. The spontaneous integration of a bacterial DNA insertion element into cloned eukaryotic DNA can result in more efficient expression of the foreign gene.

Derepression of E. coli trp operon on interfamilial transfer

THE P-group R factor RP4 originally isolated from Pseudomonas aeruginosa can be transferred to many Gram-negative bacteria1–3. In spite of its large molecular weight, RP4 has only one site susceptible to each of EcoRI (ref. 4), HindIII (ref. 5) and BamNI (data not shown) restriction endonucleases. We have constructed an RP4-trp plasmid consisting of RP4 DNA and the complete tryptophan operon of E. coli obtained from bacteriophage λtrpE-A60-3 in vitro6. We reported that the tryptophan synthetase activity in the strain carrying RP4-trp plasmid was repressed by exogenous tryptophan7. Manson and Yanofsky have reported that the E. coli trp operon on the colVB plasmid was similarly regulated in other enteric species8. We show here that the RP4-trip plasmid in E. coli can be transferred by conjugation to Pseudomonas aeruginosa which belongs to a different family from E. coli, converting Pseudomonas Trp− phenotype to Trp+. The levels of tryptophan-synthesising enzymes in P. aeruginosa carrying RP4-trp plasmid are much higher than those in the wild strain, and they are not controlled by the repression system in P. aeruginosa cells.

Protoplastengewinnung bei Mutterkornpilzen

Summary A method has been developed for the preparation of protoplasts from Claviceps . The DNA content, the oxygen uptake as well as the activity of DAHP-synthetase, chorismate mutase, anthranilate synthetase and tryptophan synthetase were measured. After regeneration the mycelium was again able to produce alkaloids.

Production of alkaloids and differentiation in a submerged culture ofClaviceps purpurea (Fr.) Tul

The submerged culture of the Claviceps purpurea strain studied was polymorphous. In the process of alkaloid synthesis, cytodifferentiation preceded biochemical differentiation. The onset of the alkaloid phase was characterized by: predominance of chlamydospores in the culture, the presence of vegetative cells with reduced or arrested proliferation, maximum acetylCoA carboxylase activity, the maximum amount of total fatty acids, an over-average cell pool tryptophan level and minimum tryptophan synthetase activity. Intracellular ricinoleic acid was an indicator of differentiation of the culture towards alkaloid formation and also of alkaloid synthesis. The cytodifferentiation period in the initial phases of fermentation, when the cell has several alternative possibilities of development, is regarded as the most sensitive sector of the regulatory mechanisms of alkaloid formation.

Genetic basis of nutritional requirements in Lactobacillus casei.

In a study of the genetic basis of multiple nutritional requirements in Lactobacillus casei, systematic attempts were made to isolate mutants that can grow in the absence of a specific nutrient required by the parental organism. Such mutants have successfully been isolated with respect to seven of twelve amino acids (aspartic acid, leucine, isoleucine, lysine, methionine, serine, and threonine) and three of four vitamins (pantothenic acid, nicotinic acid, and pyridoxal) tested, after extensive screenings employing various mutagens. Mutants that can grow without tryptophan were not isolated, but those that can grow on anthranilate or indole as well as on tryptophan were obtained at a frequency expected for single-step mutations. Activity of tryptophan synthetase was demonstrated in extracts of these anthranilate-utilizing mutants, but not in the parental strain. These results suggest that the multiple nutritional requirements of L. casei are often, if not always, due to one or a few small lesions such as base substitution mutations rather than large deletions affecting the genes involved in each biosynthetic pathway. The data would also imply that many of the biosynthetic pathways that are not fully functional in L. casei were active at one time and became nonfunctional during evolution of the present species.

Synthesis of L-Tryptophan from Indole and DL-Serine by Tryptophan Synthetase Entrapped in Fibres

Optimal culture conditions for microbial production of tryptophan synthetase were studied. It was found that on cultivation of Escherichia coli 476, a tryptophan auxotroph, in a medium containing 5g/liter glycerol as C source, supplemented with 1 g/liter of acid-treated peptone, cells with high tryptophan synthetase activity could be obtained.The enzyme was extracted from cells and 3-fold purified by heat treatment and ammonium sulfate precipitation. The overall yield of the isolation procedure was 60%.The partially purified tryptophan synthetase was entrapped in cellulose triacetate fibres. Under storage conditions, in refrigerator, the entrapped enzyme was stable at least for 6 months. The activity of the entrapped enzyme was about 75% with respect to the free enzyme.Similar behaviour for the free and entrapped enzyme was observed as to the effect of temperature and pH on the enzymic activity. The operational stability of the entrapped tryptophan synthetase was very good (activity unchanged after 50 day...

Expression of the trp operon in phi80trp transducing phages. Orientation of transcription and an artificial high-efficiency promotor in phage lambda h +phi80 pt5-2AB.

The presence of regulatory elements belonging to the trp operon of Escherichia coli in various trp transducing phages of ϕ80 has been investigated by measuring the synthesis of trp enzymes following infection of ϕ80 immune bacteria. In phages which lack trp regulatory elements, synthesis of trp enzymes usually is controlled by phage regulatory elements (Franklin, 1971) but for two of these phages, iλh+ϕ80pt5-2AB and ϕ80pt1 it was found that the expression of trp genes is not controlled by phage regulatory elements. Our results suggest that phage iλh+ϕ80pt5-AB contains an artificial promotor which presumably is located just ahead of the trpB gene. Transcription at this promotor is initiated with high efficiency as judged by the high amounts of trp enzymes synthesized in immune cells infected with this phage. The low activity of tryptophan synthetase found in immune cells infected with phage ϕ80pt1 might be explained by the existence of a low-efficiency promotor within the trp operon (Morse and Yanofsky, 1968).

Physiological Studies on Ergot: Further Studies on the Induction of Alkaloid Synthesis by Tryptophan and Its Inhibition by Phosphate

The failure of l-leucine to stimulate ergot alkaloid production in a synthetic medium indicates that the previously observed stimulation by tryptophan and tryptophan analogues does not merely represent a nutritional effect. Tryptophan, but not mevalonate or 5-methyltryptophan, is able to overcome the inhibition of alkaloid synthesis by high levels of inorganic phosphate. Therefore, high phosphate levels seem to limit the synthesis of tryptophan; they may, in addition, prevent induction of alkaloid synthesis by preventing accumulation of tryptophan. Experiments which indicate a 2- to 3-fold temporary increase of intracellular free tryptophan and a 20- to 25-fold increase of tryptophan synthetase activity during the transition period between growth and alkaloid production phase are in agreement with the previously postulated induction of alkaloid synthesis by tryptophan. The latter experiments also indicate 4- to 6-fold repression of this enzyme by tryptophan.

Documentation of auxotrophic mutation in blue-green bacteria: characterization of a tryptophan auxotroph in Agmenellum quadruplicatum.

A tryptophan-requiring auxotroph of Agmenellum quadruplicatum strain BG1, a species of blue-green bacteria, was isolated by means of a nitrosoguanidine-penicillin procedure. Its growth characteristics were determined, and the enzymological block was identified in the A activity of tryptophan synthetase. Starvation of the auxotroph for tryptophan resulted in the derepression of the synthesis of all five enzymes. The first four enzymes derepressed 2- to 3-fold, and tryptophan synthetase B derepressed 20-fold. In the parental prototroph, BG1, anthranilate synthetase was active in crude extracts with ammonia as the amino donor reactant, but not with glutamine.

Influences of various amino acids on tryptophan-mediated control of the tryptophan biosynthetic enzymes in Escherichia coli.

Lysates of Escherichia coli Ymel obtained from cultures grown in the absence of tryptophan in minimal medium supplemented with 0.1% casein hydrolysate show an approximate fivefold increase in steady-state specific activity of both anthranilate synthetase and tryptophan synthetase A protein relative to cultures grown in nonsupplemented medium. In the presence of repressing levels of exogenous tryptophan, growth of cultures in casein hydrolysate-supplemented medium results in a noncoordinate enhancement of repression of 10-fold for anthranilate synthetase and twofold for tryptophan synthetase A protein. Similar, but less pronounced, effects are shown for strain W3110. Strains possessing tryptophan regulator gene mutations do not exhibit this first effect, but do yield an approximate twofold decrease in specific activity of both enzymes when grown in medium supplemented with tryptophan and casein hydrolysate. A stimulation of derepression of both enzymes in strain Ymel equivalent to that induced by casein hydrolysate can be reproduced by growth in minimal medium supplemented with threonine, phenylalanine, tyrosine, serine, glutamic acid, and glutamine. Doubling time in this medium is not significantly different from that in minimal medium. An enhancement of repression which partially mimics that observed on growth in medium supplemented with tryptophan plus casein hydrolysate is obtained when Ymel is grown on medium supplemented with tryptophan plus methionine. Threonine or phenylalanine plus tyrosine as separate medium supplements are independently capable of producing a 1.4-fold or 3.4-fold stimulation, respectively, but in combination only the phenylalanine plus tyrosine effect is manifested unless serine and glutamic acid or glutamine are included. Our data show that expression of the tryptophan biosynthetic enzymes can be significantly influenced in vivo as a result of growth in medium supplemented with a variety of amino acids.