CS258259B1 - A method for producing L-aspartic acid using heterogeneous biocatalysts - Google Patents

Abstract

The solution relates to a method for producing L-aspartic acid using heterogeneous biocatalysts based on microbial cells with aspartase activity. The method consists in contacting an aqueous solution of ammonium fumarate with biocatalysts, which are prepared in advance by the action of octylphenol polyethylene glycol ether or nonylphenol polyethylene oxide or directly with a solution of ammonium salt of fumaric or aspartic acid with the addition of ammonia on microbial cells containing the enzyme L-aspartate ammonia lyase, embedded in a gene-carrageenan gel, then after the conversion is completed, L-aspartic acid is isolated from the separated liquid portion. This amino acid can be used in the production of medicines and in the production of artificial sweeteners.

Description

The invention relates to a process for the production of L-aspartic acid from ammonium fumarate by means of microbial cells with aspartase activity into gene-carrageenan.

L-aspartic acid is increasingly used in the pharmaceutical and food industries and is an important component of the aspartame sweet dipeptide and an important component of some drugs. For these reasons, there is an increasing interest in the economical mass production of this important amino acid. Since only the L-stereoisomer of this acid is applicable for a number of applications, it is advisable to produce the biologically active L-stereoisomer of the acid, for economic reasons, even when both antipodes can be applied concomitantly using only the L-form. aspartic species primarily via a biological pathway allowing selective production of the desired enantiomer.

The transformation of fumaric acid to L-aspartic acid can be accomplished using cells of microorganisms of various strains of the genera Alcaligenes, Corynebacterium and Escheriohia, which are characterized by aspartase activity. A single submerged culture of the cultured cells is either suspended with the culture medium or after separation from the culture medium in a substrate solution, i.e., ammonium fumarate, which is converted to the ammonium salt of L-aspartic acid at appropriate pH and temperature with stirring. After cell separation and subsequent acidification, L-aspartic acid is isolated.

In this process, new cells must be grown for each production batch in large-scale fermentation tanks, which is economically disadvantageous in terms of raw material and energy costs, including the need for a large investment-intensive plant.

For this reason, a number of procedures have been developed / shoemaker F. et al., Chem. Leaves 74, (1980), which allow multiple or continuous use of the aspartase enzyme (L-aspartate: ammoniaclyase E.Cj 4.3.1.1) either bound to a suitable carrier after isolation from the relevant microorganism or directly in the form of immobilized cells with aspartase activity . The use of microbial cells in the immobilized form has a number of advantages over the immobilized enzymes, primarily of an economic nature, since the expensive isolation of the enzyme is often eliminated, which results in a lower cost of the prepared biocatalyst. Biocatalysts prepared by cell immobilization usually have also higher operational stability / Marek M. and Káš J .: Chem. Industry 34, 546 (1984) //.

Two principally different procedures have been developed for the immobilization of cells with aspartase activity - the method of incorporation of cells into different types of polymer matrices and the method of covalent cross-linking of the intracellular contents of individual microbial cells or chemically bound cells using bifunctional cross-linking agents, in particular glutaraldehyde. Certificate No. 209 265 /.

The basis of industrial continuous production of L-aspartic acid became in the first half 7). elaborated procedure of cell immobilization into polyacrylamide / Chibata I. et al .: Appl. Microbiol. 27, 878 (1974). Radical polymerization of acrylamide with Ν, Ν'-methylene-bis-acrylamide / US Pat. No. 3,791,926, a cross-linked synthetic gel with embedded cells having an operating half-life of up to 120 days is prepared. However, this process also has a number of disadvantages.

It is not yet known to carry out a polymerization process according to which it is possible to prepare gel particles of spherical shape and at least approximately the same size, which would guarantee optimal hydrodynamic properties of the biocatalyst. However, the greatest drawback of this process is the fact that even short-term contact of the cell material with momoners acrylamide with Ν, Ν'-methylene-bis-acrylamide reduces aspartase activity. In addition, polymerization releases heat, which also reduces the enzymatic activity of immobilized cells.

Another disadvantage of this process is the danger that the substances produced using the biocatalyst prepared in this way may contain toxic residues of unpolymerized monomers, which is unacceptable from a hygienic and medical point of view.

For this reason, a new process for the production of L-aspartic acids by a biocatalyst prepared by incorporating cells with aspartase activity into kappa-carrageenan was developed and technologically implemented in the late 1970s (Sato T. et al .: Biochim. Biophys. Acta 570, 179/1979 // Kappa-carrageenan is a polysaccharide which is obtained by hot water extraction of seaweed Chondrus crispus and subsequent separation from lamba-carrageenan by fractional precipitation with potassium ions (Rees DA: J. Chem. Soc. 1963, 1821).

Using potassium, ammonium, calcium and other ions / Tosa T. et al., Biotechnol. Bioeng. 21, 1697 (1979), it is possible to form gel-like particles of different physical properties and shapes from kappa-carrageenan, which have excellent hydrodynamic properties in addition to sufficient strength. By incorporating cells with aspartase activity into the kappa-carrageenan gel, a biocatalyst is then prepared which, in addition to a high proportion of retained enzyme activity, also has a very high stability. In literature / Nishida Y. et al.: Enzyme Microb. Technol. 6, 85 (1984)], an operating half-life of up to 680 days is reported.

Gene-carrageenan can also be used to prepare a biocatalyst with analogous properties, i.e. a crude carrageenan preparation isolated from algae of the genera Gigartinaceae and Solieriaceae (Genu-carrageenan: A / S Kobenhavns Pektinfabrik, Denmark). Unlike isolated pure kappa-carrageenan, it is a technical mixed carrageenan preparation commonly used in the food industry, which is approximately 40 times cheaper kappa-carrageenan. By incorporating cells with aspartase activity into the gene of carrageenan, highly stable biocatalysts with defined particle size of suitable physical and hydrodynamic properties are prepared.

Based on the above-mentioned findings regarding the properties of gene-carrageenan, a process for the production of L-aspartic acid using heterogeneous biocatalysts according to the invention has been developed, characterized in that an aqueous solution of ammonium fumarate having a concentration of 0.75 to 22.5 wt. % with a pH of 6.5 to 9.5, with the addition of magnesium ions, preferably at a concentration of 0.002% by weight. is contacted at a temperature of 10 to 50 ° C with a heterogeneous biocatalyst which is prepared; microbial cells containing the enzyme L-aspartate: ammonia lyase, incorporated into the gel-carrageenan gel, are treated with octylphenolpolyethylene glycol ether or nonylphenolpolyethylene oxide or directly with ammonium fumaric or aspartic acid ammonium salts with ammonia addition, after which the acid is recovered asparagová.

Various microbial species and strains or high-production mutants of various aspartase synthesizing microorganisms, for example various strains of the genera Escherichia, Alcaligenes and Corynebacterium, preferably the microbial strain Escherichia alcalescens CCEB Asp 24, cultivated according to CCEB Asp 24, can be used for the process of the present invention. . 220 460.

The main advantages of the process for producing L-aspartic acid according to the invention are associated with the ease of preparation of the biocatalyst, in terms of cost-effectiveness for the cell immobilization of the gene-carrageenan used, the high degree of cell aspartase activity during biocatalyst preparation, and last but not least. The high operational stability of the biocatalyst is achieved especially when the cells are immobilized into gene-carrageenan in the presence of compounds containing a high proportion of hydroxyl groups such as tannin, possibly also after subsequent solidification of the gel particles with 1,6-diaminohexane and glutaraldehyde. The half-life of the biocatalysts thus prepared is at least 180 days.

The process for the production of L-aspartic acid according to the invention is particularly advantageous for the constitutional arrangement, since the biocatalyst used has excellent sedimentation and hydrodynamic properties for use in fixed or fluidized bed columns. From the filtrate or the liquid portion of the reaction mixture separated from the biocatalyst, L-aspartic acid is obtained by adjusting the pH with a mineral acid to 2.8 and subsequent crystallization at a temperature of 10 to 20 ° C.

The crystallized L-aspartic acid is filtered off, washed with cold water and dried, if necessary further.

The L-aspartic acid prepared by the process of the invention is very pure, useful in particular for the preparation of various salts used as medicaments, for example the cardiotonic agent Cardilan (Spofa), i.e. the magnesium-potassium salt of L-aspartic acid.

In the production of L-aspartic acid according to the invention, the conversion efficiency is 100% while providing optimum conditions, i.e. the amount of biocatalyst, initial substrate concentration and flow rate. The yield of isolated pure L-aspartic acid is about 94 to 96% by weight. theoretical profit.

In the following, the invention is illustrated in more detail by the examples without limiting them.

Example 1

The production strain of Escherichia alcalescens Asp 24 was inoculated from a slanted agar into a 250 ml Erlenmeyer flask containing 70 ml of sterile nutrient medium of the following composition:

% wt. yeast autolysate, 0.5 wt. peptone, 0.9 wt. fumaric acid and 0.75% by volume, beef broth 5 x concentrated, pH 7.0. The vegetative inoculum was cultivated on a rotary shaker for 16 hours at 37 ° C. The obtained inoculum was used to inoculate own production flasks.

The obtained inoculum was inoculated with 10 750 ml round-bottom flasks containing 100 ml of nutrient medium of the above composition. 1 ml inoculum per 100 ml nutrient medium was used. Cultivation was carried out on a rotary shaker for 16 hours at 37 ° C.

At the end of the culture, the cell suspensions were decanted, the enzyme activity was determined to be 31 µg / g wet cells, the cells were centrifuged and the sediment obtained was used to prepare a heterogeneous biocatalyst.

The wet cell paste was mixed in physiological saline to form a 20 wt% slurry. of wet cells. The slurry was heated to 50 ° C and then an equal amount of a 5% by weight solution of 55 ° C gene-carrageenan was added. The mixture was thoroughly mixed, cooled to 10 ° C and overlaid with 2.24 wt. potassium chloride solution at 10 ° C. After 24 hours, the resulting heterogeneous biocatalyst was mixed in a mixer, the biocatalyst suspension was centrifuged and the sediment was used for batchwise preparation of L-aspartic acid.

A heterogeneous biocatalyst having an activity of 1.7 µg / g wet gel in an amount of 50 g wet weight was suspended in 1 liter of 14.9 wt%. ammonium fumarate solution, pH 8.5, containing 0.08% by volume of nonylphenolpolyethylene oxide. The suspension was stirred at 37 ° C.

100% conversion to ammonium aspartate occurred in 4 hours. After this time, the reaction mixture was centrifuged, the biocatalyst sediment was resuspended in 1 L of 14.9 wt. of ammonium fumarate solution and the procedure was repeated.

The conversions were repeated a total of 9 times without noticeable loss of enzyme activity of the heterogeneous biocatalyst. L-aspartic acid was isolated from the supernatants by adjusting the pH to 2.8 with 70 wt% sulfuric acid. and cooling to 10 ° C. The total yield was 1120 g of L-aspartic acid, i.e. 94.2% by weight. theoretically recoverable quantity

Example 2

The cells of the strain Escherichia alcalescens Asp 24 were prepared in the same manner as in Example 1. The cell paste obtained was mixed in physiological saline to make 40% by weight. suspension of wet cells. The slurry was heated to 45 ° C and then a 3-fold amount of a 4% by weight 50% C-carrageenan solution was added.

The mixture was thoroughly mixed and dripped into a cold 2.24% potassium chloride solution while warm. coated with a layer of paraffin oil. The formed heterogeneous biocatalyst beads were left for 24 hours in 2.24% potassium chloride solution. and a temperature of 10 ° C. Their surface was reinforced by the following procedure.

Beads of a heterogeneous biocatalyst having a total weight of 100 g and a diameter of about 3.5 mm were cooled to 5 ° C and overlaid with 113 ml of a solution containing 0.5 M phosphate buffer pH 7.0, 2.24% potassium chloride. and 1,6-diaminohexane at a concentration of 0.99% by weight.

The solution had a temperature of 5 ° C. The mixture was stirred gently for 5 minutes and then 48.6 ml of 0.5 M phosphate buffer, pH 7.0, 2.24 4 wt. and glutaraldehyde at a concentration of 0.95 wt. Stir again at 5 ° C for 30 minutes. The red colored beads of the biocatalyst were washed several times with a sufficient amount of 10 ° C potassium chloride solution at a concentration of 2.24% by weight. A portion of the beads were used to determine enzyme activity and 90 g of the biocatalyst was used to semi-continuously convert ammonium fumarate to L-aspartan ammonium.

Beads of a heterogeneous biocatalyst having an activity of 0.85 µg / g wet biocatalyst were overlaid with 1 L of 14.9 4 wt.% Ammonium fumarate, pH 8.5, containing 0.2 4 vol. Octylphenolpolyethylene glycol ether. The mixture was gently stirred at 37 ° C. 1004 conversion to L-aspartan ammonium occurred in 9.5 hours. The ammonium aspartan solution was carefully aspirated and the conversion was repeated 15 times as described above. The loss of activity of the biocatalyst after completion of all conversions did not exceed 2.5 4 of the original activity.

1,880 g of L-aspartic acid were isolated from the combined aspartan ammonium aspirate solutions as described in Example 1, which represents 95% of the theoretical yield.

Example 3

The cells of the strain Escherichia alcalescens Asp 24 were obtained as described in Example 1. The cell sediment was mixed in physiological saline to give a suspension of 42.5% by weight. of wet cells. The mixture was heated to 45 ° C and then a 3-fold amount of 50 ° C warm 4-wt.

After thorough mixing, a solution of tannin in saline at a concentration of 14% by weight was added to the mixture. so that the final concentration of tannin in the gel was 0.1 4 wt. The whole was again mixed thoroughly and poured warm onto the dish so that the gel layer was at most 5 to 6 mm high.

The solidified gel was overlaid with 2.24% potassium chloride solution. and left at 10 ° C for 24 hours. The cured gel was cut into 5 x 5 x 5 mm particles and then used for continuous column preparation of L-aspartic acid.

The cubes of the heterogeneous biocatalyst with a total weight of 95 g and the activity of 0.75 _< ukat / g wet biocatalyst were packed into a 2.6 x 25 cm thermostated column at 37 ° C and the gel was activated for 48 hours at a flow rate of 140 ml / h a solution of ammonium fumarate having a concentration of 14.9 4 wt. and at pH 8.5. After activation, the ammonium fumarate flow rate was reduced to 70 ml / h. The conversion of ammonium fumarate to L-aspartan ammonium took place at 37 ° C for 40 days without any detectable change in the enzymatic activity of the biocatalyst used. A total of 67.2 L of ammonium fumarate solution was converted over the period. Isolation according to Example 1 gave 8.35 kg of L-aspartic acid, which is 94.14% of theory.

Example 4

The cells of the strain Escherichia alcalescens Asp 24 were obtained as described in Example 1. The cell sediment was mixed in physiological saline to give a suspension of 42.5% by weight. of wet cells. The mixture was heated to 45 [deg.] C. and then three times 50 [deg.] C. of a warm 4% carrageenan gene solution were added. After thorough mixing, a 1% w / w solution of tannin in saline was added. such that the final concentration of tannin in the gel was 0.1 wt. The mixture was thoroughly mixed and dripped into a cold 2.24 4 wt. and a temperature of 10 ° C.

Beads of 1.35 ukat heterogeneous biocatalyst per 1 g wet biocatalyst were packed into the column and activated at 45 ° C for 48 h with a 22 wt% ammonium fumarate solution. and pH 9.5. After activation was complete, the ammonium fumarate was converted to L-aspartan ammonium under the same conditions at a flow rate of 100 ml / h. No change in the enzyme activity of the biocatalyst used was observed after 60 days. After isolation, a total of 17.1 kg of L-aspartic acid was obtained, which is 95% of the theoretical yield.

Example 5

To the beads of the heterogeneous biocatalyst prepared according to Example 4, a 1% w / w solution of ammonium fumarate, pH 6.5, containing 0.2% by volume octylphenolpolyethylene glycol ether was added. The mixture was gently stirred at 15 ° C. 100% conversion to L-aspartan ammonium occurred in 2 h. The ammonium aspartan solution was gently aspirated and the conversion was repeated 20 times as described above. There was no loss of original enzyme activity in the biocatalyst.

Claims (1)

Process for the production of L-aspartic acid by means of heterogeneous biocatalysts by means of microbial cells with aspartase activity, characterized in that an aqueous solution of ammonium fumarate at a concentration of 0.75 to 22.5% by weight, with a pH value of 6.5 to 9.5 and an additive of magnesium ions at a temperature of 10 to 50 ° C in contact with a heterogeneous biocatalyst which is previously prepared by treatment with octylphenolpolyethylene glycol ether or nonylphenolpolyethylene oxide or directly with ammonium salt of fumaric or aspartic acid with the addition of ammonia to microbial cells containing L-aspartate of the gene carrageenan, after which L-aspartic acid is recovered from the separated liquid fraction after the conversion.