SU990814A1 - Process for producing p-tryptophan - Google Patents

Description

(54) METHOD OF OBTAINING L-TRIPTOFAN

The invention relates to the medical industry, in particular to the method of obtaining the essential amino acid L-tryptophane used in the pharmaceutical industry and the medical industry in the preparation of mixtures containing free amino acids for parenteral nutrition, for the enrichment of protein hydrolysates used in the same 1 {x, It can also be used as an additive for balancing the composition of fodder in live-breeding. Of the known microbiological methods for producing L-tryptophan, the most cost-effective method is the use of microorganisms capable of producing tryptophan based on direct enzyme extraction from common sources of carbon and nitrogen without introducing TfMoi precursors into the medium, for example, indole or anthranic acid. lots. At the same time, molasses, gndrol or technical sugar are used as a source of carbon, urea, ammonium salts as well as necessary growth supplements and mineral salts are used as a source of nitrogen. Upon receipt of high purity crystalline L-trnptophan for medical purposes, the technology of its isolation becomes much more difficult, which leads to a decrease in the final yield of the product and an increase in the cost of its additional purification, and therefore the effectiveness of any method for producing tryptophan is taken into account fermentation and stages of crystallization of the drug. A known method for producing tryptophan II) is the cultivation of mutant strains of Nebacterium glutamicum on media not supported by the precursors of this amino acid for the growth of biotin, phylalanine, tyrosine, and resistant to at least one of the tryptophan analogues and one of the phenylalanine or tyrosine tyrosine analogues of phenylalanine or tyrosine. On a medium with glucose and a source of growth factors (NZ-amine), the level of education is. nor t {zhttofana with cultural zhndkoi

reaches 3.6 g / l in 4 days of cultivation. On a medium with molasses, corn extract and hydrolysis of soybean latitude, the level of tryptophan formation is reached 3.4–16.8 g / l in 3-4 days of cultivation. Crystalline tryptophan in a known process is isolated using a strongly acidic cation exchanger in 50% yield from the culture fluid containing glucose as a carbon source. For media containing molasses, indicators characterizing the process of tryptophan separation are absent, apparently due to insufficiently effective purification of tryptophan from other amino acids that accumulate during fermentation or are present in the original fermentation medium (for example, if the medium contains molasses).

There is also known a method to isolate tryptophan from a culture fluid, including selective sorption of tryptophan on a weakly basic anion exchanger, followed by its desorption with water or a weak acid solution and re-sorption from this solution to concentrate on sulfocatonite in H-form, and providing crystalline product yield up to 70% depending on the desired purity of the final preparation 2.

The disadvantages of the known methods for producing L-tryptophan and the method for its isolation from the microorganism liquid are:

the need for several growth factors and Cor strains, glutamicum, which necessitates the introduction into the nutrient medium of the sources of these factors, in particular the hydrolysates;

the long duration of the process of cultivation of microorganisms is 72-96 h;

The low level of tryptophan accumulation on standard carbon sources (glucose, sugar), which are most suitable for efficient recovery of crystalline tryptophan, whereas molasses and hydrol are types of raw materials with an unspecified composition that affect microorganism growth and biosynthesis, do not provide stable indicators at the stage of fermentation, and reduce the effectiveness of the selection of the crystalline product of high purity due to significant impurities of pigments, mineral salts, amino acids and o-xi acids, etc .;

relatively low yield (50%) of crystalline tryptophan at the stage of isolation from the culture fluid.

The closest in technical essence and the achieved effect to the proposed

is a method for producing L-tryptophan by submerged cultivation of Bacillus subtilis strains Gene-557 / p or 2282 on a nutrient medium containing carbohydrates as a carbon source, sources of nitrogen, phosphorus, growth substances and other essential mineral salts under conditions of aeration of the medium followed by isolation of L-tryptophan by one of the known methods using ion exchange and crystallization of amino acid 3.

The disadvantages of this method are the low accumulation of tryptophan on glucose and sugar and longer fermentation time (on a medium with technical sugar, the level of tryptophan formation reaches 4.6 g / l, on a medium with molasses or hydrol, 8.2-10.1 g / l for 72 h of cultivation).

The aim of the invention is to increase the yield of L-tryptophan.

The goal is achieved by the fact that according to the method of producing L-tryptophan, which involves the submerged cultivation of microorganisms producing it, of the Bacillus subtilis species under aeration conditions on a nutrient medium containing carbohydrates as a carbon source, nitrogen sources, phosphorus, growth substances and necessary mineral salts, the subsequent allocation of the target product from the culture fluid using ion exchange and crystallization, from microorganisms of the species Bacillus subtilis use the strain Bacillus subtilis ARRIAS-15, In the process of cultivation, a concentrated nutrient supplement containing a source of carbon, inorganic nitrogen and growth substance is introduced into the medium in an amount that maintains the concentration of dissolved oxygen in the medium of 10-40%.

The essence of the method is to cultivate a new schm of Bacillus subtilis VNIIgenetika - 15 on a nutrient medium containing sources of carbon, nitrogen, mineral salts, under aeration conditions when feeding in the process of cultivation of the nutrient mixture followed by the generation of the resulting culture. L-tripto liquids. fan-ion exchange method. As a carbon source, use technical sugar. At the final growth phase, 12-30 hours after the start of fermentation, a nutrient supplement is added to the fermenter, containing a nitrogen source of carbon and a growth substance so that the dissolved oxygen concentration does not exceed 40%. The use of a new strain of the All-Russia Scientific Research Institute of Genetics-15 in combination with the introduction of feed during fermentation allows to increase the rate of biosynthesis of L-tryptophan and increase its level in the medium using sugar (or sucrose) as a carbon source. After 48 hours of cultivation at 37 ° C, the level of accumulation of L-tryptophan in the culture fluid reaches 10.8 g / l. When l-tryptophan is used, the weakly basic condensation-type anion exchanger and the sulfonic cation in the H-form are used in series. The yield of crystalline L-tryptophan is 65-75%. A new strain of you. subtil is VNIIgenetica-15 obtained from a known strain You. subtilis Gene-3557 by using a stepwise selection, including treating the cell suspension with a mutagenic factor N-methyl-M-nitroso-M-guanidine (concentration 200 mg / m 20 min exposure) and obtaining mutants resistant to structural analogs of amino acids. At stage 1 of the selection, mutagen-treated cells of the Gene-3557 strain were seeded on minimal medium containing the histidine analogue 1-L-methylhistidine at a concentration of 5 mg / ml. Among the grown resistant colonies, a strain 924 would be selected, the cells of which, after exposure to a mutagen, were cultured on a medium containing the following mixture of tryptophan, phenylalanine, and tyrosine analogues: 5-fluorotryptophan (2 mg / ml), phenylalanine hydroxamate (0.5 mg / ml) and tyrosine hydroxamate (2.5 mg / ml). From among the colonies, resistant and the specified mixture of analogs of amino acids, the strain 22-13 was selected. The cells of strain 22–13 were again treated with the mutagen and plated on medium containing a mixture of the same amino acid analogs, but at a concentration of 2.5 mg / ml, 0.5 mg / ml, 2.5 mg / ml, respectively. From among the colonies that are resistant to this mixture of analogues, the VNIIgenetika-15 strain was isolated. In tab. Table 1 gives comparative data on the level of tryptophan accumulation during fermentation in flasks on medium with sucrose in the original strain of gene-35 5 7 and in the new strain of vniIgenetstsa-15. Under these conditions, the yield of tryptophan at the end of the fermentation in the new strain of the VNIIgenetik-15 exceeds the yield of the amino acid in the original strain by 45%. A high level of accumulation of L-tpShlofan (9-11 g / l) in the VNIIgenetika-15 strain can be achieved in 48 h when cultivated in a fermenter with the introduction of nutrient feed during fermentation according to the proposed method. The Bacillus subtlMs strain of the All-Russia Scientific Research Institute of Epidemiology-1S is stored in the Central Museum of Industrial Microorganisms of the Institute of Scientific Research Institute of Genetics, where it is deposited under the number CMPM-B 306. The strain Bacillus subtitis VNIIgenetika15 has the following cultural and morphological characteristics. Gram-positive spore-forming bacillus. Colonies on mono-peptone agar after 24 hours of incubation prn 37 ° C reach. 4-5 mm in diameter, solid, round, with 1 cut edge, the surface is smooth, with weak radial along the edge and concentric in the center of striation, the color is grayish-white. Sowing, streaking on mi-peptone agar after 24 hours of incubation at 37 ° C gives moderate growth, a broad bar with a finely toothed edge, a characteristic odor, consistency is oily, and grayish-white. Growth on moc-peptone broth after 1 day. incuba-. At 37 ° C, it is characterized by moderate clouding of the medium without film formation, the smell is characteristic, the precipitate is scanty, and tingling upon shaking. On Spitsizen agar mineral medium with a glucose colony after 3 days of incubation, at 37 ° C reach 1.5-2.0 mm in diameter, solid, round, with a smooth, shiny surface, dark white color, the structure is uniform. on liquid liquid mineralic acid (24 h incubation at 37 ° C in a roll) is characterized by a weak streamy turbidity. environment. Growth of the VNIIgene.tika-15 strain on a liquid or agarized minimal medium is not stimulated by L-phenylalanine, in contrast to the original strain of Gene-3557. The method is carried out as follows. Strain you. VNIIgenetica-15 subtilis is grown for 24 hours on agar medium (MPA), subcultured in flasks with sterile nutrient medium containing sources of carbon, nitrogen, growth substances and necessary mineral salts, and grown for 18-24 h on a rocking chair, providing sufficient aeration at a temperature of 28-EO ° C under aseptic conditions. The obtained seed in the amount of 1-10% is transferred to a fermenter with a sterile nutrient medium containing sources of carbon, nitrogen, growth substances and the necessary mineral salts. As a carbon source, sucrose or substrates containing it are used, for example, technical sugar, as a nitrogen source — moquine, as a source of growth substances — corn extract. 7 The fermenter is equipped with a dissolved oxygen concentration sensor, a device for aeration, mixing, maintaining temperature, sampling and supply of additional nutritional supplement during fermentation. Fermentation is carried out for 48 hours at a temperature of 35-40 ° C, pH 6-8, continuous Noah aeration and mixing. At the beginning of the fermentation process, due to the intensive growth of the biomass, the concentration of dissolved oxygen decreases and then begins to increase. 12-30 hours after the fermentation with an increase in the concentration of dissolved oxygen pOj above 40% of air saturation at atmospheric pressure, a concentrated nutrient supplement containing a source of carbon, nitrogen, and growth substances is introduced into the enzyme medium. The additive is introduced in such a way that the dissolved oxygen concentration pOj does not exceed 40%. Within 48 hours of fermentation, the concentration of L-triggtophane in the culture fluid reaches 9-11 g / l. The resulting culture fluid is treated with followers of lime milk and sulfuric acid in order to separate the biomass by filtration or centrifugation. Then, the isolation of crystalline L-tryptophan is carried out in a known manner, with the successive sorption of L-tryptophan on a weakly based condensation-type anion exchanger and sulfo cation exchanger in the H-form. Example .1 (control). For the preparation of the sowing material daily culture of the strain You. subtil is BftHMi enotic 15 grown on agar medium (MPA), transferred to 750 m flasks with a content of 40 ml of sterile inoculum. The culture medium has the following composition,%: Technical sugar Corn extract (according to technical KH2 Р04 К2НРО4, 0-7.2 The medium is sterilized at 0.8 atm for 30 minutes, the seed is grown for 18-20 hours on a rocking chair (200-300 vol / mts) at 28-30 ° C. For seed of the fermentation medium, the seed is added in an amount of 5%. has the following composition,%. Technical sugar 10,0 Corn extract (according to the technical ECU) 3.0 KN, U40.06 4 K2HP040.14 MgSO47HjO0.1 NaCI0.015 Urea 0.5, 0-7.2 Fermentation medium without urea is sterilized at 0.8 atm for 30 minutes. Urea as 25% - The solution was sterilized separately at 0.5 atm for 30 minutes and added to the basic medium before sowing. Fermentation was carried out by the method of deep cultivation in Erlenmeyer flasks (capacity 250 ppm with two baffles) containing 20 ml of medium on rocking chairs (400 r / min) at t 37 ° C for 48 hours. After 48 hours, 7.5 g / l of L-tryptophan accumulates in the culture fluid. Example 2. The producer strain, the composition of the inoculum and fermentation media is the same as in Example I. In contrast to the fermentation management method described in Example 1, the cultivation is carried out with a fractional feed recharge depending on the content of dissolved oxygen in the medium. The feed has the following composition,%: Technical sugar 50.0 Corn extract 20.0 Urea 2.25, 2-7.3 The feed is sterilized at 0.8 atm during 30 minutes. Urea is sterilized separately as a 25% solution at 0.5 atm for 30 minutes. During fermentation, the content of dissolved oxygen (pO3) in the medium decreases to zero, then increases. When pOj is increased to 40%, the amount is fed in an amount that causes a decrease in pOj. Feed start after 19-30 hours of fermentation. The volume of spent recharge 44 ml. The dynamics of tryptophan accumulation in the culture fluid are shown in Table. 2. Example 3. The producer strain, the composition of the inoculum and fermentation medium and feed are the same as in example 2. Fermentation is carried out in a 30 l fermenter, filling factor is 0.5. Mixing and aeration conditions ensure a sulfite value of 3.5 g Oa / lh. During fermentation, the dissolved oxygen content (pOg) decreases to zero, then increases. When reaching a value of pOj equal to 40%, turn on the supply of water, which is fed into the atcharat until p02 is reduced to 15%. Feed feed start at 28 h of fermentation. The amount of feed consumed during fermentation is 450 ml. After 48 hours of fermentation, 9.7 g / l of L-tryptophan accumulates in the cupural fluid, which is recovered as follows. 1 liter of culture liquid, released from biomass, with concentration. 9.7 g / l of tryptophan is directed to a column with 0.250 l of sorbent - a weakly basic condensation-type anion exchange resin (colo-IHA). Before washing, a second column with IA-1p is connected to the outlet of column 1, And (column 2 of AA contains 0.250 l of sorbent). At the end of the washing, the column 2 of the IA is disconnected, and to the output of the column 1 of the IA, a column with sulfonic cation exchanger is connected (the column 1 of the CU contains 0.1 l of sorbent dates) and is eluted. Washing column 1IA and elution of tryptophan from it is carried out with water, passes of 0.5 and 2.5 l of water, respectively. Further elution of tryptophan from column 1 of the AI is carried out with a solution leaving the column 1 of the CU. The volumetric flow rate of the solution circulating through the two-column system is 0.25 l / h, the duration of this operation is 10 hours. The next batch (1 l) of the liquid liquid is transferred to the TI column 2. Before washing, an IA column 1 is connected to the column inlet, and a third column with an IA-1P is connected to the outlet (column 3 IA. Contains 0.25 l of sorbent). After the completion of the washing of the column 2 with water with a water column, the SIA column is disconnected, and the column 1 of the KU is connected to the output of the column 2AA. Eluent (water), taken in the amount of 2.5 l, is passed through successively connected columns 1 AI, 2 IA and 1 CU. Further elution (resorption) of tryptophan from column 1IA and 2IA to column 1 KU is carried out by connecting the column 2IA and 1 KU to a closed system. Sorben in column 1 IA is subjected to regeneration. The next batch of culture liquid (1 l) is treated similarly to that described above using columns 3 IA 1 IA (resin recovered, column Connected to the outlet from column 3 IA during washing), 2 IA (connected to the inlet of the wheel 3 IA during washing and eluir vash ) and 1 КУ (it is connected to the output of the column 3 ИА when zyuirovanie). The adsorption of tryptophan is carried out by connecting to the outlet of the 1 KU column another saline salt with KU-2-8 sorbent (the HCU column contains 0.1 l of sorbent). Thus, the solution is circulated through a system consisting of successively connected columns 3 IA, 1 KU, and 2 KU (column 2 IA is disconnected and the sorbent is regenerated). The elution of trstofan from the 1 KU column is carried out with a water-ethanolic ammonia solution at a temperature of 70 ° C. After treatment with a rich fraction of the acidic acid with acetic acid and cooling to 0 ° C, an L-tryptophan crystal is obtained. After drying at 50 ° C, 17.2 g of L-tryptophan are obtained, with a basic substance content of more than 99%. After the described operations for extracting tryptophan from three liters of culture liquid. On columns 3 IA and 2 KU, sorbed trhigophane remains in an amount of 3.5 g. Tryptophan is also contained in poor fractions of ammonium eluate and in the ammonia-ethanol mother liquor obtained after separation of tryptophan crystals in the amount of 3.5 g. With regard to tryptophan, which can be further separated in the subsequent technological cycles, the total yield of tryptophan is 70%. The advantages of the proposed method are that the method achieves significant savings in desalted water, ammonia and ethanol, due to the fact that the low concentrated fractions of the eluate are used as eluent in subsequent teriological cycles for desorption of L-tryptophan from anion exchanger and sulfonic cation exchanger. In this way, it is also possible to reduce product losses during discharge. The resulting rich fractions of the eluate are neutralized with acetic acid upon cooling, and the tryptophan is precipitated as crystals, which are isolated by filtration and dried. The mother liquor is sent for sorption onto an anion exchange column. The total yield of tryptophan reaches 70%. The content of the basic substance in the resulting preparation is not less than 96%, and after recrystallization not more than 99%. The proposed method for producing 1.-tryptophan has the following advantages in comparison with known methods: a higher level of tryptophan accumulation on media with a standard source of carbohydrates — sucrose or sugar (9–11 g) compared to 3.6–4 | 6 g / l ); reduced fermentation period achieved by the introduction of nutritional supplements (48 hours compared to 72 hours); In the method, the step of isolating and purifying β-tryptophan has been improved, which makes it possible to significantly reduce the consumption of desalted water, ammonia and other reagents at this stage.

99081412

The output of L-tryptophan after 48-68 h

fermentation in 250 ml flasks at 37 ° C in medium with 10% sucrose,

, 6,16,6

7.39.6

{0 Table2

 2136 I 48

2.0 .6,48,610,7

0,170,290,2320,22

Note, t is the time of fermentation;

Claims (3)

T is the accumulation level of L-tryptophan; T / t is the rate of accumulation. The invention of the method for producing L-tryptophan by deep cultivation of its producing microorganisms of the Bacillus subtil species is under conditions of aeration in a nutrient medium containing carbohydrates as a source of carbon, nitrogen sources, phosphorus, growth substances and necessary; mineral salts, followed by separation of the target product from the culture fluid using ion exchange and crystallization, characterized in that, in order to increase the yield of L-tritofan, from Bacillus subtilis species using microorganisms, the Bacillus subtilis strain VNIIgenetika-15 is used, Table 1 10 ml of medium, g / l 48 hI68 h at the same time, in the course of cultivation, a concentrated nutritional supplement containing sources of carbon, inorganic nitrogen and a growth substance is introduced into the medium in an amount that maintains the concentration of dissolved oxygen in the medium 10-40%. Sources of information taken 80 attention in the examination 1. US patent number 3849251, cl. 195-29, published. 1974. 2. USSR author's certificate number 745889, cl. from 12 P 13/22, 1977. 3. USSR author's certificate number 480758, cl. from 12 P 13/22, 1972.