Research article · 2019 · Full text
International Journal of Biology SciencesTryptophanase and tryptophan synthetase in Escherichia Coli
Volume 1(2) · Pages 06–11 · DOI: 10.33545/26649926.2019.v1.i2a.9 ↗
Research areasAmino Acid Enzymes and MetabolismMicrobial Metabolic Engineering and BioproductionBiochemical Acid Research Studies
Abstract
Author-writtenThe 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.
Summary
Source-checkedPlain-language reading of the full paper, prepared with AI and checked against the text. Each point links to the authors' own words.
Why it matters
- Bacteria like E. coli regulate tryptophan production because synthesizing the amino acid is energy-intensive, making it advantageous to stop production when tryptophan is available externally. View evidence ↗
- The enzyme tryptophanase is responsible for breaking down tryptophan into indole, pyruvate, and ammonia. View evidence ↗
- Tryptophan synthetase performs the opposite role by catalyzing indole and serine to synthesize tryptophan and water. View evidence ↗
The question
- The study aimed to determine how glucose and tryptophan induce or repress the activity of tryptophanase and tryptophan synthetase in E. coli over various incubation periods. View evidence ↗
What they did
- E. coli cells were grown in four different media: basal medium (Cells A), basal medium with DL-tryptophan (Cells B), basal medium with glucose (Cells C), and basal medium with both tryptophan and glucose (Cells D). View evidence ↗
- Tryptophanase activity was measured by adding tryptophan to the cells and incubating them for 10, 20, and 60 minutes. View evidence ↗
- Tryptophan synthetase activity was assessed by adding DL-serine to the tubes and incubating for 10, 20, and 60 minutes. View evidence ↗
What they found
- Glucose repressed tryptophanase activity, with Cells C (1.00±0.35 µg/mL) and Cells D (1.63±0.88 µg/mL) exhibiting the lowest activity. View evidence ↗
- Tryptophanase activity increased with longer incubation times in Cells A, B, and D. View evidence ↗
- At 10 minutes of incubation, Cells C (grown with glucose) exhibited the highest tryptophan synthetase activity compared to the other groups. View evidence ↗
- After 60 minutes, Cells B showed the highest tryptophan synthetase activity, which was significantly higher than that of Cells A. View evidence ↗
- Tryptophan synthetase activity was significantly higher at 60 minutes of incubation compared to 20 minutes, though not compared to 10 minutes. View evidence ↗
What it means
- The authors concluded that L-tryptophan induces tryptophanase activity and that this activity increases over time. View evidence ↗
- The authors concluded that tryptophan synthetase activity increased with prolonged incubation and appeared higher in tubes without DL-serine. View evidence ↗
- The authors noted potential errors in the synthetase experiment, as initial indole concentrations were often lower than final concentrations, leading them to report 'unutilized indole' instead of 'utilized indole'. View evidence ↗
- The authors stated that the results for Cells B were not ideal because tryptophan is known to repress synthetase activity, yet high activity was observed. View evidence ↗
Figures & tables
From the original paper
Table 1A
Table 1B
Table 1C
Table 1D
Table 2
Table 3
Table 4A
Table 4B
Table 4C
Table 4D
Table 5
Table 6Introduction
In the control of metabolism in bacteria, small molecules can regulate the physiological processes by adjusting the enzymatic constitution of the cell [1] . According to Davis [2] , this control is happened via the stimulation or inhibition of enzyme activity or formation. Many enzymes concerned with degradative and synthetic pathways have been investigated in attempts to discover the mechanisms involved in these control systems. However, until present, there is still a need to elucidate the regulation of enzymes involved in both the synthesis and dissimilation of a single physiologically important compound. Escherichia coli can use a number of mechanisms in order to regulate the expression of its tryptophan (trp) operon and control the rate of tryptophan biosynthesis. According to Yanofsky and Crawford [3] , these mechanisms include repression, transcription attenuation, and feedback inhibition. The combined actions of these mechanisms permit the bacterium to vary the rate of tryptophan production over a several thousand-fold range. Since tryptophan is costly to produce, efficient shutdown of synthesis is advantageous to the bacterium whenever the amino acid is present in its environment. As compared with other bacteria, E. coli has the capability to transport tryptophan from its environment. Trough the action of the inducible enzyme tryptophanase, E. coli can use tryptophan as a sole source of carbon or nitrogen. Tryptophanase degrades tryptophan to indole, pyruvate and ammonia. This enzyme can also catalyze the synthesis of tryptophan from indole and serine or cysteine [4] . 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 E. coli under different incubation time.
Materials and Method
2.1. Preparation of culture E. coli was inoculated in the following media: (1) Cells A = Basal media (mineral salts-hydrolized casein); (2) Cells B = Basal medium + DL-tryptophan (0.0025 M, 51 mg/100 mL media); (3) Cells C = Basal medium + glucose (0.1 M, 1.8 g/100 mL media); and (4) Cells D = Basal medium + tryptophan + glucose. The inoculated media were incubated at room temperature for 24 hours with shaking. Around 30 to 40 mL of each growth medium was transferred in falcon tube for centrifugation (5,000 rpm, 15 minutes). The resulting pellet after two times centrifugation was resuspended in 5 mL sterile distilled water.
Induction/repression of tryptophanase
The below components were prepared in duplicate tubes (all volumes given in mL): DOI: https://doi.org/10.33545/26649926.2019.v1.i2a.9
The cells, buffer and water were added in the tubes. The mixture was allowed to equilibrate for about 10 minutes in a 37 °C water bath. Afterwards, the tryptophan was added in the tubes and incubated for 10, 20 and 60 minutes as follows: tubes 1 to 8 = 10 minutes incubation; tubes 9 to 16 = 20 minutes incubation; and tubes 17 to 24 = 60 minutes incubation. In order to stop the reaction, 0.2 mL of 25% trichloroacetic acid was added in each tube. The amount of indole produced was then analyzed.
Induction/repression of synthetase
The below components were prepared in duplicate tubes (all volumes given in mL): 0.5 0.5 ------------------Cells B ------0.5 0.5 ------------Cells C ------------0.5 0.5 ------Cells D ------------------0.5 0.5 H2O 1.7 1.6 1.7 1.6 1.7 1.6 1.7 1.6 DL-serine (0.1 M) ---0.2 ---0.2 ---0.2 ---0.2
The cells, buffer, indole and water were added in the tubes. The mixture was allowed to equilibrate for about 10 minutes in a 37 °C water bath. Afterwards, the serine was added in the tubes and incubated for 10, 20 and 60 minutes as follows: tubes 1 to 8 = 10 minutes incubation; tubes 9 to 16 = 20 minutes incubation; and tubes 17 to 24 = 60 minutes incubation. To establish the initial level of indole, a set of four tubes were prepared; the components were the same as tubes 2, 4, 6 and 8 except that 0.2 mL of 25% trichloroacetic acid was added before the addition of other components. In order to stop the reaction, 0.2 mL of 25% trichloroacetic acid was added in each tube. The amount of indole produced was then analyzed.
Determination of indole
Two millilitre of toluene was added in each tube in order to extract the indole. The contents were thoroughly mixed and made it stand for 15 minutes. Upon the separation of layers, 0.5 mL of the toluene layer (upper layer) was transferred to another tube. One millilitre of Ehrlich's reagent and 8.5 mL of acid alcohol were added in the new tube. The mixture was set aside for 15 minutes to allow color development. The absorbance was read at 540 nm. The amount of indole produced was calculated by using a standard curve prepared by toluene extraction of indole standard.
Preparation of indole standard curve
The below components were prepared in test tubes (all volumes given in mL): The indole was extracted as described above. The standard indole curve was plotted and the linear portion of the curve was used to determine the indole produced (trytophanase) or utilized (synthetase)
Results and Discussion
Results and Discussion
Induction/repression of tryptophanase
The tryptophanase activity (amount of indole produced, µg/ml) in general was higher in tubes with added Ltryptophan during the assay (Tables 1a to 1d). In Cells A, tubes with L-trytophan (2, 10, 18) had 10 to 13 times higher amount of indole produced than tubes without L-tryptophan (1, 9, 17); pairwise comparison showed significant difference (p<0.05) (Table 1a). The same result was obtained in Table 1b, wherein tubes with added L-tryptophan (4, 12, 20) had 15 to 27 times higher indole than tubes without L-tryptophan (3, 11, 19) and statistical analyses showed significant difference (p<0.05). In Tables 1c and1d, no significant difference resulted when indole production was compared between tubes with L-tryptophan and without L-tryptophan. In tubes with added L-tryptophan, the highest amount of indole produced was in Cells B (grown in basal medium + DL-tryptophan) (Table 1b) and followed by in Cells A (grown in basal medium only) (Table 1a). It appeared that the addition of Ltryptophan had a positive effect in the induction of tryptophanase activity. The enzyme tryptophanase is responsible for the degradation of tryptophan to indole, pyruvic acid and ammonia, thus, higher availability of tryptophan in the tubes means more indole to be produced in the degradation process. Monod and Bazire [5] found out that tryptophanase was increased more than 40-fold in the presence of 5 x 10 -3 M DLtryptophan. Also, excellent tryptophanase activity was obtained when the bacterial cells were grown in basal media containing indole, L-, DL-, or glycyl-L-tryptophan. Some tryptophanase formation was also evident when 5 hydroxytryptophan, anthranilic acid, shikimic acid, or indole-3-acetic acid were included in the growth medium. The authors also found out that little or no enzyme activity was manifested unless L-tryptophan was present, suggesting that the capacity of these compounds to induce tryptophanase during growth is due to their conversion by the bacterial cells to indole or tryptophan. Direct measurements of the amino acid pool of E. coli showed that the free-tryptophan content of cells grown without glucose was considerably higher than of those grown with the carbohydrate [6] . The enzyme tryptophanase, which degrades tryptophan to indole, pyruvic acid and ammonia, is repressed by glucose [7] . According to Freundlich and Lichstein [8] , glucose normally exhibits the most marked inhibition of tryptophanase activity. The inhibition by glucose and other carbohydrates of induced enzyme formation has been attributed to repression by intermediates of carbohydrate metabolism [9] . Levin and Magasanik [10] have mentioned that these compounds are thought to be the "metabolite moieties" of the repressor molecule which prevent enzyme synthesis by competing with the inducer at the enzyme-forming site. Yanofsky 6 has hypothesized two possible reasons for the depletion of tryptophan in the presence of glucose: (i) glucose is metabolized to a compound which blocks the endogenous formation of tryptophan, and (ii) tryptophan is metabolized so rapidly in the presence of glucose that there is little chance for the amino acid to accumulate. There would be an increased requirement for protein building materials in culture containing glucose, thus, it is acceptable that there would be a smaller pool of free tryptophan [11] . Since glucose is a principal product of the β-galactosidase reaction, a hexose proximally derived from glucose would be most likely to be responsible for effecting catabolite repression. Similarly, since pyruvate is the product of catabolic significance in the tryptophanase reaction, pyruvate or a closely related compound would play a primary role in the catabolite repression of tryptophanase [12] . In addition, data of Freundlich and Lichstein [12] has suggested that glycerol, pyruvate and glucose can provide a compound which can cause catabolite repression either directly or indirectly. Alternatively, the mechanism for catabolite repression of tryptophanase may be multivalent with a whole variety of compounds capable of causing repression. The addition of glucose to cultures of E. coli causes the excretion of cAMP 13 . It has been suggested that the loss of cAMP is responsible for catabolite repression [14,15] and that various physiological conditions affect the internal levels of cAMP. Pastan and Perlman [16] found out that cyclic AMP enhanced tryptophanase production and relieved transient repression when it was present before RNA synthesis was inhibited, but not when it was added in the presence of the inhibitor. Those results suggested that cyclic AMP stimulated the transcription of the tryptophanase gene; however, since translation was also operating during induction the stimulation of transcription by cyclic AMP might have been secondary to the effect on translation.
Induction/repression of synthetase
Since the initial concentrations of indole (Cells A = 33.89 µg/mL; Cells B = 35.61 µg/mL; Cells C = 27.61 µg/mL and Cells D = 15.28 µg/mL) were most of the time lower as compared to the final concentrations, negative values of tryptophan synthetase activities were obtained in most of the time. It was hypothesized that something was done wrong in the conduct of this part of exercise. In order to have a better discussion, the data were expressed as unutilized indole rather than utilized indole wherein the final indole was not deducted to the initial indole. In Table 4a, it appeared that tubes without DL-serine had higher tryptophan synthetase activity (lower unutilized indole) as compared with tubes with DL-serine. There was no significant difference when the two groups were compared (p>0.05). For Table 4b, tubes with DL-serine in general had higher synthetase activity as compared with tubes without DLserine. Significant difference was observed in tubes incubated at 20 and 60 minutes but results were contradicting. Synthetase activity was generally higher in tubes with DLserine in Table 4c but opposing result was obtained in tubes with DL-serine in Table 4d. 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. Indole and serine are being catalyzed by tryptophan synthetase to produce tryptophan and water. In Cells A, Cells B, Cells C and Cells D (Table 5), it was observed that tryptophan synthetase activity has increased as the time of incubation increases. This will give more time for the enzyme synthetase to catalyze serine and indole to produce tryptophan. The synthetase activity during 60 minutes incubation was significantly higher as compared to 20 minutes incubation but not with 10 minutes incubation. Meanwhile, synthetase activity in Cells B during 60 minutes incubation was significantly higher as compared to 10 and 20 minutes incubation (p<0.05). Highest synthetase activities in Cells C and Cells D were also recorded during 60 minutes incubation but not significantly different when compared to 10 and 20 minutes incubations (p<0.05) (Table 5). During 10 minutes incubation, Table 6 showed that Cells C had the highest tryptophan synthetase activity as compared to Cells A, Cells B and Cells D. No significant difference was observed when the amount of unutilized indole was compared (p>0.05). Meanwhile, tryptophan synthetase activity in Cells C during 20 minutes incubation was significantly higher as compared to Cells A, Cells B and Cells D (Table 6; p<0.05). During the 60 minutes incubation, it appeared that cells b had the highest tryptophan synthetase activity and it was significantly higher as compared to Cells A only (Table 6). Result of Table 6 was not ideal since tryptophan is known repressor of synthetase activity. According to Pardee [1] , the physiological significance of the glucose inhibition of tryptophanase becomes clearer when this is considered in conjunction with the stimulatory effect of glucose on tryptophan synthetase. Thus, the function of repression in tryptophan biosynthesis appears not to decrease the amount of tryptophan produced in the presence of excess exogenous tryptophan, but, rather, to provide for economy [1] and flexibility [17] in the formation of the synthetase enzyme. It is likely that cells growing in the presence of glucose would need more of an enzyme synthesizing an amino acid essential for increased protein formation. This argument is supported further by the fact that other environmental factors that increased growth also increased the level of tryptophan synthetase. In the case of the degradative enzyme, it is speculated that repressors of tryptophanase formation are produced from carbohydrate dissimilation. On the other hand, the increased metabolic rate caused by glucose and other carbon sources reduces the intracellular level of tryptophan, the repressor of tryptophan synthetase [18] . In study conducted by Freundlich and Lichstein [12] , it was revealed that compounds which increased the level of tryptophanase decreased the formation of synthetase; L-, DL-, and glyCyl-L-tryptophan were equally effective in repressing the enzyme.
| Media | Unutilized Indole (µg/mL) | — | — |
|---|---|---|---|
| 10 mins. incubation | 20 mins. incubation | 60 mins. incubation | |
| Cells A | 28.17±0.24 (tube 2) | 47.39±0.55a (tube 10) | 27.56±0.00a (tube 18) |
| Cells B | 35.11±1.73 (tube 4) | 43.33±0.00a (tube 12) | 20.00±0.94b (tube 20) |
| Cells C | 27.33±4.71 (tube 6) | 30.44±3.77b (tube 14) | 23.56±0.00ab (tube 22) |
| Cells D | 39.89±9.74 (tube 8) | 40.06±2.44a (tube 16) | 25.00±2.67ab (tube 24) |
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| Time (min.) | Unutilized Indole (µg/mL) | — | — | — |
|---|---|---|---|---|
| Cells A | Cells B | Cells C | Cells C | |
| 10 | 28.17±0.24b (tube 2) | 35.11±1.73b (tube 10) | 27.33±4.71a (tube 16) | 39.89±9.74a (tube 8) |
| 20 | 47.39±0.55a (tube 10) | 43.33±0.00a (tube 12) | 30.44±3.71a (tube 14) | 40.06±2.44a (tube 16) |
| 60 | 27.56±0.00b (tube 18) | 20.00±0.94c (tube 20) | 23.56±0.00a (tube 22) | 25.00±2.67a (tube 24) |
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Conclusion
The tryptophanase activity in general was 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. It appeared that tubes without DL-serine had higher tryptophan synthetase activity as compared with tubes with DL-serine. There was also observed increase in tryptophan synthethase activity as the time of incubation was prolonged.
Figures and tables from the paper
| Without Tryp | Indole Produced (µg/mL) | With Tryp | Indole Produced (µg/mL) |
|---|---|---|---|
| 1 | 1.00±0.00 | 2 | 13.31±0.62* |
| 9 | 1.44±0.44 | 10 | 13.69±1.15* |
| 17 | 1.25±0.18 | 18 | 15.38±1.40* |
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| Without Tryp | Indole Produced (µg/mL) | With Tryp | Indole Produced (µg/mL) |
|---|---|---|---|
| 3 | 2.25±0.35 | 4 | 33.81±3.27* |
| 11 | 1.5±0.35 | 12 | 40.75±2.47* |
| 19 | 2.31±0.62 | 20 | 56.88±3.71* |
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| Without Tryp | Indole Produced (µg/mL) | With Tryp | Indole Produced (µg/mL) |
|---|---|---|---|
| 5 | 1.88±0.17 | 6 | 1.81±0.44 |
| 13 | 1.31±0.27 | 14 | 0.88±0.00 |
| 21 | 1.19±0.27 | 22 | 1.00±0.35 |
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| Without Tryp | Indole Produced (µg/mL) | With Tryp | Indole Produced (µg/mL) |
|---|---|---|---|
| 7 | 1.00±0.35 | 8 | 0.75±0.18 |
| 15 | 1.38±0.35 | 16 | 1.38±0.18 |
| 23 | 0.75±0.18 | 24 | 1.63±0.88 |
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| Time (min.) | Indole Produced (µg/mL) | — | — | — |
|---|---|---|---|---|
| Cells A | Cells B | Cells C | Cells D | |
| 10 | 13.31±0.62 (tube 2) | 33.81±3.27b (tube 4) | 1.81±0.44 (tube 16) | 0.75±0.18 (tube 8) |
| 20 | 12.56±2.74 (tube 10) | 40.75±2.47b (tube 12) | 0.88±0.00 (tube 14) | 1.38±0.18 (tube 16) |
| 60 | 15.38±1.41 (tube 20) | 56.88±3.71a (tube 20) | 1.00±0.35 (tube 22) | 1.63±0.88 (tube 24) |
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| Media | Indole Produced (µg/mL) | — | — |
|---|---|---|---|
| 10 mins. incubation | 20 mins. incubation | 60 mins. incubation | |
| Cells A | 13.31±0.62b (tube 2) | 12.56±2.74b (tube 10) | 15.38±1.41b (tube 18) |
| Cells B | 33.81±3.27a (tube 4) | 40.75±2.47a (tube 12) | 56.88±3.71a (tube 20) |
| Cells C | 1.81±0.44c (tube 6) | 0.88±0.00c (tube 14) | 1.00±0.35c (tube 22) |
| Cells D | 0.75±0.18c (tube 8) | 1.38±0.18c (tube 16) | 1.63±0.88c (tube 24) |
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| Without Ser | Unutilized Indole (µg/mL) | With Ser | Unutilized Indole (µg/mL) |
|---|---|---|---|
| 1 | 33.72±7.46 | 2 | 28.17±0.24 |
| 9 | 28.67±0.409 | 10 | 47.39±0.55 |
| 17 | 23.28±3.38 | 18 | 27.56±0.00 |
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| Without Ser | Unutilized Indole (µg/mL) | With Ser | Unutilized Indole (µg/mL) |
|---|---|---|---|
| 3 | 40.94±7.94 | 4 | 35.11±1.73 |
| 11 | 39.78±0.16 | 12 | 43.33±0.00* |
| 19 | 41.67±0.00* | 20 | 20.00±0.94 |
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| Without Ser | Unutilized Indole (µg/mL) | With Ser | Unutilized Indole (µg/mL) |
|---|---|---|---|
| 5 | 33.78±0.00 | 6 | 27.33±4.71 |
| 13 | 27.78±8.64 | 14 | 30.44±3.77 |
| 21 | 23.78±0.63 | 22 | 23.56±0.00 |
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| Without Ser | Unutilized Indole (µg/mL) | With Ser | Unutilized Indole (µg/mL) |
|---|---|---|---|
| 7 | 33.00±5.19 | 8 | 39.89±9.74 |
| 15 | 32.00±15.24 | 16 | 40.06±2.44 |
| 23 | 30.33±8.64 | 24 | 25.00±2.67 |
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