SU1693056A1 - Strain of bacteria bacillus subtilis - a producer of l-phenylalnine - Google Patents

Description

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(21) 4716354/13 (22) 07.06.89 (46) 11/23/91. Bul №43 (71) All-Union Scientific Research Institute of Genetics and Breeding of Industrial Microorganisms (72) T. A. Yampolska, G. A. Velikzhanina. N. I. Zhdanova, T. A. Bachina, N. A. Vasilyeva and A. K. Sokolov (53) 663.15 (088.8)

(56) US Patent

No. 4591562. cl. C 12 R 13/22, 1986. USSR Copyright Certificate No. 1380212 / class. C 12 R 13/22, 1986. (54) STRAINS B-ACTERIA BACILLUS SUBTILIS - PRODUCTED-FENILALANINE

(57) The invention relates to biotechnology and concerns a new strain, the essential amino acid L-phenylalanine, which is used in medicine, and can also serve as a starting material in the synthesis of aspartame, a peptide, used as an intensive sweetener for food. The aim of the invention is to obtain a bacterial strain Bacillus subtills VKPM-4761 (264-T), capable of synthesizing up to 17 g / l of L-phenylalanine with growth on simple compositional media. When grown on a medium with corn extract, ammonium nitrogen, mineral salts and while maintaining the sugar concentration in the medium at a level close to the limiting one, after 80 hours, 17 g / l of L-phenylalanine accumulates in the culture fluid. The conversion rate of sugar to phenylalanine is 12.2%.

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The invention relates to biotechnology and relates to a new strain - the producer of the essential amino acid L-phenylalanine, which is used in medicine, and can also serve as the initial substance in the synthesis of aspartame - a peptide used as an intensive sweetener for food products.

The aim of the invention is to obtain a bacterial strain Bacillus subtills of VKPM B-4761 (264-T), capable of synthesizing up to 17 g / l of L-phenylalanine with growth on simple compositional media.

The strain was obtained as a mutant of the B. subtills strain of VNIIgenetika-19p by mutagenizing cells with nitrosoguanidine and selecting mutants that are auxotrophic for tyrosine.

The strain is stored in the columns of Hottinger semi-liquid agar (0.7%) with 2-5% maltose under a layer of vaseline oil at 2-4 ° C or in a lyophilized state in sealed glass ampoules at room temperature.

The strain has the following characteristic.

Cult ral morphological signs. Gram-positive spore-forming sticks (2.5–4.5) xO, 7 µm in size. The size of the dispute (1.0-1.2) x (0.6-0.7) microns.

M saltpton agar and Hottinger 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, cream-colored.

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Spicizen agar medium with 40 µg / ml of tyrosine and 10 µg / ml of adenine: after 48 hours incubation at 37 ° C, colonies are 1 mm in diameter, the surface is smooth, shiny, grayish-white,

Physiological and biochemical signs. Relation to carbon sources: uses sucrose, glucose, maltose, mannitol, glycerin, acetate.

Relation to nitrogen sources: uses urea, ammonium salts.

Does not grow on milk, dilutes gelatin weakly, grows well on starch.

Relation to amino acid analogs: resistant to p-fluorophenylalanine (9 mg / ml) and D-tyrosine (0.075 mg / ml).

Growth factor requirement: requires 40 µg / ml tyrosine for growth, growth is stimulated by adenine.

Tyrosine auxotrophy allows the strain to expend the entire pool of prephenic acid (a common precursor of phenylalanine and tyrosine) for the synthesis of phenylalanine alone. In addition, Z-deoxy-D-arabinoheptuloose-7-phosphate synthetase (DAGF-c), a key enzyme for the biosynthesis of aromatic amino acids, is repressed in the original strain in the presence of tyrosine and phenylalanine. The absence of tyrosine in the pool of the proposed strain leads to a decrease in the repression of DAGF-c, which ensures that the strain has a higher level of phenylalanine (up to 17 g / l) than the original strain.

Another advantage of the proposed strain in comparison with the known one is the complete absence of tyrosine in the culture fluid, which greatly facilitates the process of isolation of the target product. At the same time, the need for tyrosine is provided by the presence in the fermentation medium of corn extract, which is added to the medium and to a known strain. The strain grows at 28-3 ° С: the optimum temperature is 32 ° С. Growth is possible at pH 5-9, the optimum pH is 7.0-7.2.

The preparation of L-phenylalanine using the proposed producer strain is carried out as follows.

Cells strain you. The subtills of VKPM B-4761 are grown for 1 day on Hottinger's agar medium, subcultured in flasks with a sterile nutrient medium containing carbon sources, nitrogen, growth substances and necessary mineral salts, and cultured for 18-24 h on a rocking chair at 220 rpm and 28-37 ° C. The obtained seed is introduced into flasks or fermenters with a sterile nutrient medium containing carbon sources.

nitrogen, growth substances and mineral salts. As a carbon source, sucrose or substrates containing it are used, for example, process sugar, urea or ammonium salts as a nitrogen source, and corn extract as growth substances. Fermentation is carried out for 46-72 hours under aeration conditions at 28-37 ° C, pH 6-8, in flasks on a shaker.

0 or in a fermenter. At the end of the fermentation, up to 17 g / l of phenylalanine accumulates in the culture liquid.

The L-phenylalanine preparation from the fermentation solution is prepared as a feed preparation or is isolated in crystalline form by known methods.

Example 1 Strain you. subtilis VKPM B-4761 grown for 18 hours at 37 ° C

0 on Hottinger's agar medium. A loop is then seeded with 250 ml flasks containing 30 ml of inoculum. The composition of the sowing medium, g / l: sucrose 50; corn extract 20: K2HP04 1.4; KH2PO-J 0.6;

5 tap water to 1 l. The pH before sterilization is 7.2-7.5, after sterilization 7.0-7.2. The seed medium is sterilized in an autoclave at 0.8 atm for 30 minutes. After seeding, the flasks are mounted on a circular rocking chair.

0 (220 rpm) and incubated at 30 ° C for 18 hours. The finished seed was introduced in an amount of 5% by volume into 750 ml flasks with 30 ml of fermentation medium. The composition of the fermentation medium, g / l: sucrose 130;

5 corn extract 30; PRC 1.4; KH2P04 0.6; NaCl 0.5; MgSO 1.0; urea 5.0; water supply 1 l. pH of 7.2. Urea is added to the prepared medium in the form of a 40% solution, sterile at 0.5 atm for 30 minutes. After seeding, the flasks are placed on a rocking chair. After 72 hours of growth at 30 ° C, 12 g / l of phenylalanine accumulates in the culture fluid,

5 In addition to phenylalanine, the following amino acids are contained in the culture fluid, g / l: 0.56; asparagine; 0.042; threonine 0.0088; series 0,215; glutamic acid 0.119; alanine 0.098; isoleucine 0.056: lei0 qing 0.171; histidine 0.164; lysine 0.116. Aromatic amino acids tyrosine and tryptophan in the culture fluid are absent.

The original B. subtilis strain of the All-Russia Scientific Research Institute of Tene-19p under similar conditions produces 9.1 g / l of L-phenylalanine.

For example 2. The seed of the VKPM B-4761 strain is grown as in Example 1. 650 ml of fermentation medium are inoculated with the prepared seed.

The composition of the medium, g / l: corn extract 30; NaCI 0.5; (5; MgSCM 1; KH2P04 0.6; KH2P04 1-4; tap water up to 1 l. Cultivation is carried out at 32 ° C in a laboratory enzyme equipped with an automatic pH-setting system and a peristaltic pump for feeding sterile sugar solution into the fermenter, the flow rate of air through the culture fluid is 600 ml / min and the rotation speed of the stirrer is 750 rpm. The pH value of the culture fluid is maintained at 7.0-8.1 using 6.9N ammonia water solution. at a level close to the limiter For this, after 1 h after seeding, the sugar solution of a concentration of 700 g / l is fed into the fermenter, first at a rate of 1 ml / h, then after 15 and 21 h of cultivation the feed rate is increased to 2.0 and 2.5 ml / h, respectively, and then not In this mode, the concentration of sugar in the medium during the fermentation process does not exceed 0-5 g / l. The cultivation is carried out for 80 hours. As a result, 17 g / l of phenylalanine accumulates in the culture liquid. conversion rate of 12.2%. Strain-prototype under similar conditions allocates 9.5 g / l of phenylalanine, the conversion rate is 7%.

Example 3. The growth conditions of the seed, the initial composition of the fermentation medium and the fermentation conditions as in Example 2. The difference is that the fermentation medium contains 15 g / l.

(NH4) 2SO4 instead of sugar, molasses solution with carbohydrate concentration of 450 g / l is fed into the fermenter. The feed rate is 1.7 ml / h, after 15 cultivation the feed rate is increased to 3.5 ml / h and then not changed. Cultivation is carried out at 32 ° C for 80 hours. At the end of the process, up to 12 g / l of L-phenylalanine accumulates in the culture fluid. The conversion ratio of carbohydrate molasses to phenylalanine is 8.2%.

Example 4. Fermentation is carried out in a 10 m3 apparatus. The composition of the fermentation medium, as in example 2. The initial volume of the medium is 5 m3. Seeds grown in flasks as described in Example 1 are added in an amount of 1 l. Fermentation is carried out at 32-33 ° C, stirring at 260 rpm and blowing air at 5 m / min. The pH is maintained at the level of 7 automatic feeding of a 20% aqueous solution of ammonia. After 12 hours of fermentation, a sterile sugar solution with a concentration of 550 g / l is started to be supplied to the apparatus. Initially, the feed rate is 8 l / h, after 10 h it is increased to 23 l / h and then not changed. The total amount of feed fed is 1,300 liters (720 kg of sugar). After 75 hours of fermentation, the content of phenylalanine in the culture fluid is 13 g / l. The conversion rate of sugar to phenylalanine is 10.8%.

Formula of the invention The bacterial strain Bacillus subtilis VKPM B-4761 is a producer of L-phenylalanine.

Claims (1)

Claim The bacterial strain Bacillus subtilis VKPM B-4761 is a producer of L-phenylalanine. Compiled by L. Mineeva Editor O. Yurkovetskaya Tehred M. Morgenthal Corrector T. Malets Order 4051 Circulation Subscription VNIIIPI of the State Committee for Inventions and Discoveries under the State Committee for Science and Technology of the USSR 113035, Moscow, Zh-35, Raushskaya nab .. 4/5 Production and Publishing Combine Patent, Uzhgorod, 101 Gagarin St.