FR2723379A1 - Enzymatic prodn. of L-tryptophan from indole, pyruvate and ammonium salt - Google Patents

Abstract

Enzymatic prodn. of L-tryptophan (I) comprises reacting indole (II), pyruvic acid (III) and/or its salt, and an NH4 salt (IV) in presence of tryptophanase (V) in an automatic, computer-controlled system consisting of a bioreactor and device for direct determn. of (II). The computer regulates addn. of reactants and controls the reaction by feedback according to the measured (II) concn. Addn. of (II) and (IV) is controlled according to a predetermined profile, corresp. to the activity profile of (V), and (II) or its salt is added at a specific time. Also new is the automatic, computer-controlled system.

Description

The present invention relates to a method for the enzymatic synthesis of L-

  tryptophan using an automatic controlled bioreactor system

  by computer as well as this computer-controlled bioreactor system.

  Tryptophan is one of the essential amino acids that make up the body of animals and is an important amino acid as a medicine, nutrient or additive for animal feed. The known processes for the production of tryptophan involve fermentation processes and enzymatic processes. For fermentation processes, tryptophan can be produced from its precussors by adding anthranilate or indole and sugar to a culture or by direct fermentation of glucose in the culture. For enzymatic processes, tryptophan can be produced from indole, pyruvic acid and ammonium ions using a tryptophanase produced by microorganisms. The advantage of an enzymatic process is that it is easier to purify tryptophan and that it is possible to obtain a tryptophan of higher purity so that it is now a process of choice for produce

  Medicinal grade L-tryptophan.

  In the production of L-tryptophan using bacteria that produce high levels of tryptophanase, indole, which is one of the substrates, is toxic to bacteria and causes inhibition by the substrate even at low concentrations (18 mM). It is therefore not advantageous to use the concentration of indole as the cause causing the synthesis reaction. In addition, tryptophan concentrations above 50 mM cause inhibition by the product and the sodium pyruvate decomposes in the presence of NH4 + ions at alkaline pH (pH 9.0). In view of the above, it is possible to develop methods for producing high concentration L-tryptophan by continuously or discontinuously introducing concentrated indole to maintain a limited concentration of indole, and it is possible to obtain a higher concentration of L-tryptophan (> 80 g / l) thanks to the precipitant of

tryptophan which is inosine.

  Many processes are known for producing L-tryptophan using tryptophanase. Japanese Patent No. 56085291 describes the production of L-tryptophan by the reaction of indole with serine or pyruvic acid and

  ammonium ions by means of a strain of the genus Aeromonas, Vibrio or Bacillus.

  US Patent No. 4,349,627 describes a process in which L-tryptophan is produced from indole and serine or from indole, pyruvic acid and ammonium ions with a particular microorganism of the genus Enterobacter. Tryptophanase producing strains of the species Proteus rettgeri, Proteus vulgaris, Proteus mirabilis, Proteus morganil and Escherichia coli were used to prepare L-tryptophan according to Japanese patent no. 62134094. Japanese patent no. 63137689 describes the use of heat treated organisms, including different species of Escherichia coli, to prepare L-

  tryptophan from indole, pyruvic acid and ammonium ions.

  Japanese Patent No. 1,104,186 describes a process for the production of L-

  tryptophan from indole and L-serine or D, L-serine using tryptophan synthetase. French Patent No. 2581 654 describes a process for reacting indole with pyruvate and ammonium ions with an immobilized tryptophanase and for collecting L-tryptophan by precipitation with inosine. Japanese patent n ° 64051093 describes a process for the preparation of L-tryptophan using tryptophanase with discontinuous supply of indole to maintain its concentration below 4 mM, this process making it possible to prepare 39 mM of L-tryptophan in 10 h of reaction. Japanese Patent No. 01191692

  describes a process involving immobilized cells to prepare L-

  tryptophan from indole and pyruvic acid, into which indole is introduced

  continuously to maintain its concentration at 17 mM and in which the L-

  tryptophan is removed continuously or discontinuously using a separator, and which makes it possible to obtain 36.49 g of L-tryptophan in 120 h of operation in a 1 1 reactor, the rate of conversion of the indole being 92% and that of pyruvate 84%. European Patent No. 381744 describes a multi-step process for the production of L-tryptophan, in which a bacterial host cell is transformed so as to contain tryptophanase which is then used in a subsequent bioconversion step to cause the accumulation of L-tryptophan by controlled introduction of indole continuously to maintain its concentration below 10 mM in a discontinuous reaction, which makes it possible to produce 24 g / l of L-tryptophan in 75 min, i.e. a rate of

  conversion of indole greater than 99%.

  Although inexpensive raw materials such as ammonia can be used to synthesize L-tryptophan with indole and pyruvate in a manner which has economic advantages, there are still serious problems such as inhibition by indole, inhibition by L-tryptophan and decomposition of pyruvate. Currently, there are certain enzymatic methods for producing L-tryptophan with a relatively high yield and a relatively high speed by controlled introduction of indole and / or pyruvic acid. However, the simultaneous introduction of indole,

  pyruvate and NH4Cl controlled by online feedback is not disclosed.

  In addition to the known problems, a phenomenon has been observed in which, except under optimal conditions, tryptophanase degrades when it is incubated with two of the substrates, namely sodium pyruvate and NH4Cl. To overcome the above problems, according to the present invention, the introduction of indole, sodium pyruvate and NH4Cl is controlled by a computer equipped with an on-line indole dosing device. The computer controls the introduction of indole and NH4CI according to a predefined profile which is in agreement with the activity profile of tryptophanase. Sodium pyruvate is added at a predetermined time during the reaction. Inhibition by

  product is overcome by addition of inosine which is a precipitant of L-

  tryptophan, which is able to separate L-tryptophan from the reaction system by closing an insoluble complex with L-tryptophan. The combination of feedback control of the introduction of the substrate and the introduction of inosine into the reaction vessel leads to a very efficient process for synthesis.

enzymatic of L-tryptophan.

  Thus, the object of the present invention is to provide a process for the enzymatic synthesis of L-tryptophan by means of an automatic computer-controlled bioreactor system for controlling the introduction of indole, pyruvic acid and / or one of its salts and an ammonium salt, and a

  feedback feedback control.

  The present invention also aims to provide a method of enzymatic synthesis of L-tryptophan which comprises the reaction of indole with pyruvic acid and / or one of its salts and an ammonium salt in the presence of tryptophanase by means of an automatic computer-controlled bioreactor system comprising a bioreactor connected to a computer equipped with an on-line indole dosing device, in which the computer controls the introduction of indole, pyruvic acid and / or its salt and an ammonium salt in the bioreactor and feedback control the reaction in the bioreactor through the concentration of indole determined by the indole metering device online, in which the introduction of indole and ammonium salt is controlled by a predefined profile which is in agreement with a profile of activity of tryptophanase, and pyruvic acid and / or its salt is added at a time

predetermined.

  The present invention also aims to provide an automatic computer-controlled bioreactor system intended to be used for the enzymatic synthesis of L-tryptophan which comprises a bioreactor connected to a computer equipped with an on-line indole assay device, wherein the computer controls the introduction of indole, pyruvic acid and / or its salt and an ammonium salt into the bioreactor and feedback control the reaction in the bioreactor through the concentration of indole determined by the online indole metering device, in which the introduction of indole and the ammonium salt is controlled by a predefined profile which is in agreement with a profile of tryptophanase activity, and pyruvic acid and / or its salt is added

at a predetermined time.

  Other characteristics and advantages of the invention will appear better

  in the detailed description which follows and refers to the attached drawings, given

  by way of example only, and in which: FIG. 1 shows a profile for the introduction of substrate into the enzymatic synthesis of L-tryptophan by introduction of indole with multiple slopes controlled by computer. The initial conditions for the bioreactor are as follows: 48 mmol of NH4Cl, 60 mmol of sodium pyruvate and 72 mmol

  inosine; 1 g of wet cells on the 120 ml scale.

  Figure 2 shows the substrate introduction profile in the enzymatic synthesis of L-tryptophan by means of an automatic computer-controlled bioreactor. The initial conditions are as follows: 180 mmol of NH4Cl, 300 mmol of sodium pyruvate and 390 mmol of inosine; 100 ml of crude extract

  of enzyme (15,000 U) and 9 mmol of L-tryptophan on the 600 mi scale.

  The introduction of additional pyruvate took place for 3.3 min, 40 min

after the start of the reaction.

  Figure 3 shows an automatic controlled bioreactor system

by computer according to the invention.

  FIG. 4 is a graphic representation of an enzymatic synthesis of L-tryptophan with automatic introduction by computer and with

  feedback control based on online indole dosage.

  The present invention relates to a method for the enzymatic preparation of L-tryptophan making it possible to obtain large amounts of L-tryptophan (> 100 g / l) by an accumulation reaction catalyzed by tryptophanase in a bioreactor of the discontinuous type, in the presence inosine. The invention also relates to an automatic computer-controlled bioreactor system comprising an online indole dosage and a feedback control of the introduction and determination of the optimal reaction profiles. A typical discontinuous reaction on the 600 ml scale involving a crude enzyme gives satisfactory results. For example, approximately 66.2 g (324 mmol) of L-tryptophan was obtained from 38.3 g (327 mmol) of indole, from 50.8 g (462 mmol) of sodium pyruvate and 104.6 g (390 mmol) of inosine in 4 h. All

  the operation can be controlled automatically by computer.

  According to the present invention, a discontinuous type bioreactor comprising a continuous automatic introduction of indole, pyruvic acid and / or one of its salts and a computer-controlled ammonium salt is designed and operated to obtain the optimal conditions for the reaction. Thanks to this system, it is possible to obtain a relatively high concentration of L-tryptophan (> 100 g / l) and to maintain an indole conversion rate greater than 96% and a

  pyruvate conversion rate between 65 and 82%. The L- complex

  formed tryptophan-inosine can be easily isolated by filtration to be purified and the inosine can be recycled for reuse. It is an advantageous system

  for the industrial production of a high purity L-tryptophan.

  In the process according to the present invention, the rate of introduction of the indole is a function of time and the ammonium salt is introduced at the same time as the indole. Preferably, pyruvic acid and / or its salt is added 40 to 60 min

after the start of the reaction.

  The tryptophanase for use in the process of the present invention can be produced by a microorganism producing tryptophanase such as E. coli. In the following examples, the E. coli strain PGT 67 / N 4830 a

  been used to produce tryptophanase.

  I) Fermentation in a fermenter with the strain E. coli PGT 67 / N 1. Culture conditions: The strain of E. coli PGT 67 / N 4830 was cultivated on a Trp I agar dish containing 1% of soy peptone , 0.5% yeast extract, 0.5% sodium chloride, 1% Tween 80, mM tryptophan, 100 mg / ml ampicillin and 1.5% agar at 30C overnight. A single colony was removed to inoculate 15 ml of Trp I medium and it was cultivated overnight at 30C with vigorous stirring. Glycerol was added to the cell solution to reach a final concentration of 50%, then the cell solution was distributed in

  several small tubes (1.0 ml / tube) and stored at -70C.

  2. Fermentation in a fermenter of 5 1: 2 ml of the mother solution of cells were inoculated in ml of Trp I medium and cultured overnight at 30C with vigorous stirring (150 rpm), then the culture overnight was transferred to a 5 L fermenter containing 3.5 I of Trp I medium and cultured at 30C and 350 rpm. The temperature was raised to 42 ° C until the D.O. 600 was close to 0.6. After 5 h, the cells were centrifuged for 20 min at 8,000 rpm and 4C and then washed with 0.9% NaCl solution (the cells were resuspended in the NaCl solution at 0, 9% and centrifuged for 10 min at 16,000 rpm and

4'C) and finally stored at -20C.

  3. Fermentation in a 20 1 fermenter: 7 ml of mother culture were inoculated in 700 ml of medium containing 1% of Tween 80, 1% of soy peptone, 0.5% of yeast extract, 0.5% NaCI, 0.01% ampicillin and 0.2% tryptophan and were grown overnight at 30 C. The overnight culture was seeded in 13.3 1 medium and incubated at 30C with shaking (600 rpm), with an aeration of about 0.5 volume / volume / min (vvm: volumetric air flow rate per liter of fermenter and per min), the temperature was raised to 42 ° C when the DO reached 1.2, then the culture was extended for 5 h and the aeration was adjusted to 0.63 vvm. The cells were harvested by centrifugation at 6000 rpm for 20 min, washed once with 0.9% NaCl solution then centrifuged again at 13,000 rpm for 30 min and finally

stored at -20'C.

  I1) Preparation of a crude enzyme solution: g of E. coli PGT 67 / N 4830 cells "at rest" (harvested in the fermenter and then stored at -20C, that is to say which were not growth phase) were suspended in 200 ml of a solution containing 3% Tween 80, 20 mM potassium phosphate (pH 9.0) and 0.2 mM pyridoxal phosphate and then lysed under action of a French press. Centrifuging this solution at 13,000 rpm for 15 min allowed to obtain a

  crude enzyme solution which was stored at -20C.

  III) Determination of the specific activity of trvptophanase mg of cells were suspended in i ml of a solution containing 0.2 mM pyridoxal phosphate, 20 mM potassium phosphate (pH 9.0) and 3 % of Tween 80, then subjected to a sonication at level 14 at 4C for 30 s followed by a stop of 1 min, this process being repeated five times. The supernatant, i.e. the crude enzyme extract, was obtained by centrifugation at 13,000 rpm for 10 min. The protein concentration was determined by the Bradford method (see

  Bradford, M.B. (1976) Anal. Biochem, 72, 248).

  Distilled water (0.395 ml) and 1 M potassium phosphate (0.1 ml) (pH 9.0) were mixed in a 1 ml quartz tank and the absorbance value read was set zero by pressing the automatic zeroing button, then 0.5 ml of SOPC (S-0-nitrophenyl-L-cysteine) was added to the tank and mixed. The reaction was started by adding 5 ml

  of sample and the variation of D.O. was measured at 370 nm.

  The specific activity has been defined as the hydrolysis of

  1 / mol of substrate per minute and per mg of protein.

  IV) Synthesis of L-trptptophan: The synthesis of L-tryptophan was carried out in a batch reactor by applying different methods of introducing indole so as to obtain the accumulation of a large amount of L-tryptophan in a very short duration, based on the high enzymatic conversion of indole and sodium pyruvate. 1. Batch manual introduction: In tests 1 to 3, solid indole (tests 1 and 2) or concentrated indole (4.6 M in ethanol for test 3) was introduced manually and discontinuously in the reaction solution consisting of 0.2 mM pyridoxal phosphate, mM potassium phosphate (pH 9.0), 3% Tween 80, with or without addition of inosine, the reaction being carried out at 30 'VS

and pH 9.0.

  2. Continuous introduction to the moven of a manually actuated pump In tests 4 to 6, the conditions were the same as those described in 1, except that concentrated indole (4 M in ethanol) was introduced in the reaction solution using

  a manually operated pump.

  3. Continuous introduction controlled by computer using a multi-slope radiant In tests 7 to 12, concentrated indole (4 M in ethanol) was continuously introduced into the reaction solution, the introduction being controlled by computer by means of multi-band gradient software such as that which is

shown in figure 1.

  The conditions of tests 1 to 12 are summarized in Table I.

Table I

  Summary of test conditions 1 to 12 Pyruvate NH4CI test Ethanol Inosine ne (mM) (mM) (%) (mM)

1,500,600 5

2,100,300 5 -

3,400,500 4,400

4 400 500 -_ 350

1000 800 - 785

6 1000 1000 - 600

7 1100 1133 -_ 850

8 500 600 -_ 400

9 1200 1600 - 1100

700 900 -_ 600

1i 1,760 1,270 _ 700

12 1700 1500 - 1000

  4. Continuous introduction with feedback control 20 ml of Tween 80, 13.3 ml of 1 M potassium phosphate, pH 9.0, 13.3 ml of sodium phosphate were introduced into a 1.5 l glass reactor. 10 mM pyridoxal, 620 ml of distilled water and 60 ml of 5N NH4Cl (the pH was adjusted to 9.0 with NH4OH) and the resulting mixture was then stirred thoroughly. 174.3 g (650 mmol) of inosine and 3.06 g of L-tryptophan (15 mmol) were added to the solution, the reaction was started by adding 55.03 g (500 mmol) of pyruvate sodium and 166.7 ml of crude enzyme solution (150 U / ml). The NH 4 Cl 5 N and 4 M indole solution (prepared in ethanol) was introduced into the reaction system from the start to the end of the reaction, according to the curve shown in FIG. 2. An additional 110 ml (270 mmol) of sodium pyruvate (2.45 M aqueous solution) was introduced into the reaction solution by means of a pump over 2 min after 40 min of reaction, as shown in FIG. 2. Samples were taken at certain intervals to monitor the residual concentration of indole by HPLC. The

  device was arranged as shown in Figure 3.

  After the reaction was complete, the mixture was filtered and washed with cold water to give a solid white cake which was identified as a tryptophan-inosine complex. The gradient introduction profile shown in Figure 2 was simulated by computer to follow the degradation profile of the enzyme activity and was controlled by a Labtech Notebook program. We give below

  certain details concerning the apparatus represented in FIG. 3.

  * Computer: IBM-compatible laptop 80486-33 32 b * Interfaces: AXIOM high-speed data capture card (AX-5412-LG), channel 2 for signal input from the 8-channel N / A card detector (AX5212), channels 2 and 3 to control the supply of pumps 1 and 2, respectively amplifier / multiplexer panel (AX-751) multipurpose screw terminal block (AX-752) * Software: LTN Labtech Notebook

  Pumps: - P1: Masterflex Drive model n 7523-10 1-

  rpm with pump head 7523-10 (can

  control 8 supply channels) for intro

  duction of indole and NH4Cl, tubes 7624-22 (silicone) or 7605-26 (viton)

  - P2: Masterflex Drive model n'7520-35 1-

  rpm with pump head 7518-10 (pump head can be easily loaded) for the introduction of the sodium pyruvate solution, Masterflex 6411-16 tubes - P3: Eyela MP-3 pump working with microtubes il - P4: Pharmacia peristaltic pump P-1 -P5: Eyela MP-3 pump working with microtubes - P6: identical to P2 * Tubes & Flow rates: P1 P2 P3 P4 P5 P6 Tubes pumps * 7624-22 (0,1-06L) 96410-16 96410-14 96410-14 96410-14 96410-14

7605-26 (0.6-1L)

  Adjustment controlled by controlled by 1.5 6 4 (x10) 1.8 computer computer _ Flow controlled by controlled by 0.375 ml / min 4.05 ml / min 0.37 ml / min 3, 2 ml / min computer computer * all the tubes were of the Masterflex type The predetermined speed of introduction of the indole is a function of time according to equation (1), and the relationship between the signal

  feedback from the computer and the optical density of the photodetector is expressed by equation (3).

  F = 5.51 - 5.44 x 10-2T + 2.79 x 10-4T2 - 7.05 x 10-7T3 + 7.0 x 10-10T4 (1) S = (F x 6-33,167) / 3.7257 (2) Sfb = S + 3.492 x DO- 29.34 x DO2 + 0.0308 (3) With F: flow rate of indole introduction (mmol / min / l) T: reaction time (min) S: initial signal Sfb: feedback signal DO: optical density of the photodetector As shown in Table II given below, when a discontinuous manual introduction of indole was used, we obtained in the tests 1 and 2 a low concentration of L-tryptophan without addition of inosine, but in test 3 the accumulation of a relatively high amount of L-tryptophan (57.2 g / l or 280 mM) was observed in the presence inosine. Due to the multi-slope introduction of indole by computer in tests 4 to 6, the amount of L-tryptophan clearly increased beyond 122.6 g / l (600 mM) in 7 h of reaction and a reasonable conversion rate of sodium pyruvate (about 60%) has been observed. Finally, the use in tests 7 to 12 of a more precise indole introduction profile, predetermined as a function of the activity profile of the enzyme, made it possible to further improve the yield of biosynthesis by producing more than 102 g / 1 (500 mM) of L-tryptophan in 3.5 h of reaction

  (test 11) and to maintain a conversion rate of sodium pyruvate of 70%.

  This process also made it possible to obtain the largest amount of Ltryptophan

  (188.6 g / l or 923 mM) in test 12.

  When the introduction of the indole was combined with the feedback control via an online indole assay by photodetector, it was possible to maintain the concentration of the lower indole for the entire duration of the reaction. 0.59 g / l (5 mM) in the reaction mixture, which gave

  finally as a favorable result the accumulation of 108.3 g / l (540 mM) of L-

  tryptophan while the indole concentration was kept below 4 mM. The yield was 99.4% based on indole and 62.7% based on sodium pyruvate. The corresponding experimental data are

shown in Figure 4.

Table II

  Summary of the results of the enzymatic analysis of L-trvyptophane under different conditions Biocatalyst rate * L-Trp conversion of Inosine Accumulated test Pyruvate time Scale Temperature used n * (/ 1) (min) (%) (ml) (C) ( / 1) 1 19 90 18.6 cells / 41.4 mg 3 30

  2 14.7 80 70.0 cells / 25 mg 3 30 -

  3 57.2 250 70.0 cells / 50 mg 6 30 53.6 4 65.8 189 80.6 enzyme / 1 g 120 25 107, 3 133.2 420 59.3 enzyme / 1 g 120 25 210.8 6 127.5 428 62.4 cells / 5 g 600 25 160, 9 7 109.3 377 53.5 enzyme / S g 600 25 201.1 8 144.8 510 64.4 cells / 3 g 360 25 228 9 174.9 496 71.3 cells / 0.83 g 100 25 295 103.6 191 72.4 enzyme / 0.83 g 100 25 160, 9 11 109.5 220 70.4 enzyme / 1.3 g 160 25 187.7 12 188.6 1,173 61.5 cells / 0.83 g 100 25,295 * cells: cells at rest; enzyme: cell-free extract supernatant

Claims (14)

  1. A method of enzymatic synthesis of L-tryptophan characterized in that it comprises the reaction of indole with pyruvic acid and / or one of its salts and an ammonium salt in the presence of tryptophanase by means of '' an automatic computer-controlled bioreactor system comprising a bioreactor connected to a computer equipped with an on-line indole dosing device, in which the computer controls the introduction of indole, pyruvic acid and / or its salt and an ammonium salt in the bioreactor and feedback control the reaction in the bioreactor through the concentration of indole determined by the indole metering device online, the introduction indole and ammonium salt being controlled by a predefined profile which is in agreement with an activity profile of tryptophanase, and pyruvic acid and / or its salt being added to a predetermined time.   2. Method according to claim 1, characterized in that the device   Indole dosing line is a photodetector.   3. Method according to claim 1, characterized in that the computer controls the introduction of the indole according to the following equation: F = 5.51 - 5.44 x 10-2T + 2.79 x 10 -4T2 - 7.05 x 10-7T3 + 7.0 x 10-10T4 F: indole introduction rate (mmoUl / min / l) T: reaction time (min) 4. Method according to claim 2, characterized in that the computer controls the feedback reaction as a function of the following relationship between the feedback signal delivered by computer and the optical density of the photodetector: Sfb = S + 3,492 x DO - 29 , 34 x DO2 + 0.0308 with S = (F x 6 - 33.167) / 3.7257 and F = 5.51 - 5.44 x 10- 2T +2.79 x 10-4T2 - 7.05 x 10 -7T3 + 7.0 x 10-10T4 with F: indole introduction rate (mmol / min / 1) T: reaction time (min) S: initial signal Sfb: feedback signal   DO: optical density of the photodetector.   5. Method according to claim 3, characterized in that the salt   of ammonium is introduced at the same time as indole.   6. Method according to claim 1, characterized in that the acid   pyruvic and / or its salt is added after 40 to 60 min of reaction.   7. Method according to claim 1, characterized in that the   tryptophanase comes from resting cells or a cell-free extract.   8. Method according to claim 1, characterized in that inosine is   present during the reaction as a precipitate for Ltryptophan.   9. Automatic computer-controlled bioreactor system intended to be used for the enzymatic synthesis of L-tryptophan, characterized in that it comprises a bioreactor connected to a computer equipped with an on-line indole dosing device, in which the computer controls the introduction of indole, pyruvic acid and / or one of its salts and an ammonium salt into the bioreactor and controls the reaction in the bioreactor by feedback through the concentration of the indole determined by the online indole metering device, the introduction of the indole and of the ammonium salt being controlled by a predefined profile which is in agreement with a profile of tryptophanase activity, and   pyruvic acid and / or its salt being added at a predetermined time.   10. System according to claim 9, characterized in that the device   Indole dosing line is a photodetector.   11. System according to claim 9, characterized in that the computer controls the rate of introduction of the indole according to the following equation: F: 5.51 - 5.44 x 10-2T + 2.79 x 10-4T2 - 7.05 x 10-7T3 + 7.0 x 10-1lT4 F: indole introduction rate (mmole / min / l) T: reaction time (min).   12. The system as claimed in claim 10, characterized in that the computer controls the feedback reaction according to a relationship between the feedback signal delivered by computer and the optical density of the photodetector expressed as follows: Sfb = S + 3.492 x DO - 29.34 x DO2 + 0.0308 with S = (F x 6 - 33.167) / 3.7257 and F = 5.51 - 5.44 x 10-2T +2.79 x 10-4T2 - 7.05 x 10-7T3 + 7.0 x 10-10T4 with F: indole introduction rate (mmole / min / l) T: reaction time (min) S: initial signal Sfb: feedback signal   DO: optical density of the photodetector.   13. System according to claim 9, characterized in that the salt   of ammonium is introduced at the same time as indole.   14. System according to claim 9, characterized in that the acid   pyruvic and / or its salt is added after 40 to 60 min of reaction.