SU1380212A1 - Method of obtaining l-phenylalanine - Google Patents

Abstract

The invention relates to the microbiological propriety of the body and relates to the preparation of irreplaceable at iHO acids of phenylalanine, which varies with e medicine, and can also serve as a source for the synthesis of aspartame-peptide, used as an intensive sweetener in the food industry. The aim of the invention is to increase the yield of b-phenylalania and reduce the fermentation time. The method also consists in using L-phenylalanine producers of new Bacillus subtilis strains VKPM B-3549 OR BJOM B-3550, or VKPM B-3S51, or VKPM B-3552, which is resistant to p-fluorophenyl alanine and D-tyrosine. Strain VKPM B-3552. it is also resistant to fennlanann and tyrosine hydroxamates / and the VKPM B-3556 strain - k / z-tiannd-alanine. ffltam & l accumulate in the medium from 8.6 to 13 g / l of L-phenylapanine. 2 tab. . with (About with

Description

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The invention relates to the micro ™ biological industry and relates to the method of obtaining the essential amino acid b-phenylalanine, which is used in medicine, and can also serve as the initial product in the aspartame-peptide synthesis, which is used as an intensive sweetener in the food industry.

The aim of the invention is to increase the yield of b-phenylalanium and to accelerate the process.

The method consists in the application of new strains of Bacillus subtilis, obtained with the genetic transformation, mutagenesis and selection of mutants that are resistant to analogs of aromatic amino acids as well as to the antibiotic mitomycin C.

The initial strain is the strain you. subtilifi 92A is a producer of tryptophan, from which the fenylalanine producer is obtained by genetic methods based on the generality of synthetic routes to synthesized chlorine acids and the fact that the tryptophan producer has already created certain prerequisites in the selection process for the synthesis of fenylalanine. The original strain 924 transform the chromosomal DNA of the donor strain You. subtilie 39, which contains a defective trp gene. E, encoding the first enzyme in the tryptophan synthesis pathway and the Ago H gene, which encodes the highly active isoenzyme chorismatmutase, which is absent from the producer of tryptophan. As a result, transformants are obtained that require tryptophan for growth and have a bijicoKHri level of activity of chorium matmutase. Among them, after testing for the ability to produce phenylalanine, a transformant is selected that accumulates 3 g / l of this amino acid, from which a mutant resistant to 0.5 μg / ml of mitochloride C and producing 5 g / l of phenylalanine is obtained by selection. Next: The selection stages consist in the sequential preparation (after exposure of nitrosoguanidine) of mutants, which are resistant to aromatic analogs of amino acids 7 of p fluorothenylalanine (rFF) at a concentration of 9 mg / ml, D-tyrosine (DT) 0.075 mg / ml, tyrosine hydroxate mat (GT) 1 mg / ml, phenyl hydroxamate

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alanine (GF) 0.5 mg / ml, -tiyiyl alanine (TEA) 7.5 mg / ml. As a result, you are obtained at different stages of selection. subfilis VNIIGetika P, VNIIGenetika 33, VNIIGetika, From the strain You, subtilis VNIIgenetika II, a spontaneous revertant to tryptophane-independent Prototrophic strain VNIgenetil 19p was obtained.

The listed new producer strains of Nephe Nilapanin are you. subtiLis Vniigenetika II, you. subttlis VNIIgenetika 33, you. subtilie All-Russian Research Institute of Genetics I-II, Vas, subtilis All-Union Research Institute of Genetics 19p is stored in the All-Union Museum of Industrial Microorganisms (VKPM) and has registration numbers B-3549, B-3552, B-3550 and B-355, respectively.

The strains have the following cultural-morphological and physiological-biochemical characteristics.

The main characteristics of new strains of you. subtilis correspond to the taxonomic description of this type of microorganisms (Steinier R. and others. The world of microbes, vol. 3, p. 184, MIR, 1979).

Ytammyt you. subtil is VKPM B-3549, VKPM B-3550, VKPM B-3551 and VKPM B-3552 are gram-positive spore-forming rods (2.5-4.5) 0.7 microns in size. The size of the dispute (1.0-1.2) (0.6-0.7) microns.

M so-peptone agar. Colonies after 24 hours of incubation at 37 ° C reach 4-6 mm in diameter, solid, round, with a rugged edge, the surface is smooth,

Spicizen agar medium with 100 μg / ml tryptophan and 0 μg / ml adenine. Colonies after 48 hours of incubation at 37 ° C have I mm in diameter, the surface is smooth and shiny.

Relation to carbon sources: use glucose, sucrose, m, altose, mannose, minit, glycerin, acetate.

Attitude to nitrogen sources: assimilate urea and ammonium salts.

They do not grow on milk, they are thinned to gelatin, they grow well on starch.

Relation to amino acid analogues; all strains are resistant to p-fluorophenylalanine (rpp) and D-tyrosine (DT), the VKPM B-3552 strain is resistant to

Phenylalanine (GF) n-tyrosine hydroxamate (GT), BKIIM B-3550 stata is also resistant to / j-thienylalanium (TEA).

The need for growth factors: the growth of all strains is stimulated. Adequine, VKPM B-3549, B-3550 strains and require a trypto fan for growth, the VKPM B-2551 strain does not require tryptophan for growth. The distinctive features of the strains are presented in table. one .

In a general way, the method of obtaining L-phenylalanic is carried out in the following way, the Q. chains of new strains are produced by you. subtilis VKPM B-3549, B - 3551 and B-3552 are grown for 1 day on Hottinger's agar medium, subcultured into flasks with a sterile nutrient medium containing sources of carbon, nitrogen, growth substances and necessary mineral salts, and cultured for 18–24 h on a rocking chair at 220 rpm and 28–37 s.

Received the first seed introduced. in flasks or fermenters with a sterile nutrient medium containing sources of carbon, nitrogen, growth substances and mineral salts. Sucrose or substrates containing it are used as a carbon source, for example, technical sugar, urea or ammonium salts as a nitrogen source, tryptophan and corn extract as growth substances. Fermentation is carried out for 46-72 hours under aeration conditions. 28-37 ° C, pH 6-8, in Flasks on a rocking chair or in a fermenter, at an flow rate of 400 ml / min. Of air flow through the culture liquid of an agitator rotation speed of 1000 rpm. As a result, up to 13 g / l of culture fluid accumulates. -phenylalanine

The determination of fenilalanine in the culture liquid is carried out using paper or fine-scale chromatography in solvent systems butanol, acetic acid (40–10 23) and isopropanol: ammonia (7s3), respectively.

Isolation of L-feiylalanine from the culture fluid is carried out by selective sorption on a weakly basic anion exchanger, followed by desorption with water and repeated

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by sorption from a water eluate onto sulfate (to the cation exchanger in the P-form (ed. St. USSR No. 749889). Elution of b-phenychalanine and sulfonic cation exchanger with hot water and ethiol solution of l-ammonia in a highly concentrated solution of secreted amino acid, from the latter crystallizes as the solution cools and the ammonia is neutralized with acid.

Isolation of L-phenylalanine according to the described scheme provides phenlalanine output up to 60-80%. The content of the basic substance in the chromatographic homogeneous product is not less than 95%. Additional recrystallization allows the floor (It Phenylzlanine preparations with an axial content of at least 99%.

Example 1. The culture of the strain you. subtilis HCSP B-35A9 is sprayed for 18 h npti on hottinger hot medium. A loop is then seeded with 250 ml flasks containing 10-30 ml of nocepHofi medium. The composition of the sowing medium, g /: sucrose; corn extract 10; K. pN04 1.4; KH2P040.6, tryptophan 0.1, RO- doprovodnaya water up to 1 l, pI before sterilization 7.2-7.5, after sterilization 7.0-7.2. Sowing medium is sterilized in an autoclave at 0.8 atm for 30 minutes. After seeding, the flasks are placed on a circular rocking chair (220 rpm) and incubated for 18 hours. The finished seed is introduced in a quantity of 5Z (by volume) into flasks with a capacity of 2,000 mp with 75 ml of fermentation medium. The composition of the fermentation medium, g / l: sucrose I00 ;. corn extract 30, K2HP04, 4; KNORO 0.6; NaCl 0.5; MgF; 04l, 0, urea 5.0, tryptophan 0.1, water supply up to 1 l, pH 7.2. Before urea, urea is added to the prepared medium in the form of a 40% aqueous solution, sterilized at 0.5 atm for 30 minutes. After seeding, the flasks are placed on a rocking chair. After 72 hours of culture growth at 30 ° C, iO g / l 1 ..- phenyl lanine accumulates in the culture fluid.

In order to isolate b-phenyl17an 1 to 1 liter of the resulting fermentation solution, it is passed through a column with 0.4 liters of weakly basic anion exchange resin. At the end of the sorption column

npoNn-inatC T l of water, which ate by connecting to the outlet of the column from the column with Of07 l of supofthioxyte in the H-form, let the water through the system from two columns. After passing through 9 l of water, the columns of the liquid are yed, bphenylalanium, transferred from the anion exchanger IА - 1p to g atoioit KU-2-8, and eluted with an ammonia solution in 50% aqueous standard, skipping zyuent through column c. Fraction of effluent solution; containing b-feimlapenin in a concentration of more than 8.0 g / l, is treated with acetic acid to pH 6.0 and cooled. The precipitated Kristaples of L-phenyl-alapine are filtered off, watered with ethanol and dried. As a result, 6.07 g of crystalline product is obtained with a quench of L-phenylalanine of 97% (yield: 59.8%). The final product from the mother liquor and low-concentration ammonium eluate according to the described tMiue scheme (preliminarily neutralizes ammonia with acetic acid and distilled ethanol) allows to obtain to the content of E but cristoply: about the product (content of the basic substance) 95%), taking into account that the overall yield of crystalline L-fe ™ yilapanine is 74,, 6%,

. Example 2, Breeding of the seed material and cultivation of the Bac, subtilis VKGM strain was carried out as in Example I, but the fermentation medium contains 130 g / l sucrose. After 72 hours, 11.3 g / l of feilanalanin, vsheleni feiilalanin ns I l of the culture fluid are accumulated in the culture liquid, and, as a result, 7f8 g of the crystalline product with phenylalanine content of 96% is obtained. ,, 3%.

Example 3, Cultivation according to ™ of seed material and cultured strain Vas. subtilis VKPN B-355I is carried out9 as in primri I, the difference is that the seed fermentation medium does not contain tryptophan. After 72 hours of culture liquid, 9.1 g / l of pheny-palanic accumulates. You, the goal of fechalalanin from the culture of a ladder, are carried out, kz.k in example 1,

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The yield of the crystalline product is 69.5%, the content of the feilipanine is 96%. Example L. The seed material strain-producers. Fen lalanin you, subtilis VKPM B-3549, B-3550, B-3551 and B-3552 are grown as in example 1. Prepare seeds of each p tamm (20 ml) and inoculate 400 ml of fermentation medium of the same composition as in the example. For strain VKIN B-355i, the media do not contain tryptophan. Cultivation is carried out in laboratory fermenters with a capacity of 600 ml with an air flow rate of 400 ml per minute, the speed of the stirrer is 1000 ° 6 / N H, Cultivation ends as soon as sugar is exhausted from the nutrient medium. The results are shown in Table. 2. Isolation of phenylalanine is carried out as in Example I, the yield of the crystalline product is 77%, the content of feiylalanine is 98%.

Example 5. The seed material strain you. subtilis VUSH B-3550 is grown as in the example. 400 ml of the fermentation medium of the same composition as in example 1 is inoculated with the inoculated seed (20 ml), with the difference that the sugar concentration in the medium is reduced to 0.5% sulfur instead of ureas5

ammonium sulfate.

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Cultivating is carried out in a laboratory fermenter equipped with an automatic pH-setting system} at 30 ° C5 flow rate of air blown through the culture fluid 400 MP / min, the rotation speed of the stirrer is 1000 rpm. The pH value of the culture liquid is maintained at one. During cultivation, a mixture of 40% sucrose solution and 25% ammonia aka solution in a ratio of 0: 1 by volume is added to the fermenter according to the pH sensor signal. Cultivation duration of the research institute is 58 hours. The accumulation level of the phenyl-leg is equal to llj g / l.

Isolation of phenylalanine is carried out as in Example 1. The yield of the crystalline product is 70%, the content of phenyl-alanine is 98%,

The proposed method differs from the prototype by a higher level of formation of phenylalanine - up to I3 g / l (the prototype has 2.88 g / l), a shorter KIM with a fermentation period of 46-72 hours (the prototype has 96 hours). containing sucrose, nitrogen sources (urea or ammonium salts), mineral salts, and as a source of growth substances - corn extract and tri-1-tofan, if the substance used is in need of this amino acid. With the release of phenylalanine from the fermentation solution rtoro, the yield is 60-80% of the crystalline product.

B-3549 B-3552

B-3550 B-3551

p-fluorophenylalanine; D-tyrosine; phenylalanine hydroxamate; tyrosine hydroxamate; / S tishalshanin

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Claims (1)

Invention Formula A method of producing L-phenyl-lapine, which involves growing the Bacillus subtilis strains producing it under aeration conditions on a nutrient medium containing carbon, nitrogen, mineral salts and growth substances, up to the maximum accumulation of the target product, followed by In order to increase the yield of the target product and accelerate the process, VKPM B-3549 or VKPM B-3550, or VKPM B-3551, or VKPM B-3552 are used as producers. Table Needs Needs Needs Does not need Strain you. subtilis VKPM B-355 || B-3550 B-3549 | B-3552