SU659611A1 - Method of obtaining l-aspartic acid - Google Patents

Description

(54) METHOD OF OBTAINING L-ASHOLATIC ACID

The invention relates to a technique for producing L-aspartic acid and can be used in microbiology, medicine and the food industry. A method is known for producing U-ac raginic acid by displacing microorganism cells from the cell suspension, for example Rea domona 6 Eit ultaciens, from 1 M (11.6%) with a solution of ammonium fumarate and toluene followed by incubation for 48 hours at E7 ° C The disadvantage of the known method is its duration, contamination of the target product with impurities and periodicity. There is also known a method for producing L-aspartic acid by culturing microorganisms producing aspartase, followed by isolating aspartase from cells, immobilizing the enzyme on ion exchangers and passing an aqueous solution of ammonium fumarate through them. The disadvantage of this method is the need to isolate the enzyme from the cells and the rapid decrease in the activity of L-aspartic acid. There is also known a method for producing L-aspartic acid by culturing the microorganisms producing it Esche.HcH-ia coB (on a nutrient medium, separating the cell suspension from the culture fluid, washing, immobilizing cells by incorporating them into polyacrylamide gel, grinding the gel, activating the cells and washing them with ammonium fumarate, - This method is closest to the proposed - The technical essence and the achieved result. The disadvantage of the known method is its duration and insufficient efficiency due to the decrease in the activity of aspartae during cell immobilization. The aim of the invention is to accelerate the process and increase its efficiency. This is achieved by the fact that in the proposed method of obtaining L-aspartic acid Escherichia 83 or Escherichicha coEi AH 52, or Escherichi o coSi 85 strain is used as microorganisms from the Eechei tch ia coEi species, and the cells are activated after

separating them from the culture liquid by heating the cell suspension at 45-55s for 40-60 minutes, and immobilization is carried out in the presence of one of the salts of aspartic acid in an amount of 3-9% by weight, followed by cooling the mixture to 0-10 ° C with simultaneous exposure to light of the visible region with an intensity of 15-20 thousand lux for 510 minutes.

The method is carried out as follows.

The L-aspartic acid-producing microorganisms of the Esche ricbia co6i type strains Ah 52, 83 or 85 are cultured for 6 hours in a nutrient medium (BCH) at 30-37 ° C, then the cells are separated, activated by heating at 45-55 s, mostly at. for 40-60 minutes and then washed.

The subsequent immobilization is carried out in the presence of one of the salts.

Indicator

The duration of the process, h

The specific activity of aspartase included

in the gel of native cells,

Eschenichia co6i unit

activity

The specific activity of aspartase immobilized native cells of Eschenichia coEi,% of free cells

The specific activity of aspartase activated EschePich4a coEi cells,

The results of the table show that reducing the time of cultivation of microorganisms - producers of aspartase and activation time reduces the duration of the process by about 8 times.

Example, one-cell Eschef chia co8i strain B o, from the surface of the m-peptone agar (MPA), 1-2 ml of sterile liquid medium and using a sterile pipette are transferred into sterile liquid seed

aspartic acid, in the amount of 3-9 EEC,% and mixtures containing wt.%: acrylamide 10-20, methylene bisacrylamide 0.33-0.66; N, N, N; N-tetramethylTylenediamine 0.00025-0,0005; Ammonium persulfate 0.0375; .riboflavin

0,005. The cells together with the mixture are illuminated with light of the visible region with an intensity of 15–20 thousand lux and simultaneously cooled to 0–10 ° C for 5–1–0 min.

Then the gel is crushed and an ammonium fumarate aqueous solution is passed through it at a rate of 0.5-1.0. at 25-37 s.

A pure product is obtained with a yield of 88-95% of consumed fumarate.

The product is crystallized by acidifying the solution to a pH of 2.5-3.0, the crystals are separated by filtration, the precipitate is washed with ethyl alcohol and dried

on air.

The data of comparative tests on the proposed method and the known (control) are shown in the table.

The proposed method

74

187

13.1

77

173

medium of the same composition. The culture is harvested for 12–18 h in a deep-seated manner at 30-ZB C in 750 ml pumping flasks (medium volume 50 ml).

Then, seed in the amount of 2–3% is introduced into the medium of the same composition and grown in the same way for 6 hours under similar conditions in shaking flasks with a medium volume of 125 ml. Cells are separated from the medium in a C-44 centrifuge. 50 ml of the initial suspension of cells of the E5cher chta coEi strain are obtained from 4 liters of medium; B, which contain 56, 92 mg of protein in 1 ml (determined by the Lowry method and have specific and total aspartaznu activity, respectively, equal to 57 units per 1 mg of protein for 1 hour and 161500 units (1 unit equals 1 micromol of the formed, aspartic acid per 1 mg of protein for 1 h. After three times washing the suspension of cells with portions of 230 ml of sterile tap water or 0.01 M phosphate buffer with pH 7.0, followed by centrifugation for 10 min at b thousand rpm, 50 ml are obtained washed cell suspension of EH pia coEi strain B with a protein content of 29.70 mg in 1 ml and specific and total a activity equal to 180 units per 1 mg of protein per 1 h and 154850 e. Aspartase activity of free cells is determined as follows. To 1.7 MP cell suspension add 13 ml of 0.1 M phosphate buffer with pri 8.0 and 15 ml 1 M 11.6%) solution of fumarate agfuni prepared by adding to 116 g of fumaric acid a concentrated solution of ammonia aka to pH 8.5 and water to 1 l. In 1 M ammonium fumarate solution magnesium chloride is introduced in the amount of 0.001 (0.002032%). 0.3 ml of toluene is added to the reaction mixture. The reaction mixture was incubated under constant stirring at 37 ° C, 3 ml aliquots were removed after 30 minutes for 2 hours. The reaction in aliquots was stopped, and the bacteria cell was centrifuged for 10 minutes at Yutys. rpm The content of fumaric acid by absorption at 240 nm and aspartic acid by the ninhydrin method are determined in a supernatant liquid using the conventional method. When Escher-ich a coBi cells were turned on in a polyacrylamide gel, 1.1 ml of a 3% solution of methylenebisacrylamide, 0.1 ml of O, 025% solution of riboflavin, 0, were added to 2.0 ml of a 50% acrylamnd solution. 1 ml of 0.1875% ammonium persulfate solution, 0.1 ml of 5% solution of N, N, N; M-tetramethylethyl endiaMina, 150 mg of aspartic acid dipotassium salt and 1.7 ml of Escherich co6i strain B cells containing 80.7 & washed from ballast proteins. mg of protein and having specific and total aspartic activity, respectively, equal to 108 units. 1 mg of protein for 1 hour and 9500 units. The mixture was stirred rapidly, placed in a glass with ice and water and illuminated with a 300-watt lamp, placed at a distance of 14 cm, for 5-10 minutes. A block of polyacrylamide gel with bacterial cells 16 weighing 6 g incorporated in it is ground in a mortar with 2 ;; to obtain gel particles ranging in size from 0.2 to 2.0 mm, Hx is washed from free cells of microorganisms, added successively four times 30 ml distilled water and separating the washings by decantation after settling the precipitate for 5 minutes. Wash waters are collected together to obtain 90 ml with a total protein content of 33.0 MFf. 41% of the amount paid. The polyacrylamide gel includes cells of Eschenichia coSi strain B in an amount corresponding to 47.87 mg of protein or 59%. The specific activity of the immobilized cells increases to 187 units; -a 1 mg of protein per 1 hour, the total activity is 950 units, which is 94.5% of the total activity introduced. A volume of 15 ml of polyacrylamide gel particles with included Eschernclua coEi strain B, after the last wash, was quantitatively transferred into 15 ml of 1 M (11.6%) ammonium fumarate solution prepared as above with 0.3 ml. toluene. The reaction mixture was kept at 3-7C with constant stirring and after 30 min for 5 h, 5 ml aliquots were taken. The reaction in an aliquot is stopped by filtration through paper filter; gel particles filter with bacteria cells turned on. In the filtrate, the content of fumaric and aspartic acids is determined by the method described above. In all cases, the resulting aspartic acid is identified by thin-layer chromatography on Silufol plates in chloroform-methanol-ammonia-water solvent systems in a ratio of 40: 40: 7: 3 and on plates. With a 0.3 M citrate buffer, pH 3.3. The standard is L-aspartic acid chromatographically pure firg Reanol (BHP). Three identical polyacrylamide gel samples are prepared with E.coBi strain 83 cells included in it, the only difference in which is the polymerization of one gel wire in the usual way when illuminated with light in the visible region with an intensity of 1,520,000 lux and when cooled in a glass water and ice; the second is illuminated in water at room temperature; in the third, polymerization is carried out without illumination and with cooling. When illuminated and cooled to 8c, cells E, 83 that are immobilized, have a specific and a total aspartase activity, respectively, equal to 187 units. 1 mg of protein for 1 h and 8950 units. (94.5% of that), when illuminated without cooling, respectively, equal to 21.6 units. per 1 mg of protein 7 in 1 hour to 1100 units, (16.55 from BViecsH), and without illumination - 5 units, pas 1 mg of protein. For 1 hour to 230 ate, (3, introduced) and full polymerization B case proceeds 60 mik. PRI mme R 2. AH 52 strain is grown, and suspension is prepared according to example. 1 ,. The protein content is 39f368 mg per 1 ml of cell suspension, the specific activity is 25 units, per 1 mg of protein per 1 hour. Immobilization of bacteria cells was carried out. As in Example 1, in two identical samples, each , 5 mg of protein with a total aspartase activity of 1685 units. The difference between the samples is that in one case 150 mg of dipotassium salt of 5 asparone ford acid is added to the mixture for photopolymerization of a polyacrylate gel, and E is not added with the other. The aspartase responsiveness of the bacterial cells incorporated into the polyacrylamide gel is determined as in the example. Bacterial cells immobilized in the presence of an aspartic acid salt have a specific and total activity of 74.0 units. . 1 mg of protein for 1 hour and 3530 units, i.e. 210% of the original; without salt of aspartic acid, respectively 2.58 units. per 1 mg of protein for 1 hour and 130 units, i.e. 8.7% of the initial total activity. Primer, the cells of the -EvCpEJ Kp strain are grown as in the example 40 ml of a suspension of non-washed cells with a protein content of 57.0 mg per 1. ml specific activity of 57 units, per 1 mg of protein for 1 h, divided into two equal doses, 20 ml of this suspension washed from the ballast proteins, as in. of example, 20 ml of cell suspension of bacteria are obtained with the designation native and the protein content is 29.7 mg in 1 ml. Another 20 ml of suspension of unwashed bacterial cells is first heated 40 min at and then oTivibi from ballast proteins, as in Perm 1, 20 ml of suspension: cells with the designation of activated with a protein content of 27.06 mg per ml. The inclusion of bacterial adhesives in polyacrylamide gel is carried out as in Example 1.. Then, aspartic activity, free and included, is determined; native polyacrylamide gel and activated E-coB cells Kp strain, as in Example 1. I get half free and immobilized NZ, robust E.coEi Kp cells with good aspartase activity, respectively, equal to 108 and 187 units. per 1 mg of protein for 1 h, as well as free and immobilized activated cells. coB4 Cr with specific ac. : 1o, respectively, units, per 1 mg of protein U p i.: Ep 4, K. puffs E.co2 | ta; .а / Кр growing; from, as in example 1, 10 ml of cell suspension are heated 10 vjii to 50 ° С and then washed with l ballast, as in example 1. to obtain 10 ml of cell suspension containing 35.2 mgm.; ate; -; and in 1 ml and having a specific akT; -: BNNO aspstase 27.8 units per 1 mg of protein for 1 hour, 5.1 MJI of this suspension with sergean1-5em 179 mg of protein with total aspartase activity 4999 units added to the ternary number of all components for photopolymerization of polyacrylamide gel, indicated in - Example 1. U1:; loB5t photo to l; -measurement and grinding of polyacrylamide gel, like s 1 M PBS 7.0 phosphate buffer at .50 MJ; and collect 150 ml of BCvi washes, which contain 69mg b.ele and 1940. units of total activity aspartB polyacrylamide gel include. j-: j.r.ei. Soy Kr, the amount of which: p; x corresponds to 110 mg of protein or GljO-J of the initial quantity. IncludedHi: ..; e in the gel cells have a specific aspaotazkush activity 119 -un. na) - ivii; 3c,: -: h for 1. h and total 13200 units. (263 o-g rlshshennoy), All obtained 1: ol-HschestEO particles of polyacrylamide rajrw with on-h: her and his and his cells; E, coEj Kp is transferred to column 1 ,. 8x7.5 cm to a volume of 19.1 cm. A 1 liter of 1 M solution of fumarate a -1 mani was passed through a thermostated column, prepared as in npravtepc 1, at a rate of 10 ml / h, which corresponds to an elucanum specific rate of 0.525. Collect 1 l. eluate, from: with the content of asparagine to the first; Concentrated sulfuric acid is added to 1 l of the eluate to pH 3.0, a heavy precipitated precipitate is removed by filtration, washed successively with two portions of cold water to 50 ml and 50 ml of 96% ethanol and dried to constant weight in air, yielding 110 g of aspartic acid, which in b g of HC8 has (a) | +25 ,. This indicates a 100% L-form content in the target product. No fumicroic acid, a feedstock, is detected in the target product. The resulting L-asparagine KiscjpTa gives one spot when chromatographed on plates.

Silufol and Fixix, as indicated in Example 1, and one peak on the Hitachi (Japan) amino acid analyzer, exactly corresponding to the standard L-aspartic acid. The target product is not contaminated with microbial cells. Elementary microanalysis contains,%: s 4.90% H, 36.48% C, 10.49% N, which corresponds to the theoretical content of these elements in aspartic acid (5.301% I; 36, .0-95% C; 10, 52% N),

The proposed method: obtaining Laspartic acid in comparison with the well-known technical solution of the same problem provides an increase in the efficiency of the process by reducing the activation time of microbial cells and increasing their activity before mobilization, as well as increasing the elution rate of the feedstock through the column.

Claims (1)

Invention Formula The method of obtaining L-aspartic acid by culturing the microorganisms of the species EschegiCh O (Co6i) cultured on it, separating the cell suspension from the culture fluid, washing, immobilizing the cells by incorporating them into a polyacrylamide gel, grinding the gel, activating the cells and washing them with ammonium fumarate ammonium gel, washing the gel, activating the cells and washing them with an ammonium fumarate, ammonia. By the fact that, in order to speed up the process and increase its efficiency, Eschepichia co6 83 or Escher strains (coBi ÀN 52, or EscherMchia coEj 85, while activating the glue) are used as microorganisms from the species Escher chia coEi. the current is carried out after separating them from the culture liquid by heating the cell suspension at 45-55 ° C for 4060 minutes, and immobilization is carried out in the presence of one of the salts of aspartic acid in an amount of 3-9 wt.%, followed by cooling the mixture to exposure to light of the visible area with an intensity of 15-20 thousand lux for 5-10 minutes