JPS5867184A - Production of aspartase - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing aspartase, and in particular to a method for obtaining a cell-free aqueous solution of aspartase which is used for converting ammonium heptamalate to aspartic acid.

L-aspartic acid is an important amino acid used as a drug or as a raw material for certain chemical products such as L-alanine and asunolterme. This amino acid is also used as an additive to animal feed and human food. Aspartic acid (amino acids have been produced by chemical means, which generally yield Dffi and L-form racemic, s-compound bt. Only L-form aspartic acid can be bioassimilated. Therefore, Asuno As a more desirable method for producing amino acids,
Examples include a method of culturing microorganisms that produce diginate. (In this specification, the word culture is used in a broad sense,
ure, 1ncubat1on, 1noculat
1on, fermenta-tiOn, etc. ) For example, U.S. patents 8 and 2 for Chinoku Yu et al.
No. 14,845 discloses a biological process for the production of aspartic acid using fumaric acid (as the ammonium salt) as the primary carbon source. This method includes a batch type production method in which microorganisms are inoculated onto a culture medium and after culturing for a certain period of time, aspartic acid is recovered from the culture medium.

Processes for the production of aspartic acid using immobilized cell reactants have also been reported; for example, U.S. Pat. No. 8,791,926 to Chipa et al. A method for making immobilized cell reactants by bundling is disclosed. Aspartic acid is obtained by contacting the immobilized cells with ammonia fumarate. The eccentricity described above may be of batch history or continuous type. In the case of a continuous reaction, the immobilized cells are packed into a cylinder through which a solution of ammonium fumarate is passed continuously.

Immobilized cell reactants have several advantages over patch culture methods. Batch-type processes require complex techniques for product recovery, which reduces the overall output. In addition, in many cases, the resulting products are contaminated with cell metabolites contained in the culture medium. Although the use of immobilized cells eliminates many of these problems.

Therefore, it is unavoidable that the final product contains a considerable amount of cellular impurities.

Means to solve the above-mentioned recovery and contamination problems associated with the production method using patches and immobilized cells include separation methods, purification methods, and the use of enzymes that promote the desired reaction. Among these enzymes, aspartase is suitable for converting ammonium 7-malate into aspartic acid. Various attempts have been made to obtain aspartic acid directly from ammonia fumarate by immobilizing aspartase in a cylinder or on a solid support. For example, U.S. Patent Tsuru 791
．． 921F) The preceding description refers to methods of binding aspartase t-m with ion-exchangeable polysaccharides or trapping aspartase during acrylamine polymerization. These methods have been limited in their success by the difficulty of extracting and purifying the aspartase enzyme and by foregoing much of the enzymatic action during the purification, concentration, and immobilization steps.

Dialysis type reactors have also been proposed for the purpose of immobilizing the above enzymes. Dialysis here is a broad term that includes true dialysis (exchanging solute molecules between two liquids separated by a diaphragm), osmosis (conversion of solvent through a diaphragm) and ultrafiltration (pressure exchange through a diaphragm). transfer of solvent and solute from MK). In the dialysis-type reactor described above, the enzyme solution is retained predominantly on one side of the semi-permeable diaphragm.

A substrate for an enzymatic reaction is brought into contact with the other side of the semipermeable membrane. The semipermeable membrane allows the substrate and product to pass through, but not the enzyme. The substrate is thus transferred to the enzyme side of the semipermeable membrane and converted into product, which is transferred back into the substrate solution and recovered from this solution.

Dialysis-type reactors are made in a variety of configurations, such as those for sluices generally having the shape of a filter press, which consists of a number of parallel diaphragm plates separated by a plurality of membranes that allow the liquid to pass through. It has become.

1 Biotechnology and Bioengineering Link 1
In Volume 20, pp. 217-280 (197g), Kan.
G.A. and Mieller, M.L. describe the use of a hollow fiber renal dialysis device in the above-described dialysis-type reactor. In use of such a device, all of the microbial cells are held immobile Hvc on the outer shell side of the renal dialysis device, and the substrate solution is circulated through the hollow fiber tubing side of the device. The substrate passes through these fibers and reaches the cells, where the reaction takes place. The produced products are recycled again. Some of the above methods immobilize a type of fluorescent bacillus (Pseudomonas fluorescens) by converting L-histiridine into urocanic acid.

Previous attempts to use immobilized aspartase enzymes in dialysis-type reactors to obtain aspartic acid have been generally unsuccessful (see U.S. Pat. No. 8,791,920, cited above).
reference). The reason for the above failure is thought to be that the conventional methods used for separating and purifying aspartase largely result in a loss of enzymatic activity.

The present invention discloses a method for obtaining a cell-free aqueous solution of aspartase, which method comprises forming an aqueous suspension of microbial cells containing aspartase, and applying the aqueous suspension under aspartase-releasing conditions. Incubate for a sufficient period of time to neutralize most of the aspartase in the lipids to the extracellular fluid, remove solids from the cell suspension to obtain a clear aspartase-containing solution, The suspension of cells or the clear aspartase-containing solution comprises the steps of adding a sufficient amount of neutralizing aspartase stabilizing salts to bring the solution to a high osmotic pressure with respect to the microbial cells.

In a preferred embodiment of the invention, the aqueous suspension of cells is formed using a high permeability, tonic solution of the aspartase stabilizing salt.

The method according to the invention is simple and effective, and typically more than 80-80% of the cellular aspartase activity is recovered in the cell-free aspartase solution. Furthermore, it has been found that this enzyme maintains its activity for a considerable period of time in the above-mentioned solution, so that this solution can be used effectively in the dialysis-type reactor mentioned above.

Aspartase enzymes are produced by a very wide variety of microorganisms, and the invention is therefore not limited by its particular species, genus, or class. For example, the fluorescent bacillus (Pseudomonas fluorescens).

Pseudomonas aeroginosa, Pasillus saptios, Pasillus magatherium, Proteus vulgaris, Serratia marsetuscens, Escherisha coli, and Aerobacterium aerogenes have been described as being widespread in the production of aspartic acid. The specific microorganism from which aspartase was recovered was the above-mentioned Escherichia coli seed obtained using glutamate (ATCC No.
.

81976).

7 As a source of Suha k l -senobiotic [1], the cells for aspartase production are cultured in a culture medium suitable for the production of this [C and rapid growth of the cells. Methods for culturing the cells described above are well known and do not form part of the present invention.

Generally speaking, a culture medium is used which contains the necessary minerals and growth agents and is an assimilable source of carbon and nitrogen.

In particular, this culture medium contains neutralized fumaric acid and also contains toshite yeast extract, a source of nitrogen, vitamins, and growth agents, ammonia ions as an additional nitrogen source, and calcium and magnesium salts as necessary minerals. It's convenient. Potassium and sodium phosphates or carbonates are also included as buffers.

The inoculated culture medium is approximately 2 ml per liter under normal growth conditions.
It is incubated for a sufficient period of time to produce about 6 grams of solids from grams. Normally, culturing at 87°C for about 12 to 14 hours provides the required cell density.

After a suitable growth period, the cells are separated from the non-cellular portion of the culture medium (e.g. by centrifugation or filtering);
Washed. Aspartase is removed from the cells by suspending the cells in an aqueous solution and further culturing under aspartase releasing conditions. As the aqueous suspension, water or other aqueous solutions (eg, common buffers) that are not harmful to primary enzymes can be used. Preferred aqueous suspensions include high osmotic solutions (or hypertonic solutions) of aspartase stabilizing salts (described below); particularly preferred suspension solutions include high osmotic solutions (or hypertonic solutions) of aspartase stabilizing salts (described below); Pressure solutions are used. Ammonium fumarate with concentrations from about 10 molar up to saturation, particularly its 14 molar concentration, is commonly used as the suspension liquid.

The cells are suspended in the suspension at a concentration of at least about 5 grams per liter (dry cell weight), more preferably from about 10 grams to about 15 grams per liter.

The cells suspended in this manner are cultured under aspartase release conditions of F for a time sufficient to release most of the aspartase in the cells into the fumarate solution.The culture temperature is approximately 2°C. to about 70°C, more preferably about 25°C.
℃ to about 60℃ is used. A particularly desirable culture temperature is about 87°C. When the culture temperature is low, the time rate of enzyme release decreases and the action of the enzyme also decreases. On the other hand, if the culture temperature is too high, it will have a detrimental effect on the enzyme.

The culture time depends on the temperature of the culture, but it is usually about 1 hour.
0 minutes, preferably at least about 1 hour, more preferably about 1 hour
2 to 60 hours are used.

After incubation, solids are removed from the cell suspension, for example by centrifugation or filtration, resulting in a clear aspartase-containing liquid. Any separation or filtration means can be used as long as it does not impair aspartase activity. The solution obtained by such 5&C usually retains about 80% of the aspartase activity originally contained in the cells. This fluid is almost free from unwanted contamination and can maintain aspartase activity for more than 40 days at 87°C.

A sufficient amount of aspartase stabilizing salt is added to the clear solution to make it a hyperosmolar solution (or hypertonic solution). In a preferred embodiment of the invention, the aspartase stabilizing salt is used as an aqueous cell suspension, in which case addition of aspartase stabilizing salt to the clear solution is usually unnecessary.

The above-mentioned aspartase stabilizing salt maintains enzyme activity for a certain period of time and prevents its deterioration due to heat. For example 6
A comparison of the aspartase activity of solutions at 6°C showed that the enzyme extracted with buffer lost almost all of its activity after 80 minutes, while the enzyme extracted in 14 mol of ammonium heptalate lost its activity. at least 80
It retained 1G even after 2 hours of storage.

Preferred aspartase stabilizing salts are soluble salts of 7-rk acids or aspartic acid. Fumaric acid is the enzyme substrate for the production of aspartic acid (thus the use of salts of these acids obviates the need for this salt from aspartase after K).

Additionally, these salts, especially fumaric acid emonylimes, have been found to be effective in the extraction and stabilization of enzymes. Although malate and aspartate are preferred as mentioned above, any water-soluble salt that stabilizes the enzyme can also be used.

Such salt! ! 4 Usually denoted by MX, where M particularly represents a cation such as an alkali or alkaline earth metal, ammonium, lower alkylammonium, lower alkyl tetraammonium, etc., and X represents an anion such as a halogen ion, nitric acid, carbonic acid, phosphoric acid, Indicates ions such as sulfuric acid, maleic acid, 7-malic acid, and aspartic acid. The concentration of aspartase stabilizing salt in the clear solution ranges from about 0.5 molar to saturation, preferably from about 10 molar to about 14 molar TlC.
11 will be found out.

The aspartase solution obtained by this method can be used in a dialysis reactor, especially a hollow fiber reactor fllK. The cell-free solution is neutralized and introduced into the outer shell of the reactor during the day by simply applying suction to the tube side of the reactor. After the enzyme has been neutralized into the reactor, the ammonium heptamalate solution is circulated through the tube side of the device. The ammonium 7 malate substrate and the resulting aspartic acid pass freely through the dialysis tube wall and the asparagium/# is recovered from the circulating solution. On the other hand, the aspartase enzyme cannot pass through the tube wall and is neutralized and retained in the reactor. Removal of dialyzable impurities and color products takes place immediately after the start of the reaction, thus preventing contamination of the product.

The reaction conditions of the reactor used will vary depending on the IIK of the apparatus, but typically the substrate will be, for example, a concentrated solution of ammonium heptamalate, from about 1 molar to saturated and preferably about 18 molar. The reactants are incubated under aspartic acid producing conditions.

The reactor is maintained at a temperature at which effective salification of the product from the primary substrate occurs. Temperatures of about 2°C to about 70°C, preferably about 26°C to 60°C, more preferably about 87°C are commonly used. The above enzymatic reaction is an exothermic reaction.

Therefore, in large reactors where low flow rate substrates are used, a means to remove excess heat is required.

The flow rate of the substrate solution in the reactor depends on the reaction used,
It varies depending on the configuration of the substrate Noga warm reaction system (for example, llffi type or single-pass type), the level of enzyme addition, and the desired degree of heat release.

The hollow fiber reactor described above can also be operated in a recirculating batch mode in which the solution stream discharged from the device is returned to the reservoir. In this case, recycling of the substrate solution is continued until most of the ammonium heptadalate has been converted to aspartic acid. Another form of reactor operation is a single-flow type in which multiple reactors are operated in series.

The present invention provides a simple and effective method for recovering aspartase from cells that produce it. The cell-free aspartase baths suspended by this method can be used in batch or immobilized mode and are particularly suitable for use in hollow dialysis type reactors.

The cell-free aspartase solution obtained by the method of the invention has all the following advantages when used in a dialysis-type reactor compared to one containing intact cells. In order to obtain high levels of enzymatic action in those containing intact cells, the K solution must be concentrated. Concentrated cell-containing solutions are highly viscous, creating at least two problems that are not present in cell-free solutions according to the present invention. One problem is the migration of product solution neutralization impurities and colored substances from the cell slurry through the dialysis membrane.

Impurities and colored substances are leached from intact cells over a long period of time, rather than for a short period of time at the beginning of the reaction as in the cell-free case. Considered as an enzyme, the reactants and products must therefore be diffused not only through the membrane of the reactor, but also through the extracellular fluid and cellular constituents.The reaction 7 degrees in this case is therefore lower, e.g. Even when using enzymatic activity, cell-free hollow fiber reactors exhibit reaction rates approximately 45 times greater than those containing intact cells.

The present invention will be further explained in detail by the following examples, but is not limited thereto.

Example 1 Nutrient materials containing yeast extracts of various types were cultured for the purpose of obtaining aspartase. Each of these substances is 1
Contains 24-60 grams of yeast extract per liter, 80 grams of fumaric acid, 2 grams of potassium phosphate, 06 grams of sodium carbonate, 2 mmol of magnesium sulfate, Qlξ mol of calcium chloride, whose hydrogen ion concentration is determined using ammonium hydroxide. It was adjusted to about 'Z2. All materials are provided by Escherishakori (ATCC No. 81976)
) was incubated with 1 ml. The above Escherichia coli was cultured at 87° C. for 12 to 16 hours in peptone containing monosodium α6 tiglutamate. The above culture of each sample was carried out at 87°C for 12-14 hours, and then the cells were harvested. The dry weight of the cells obtained varied from about 2 to 7 grams per liter depending on the amount of yeast extract used. The aspartase production rate according to each material is as shown in Table 1.

Additionally, culture materials containing various concentrations of ammonium fumarate were prepared and cultured as described above.

Each of these materials contained 8 portions of the yeast extract and the same amounts of each of the ingredients listed above. The results of these cultures are shown in Table ①. The cell culture obtained from the culture material containing yeast extract (L4W/V) and ammonium fumarate (aO) was centrifuged and the liquid portion was decanted and discarded. The cells were then washed with water and centrifuged again.
decanted and further p14as α05 Tris-HCI
, and resuspended to 1/2 of the original volume in buffer. The resulting slurry was diluted with a L8 molar solution of ammonium fumarate (pH & 5) to give a cell suspension with a volume of V6 from the initial culture.

This suspension was divided into 6 portions, incubated at 87° C. for the periods indicated in Table 3, and then centrifuged. The liquid portion was decanted and the solids were discarded. The liquid portion was then tested for aspartase activity and the results are shown in Table ■
is shown.

Example 2 Method of Example 1 (yeast extract 24-, ammonium fumarate 8%) from K to IL 5 grams of cells (dry weight)
The resulting cell-free solution was introduced into the outer shell of a CDAK 2.5 D engineered kidney (CDAK 2.5 D) by applying suction to its tube side. After introducing the enzyme into the reactor, the shell is sealed and an 18 molar solution of ammonium 7 malate at pH &5 is added every hour to 11-1
A flow rate of 5 liters was passed through the tube side of the reactor. A recirculation system was employed in which the substrate solution was circulated back into the reactor from the reservoir. The reactor is 87
maintained at ℃. The fumarate remaining in the contents of the reservoir was measured periodically and the results are shown in Table 3. Aspartic acid was adjusted to pH'2.
8, filtered, washed and dried. Fumaric acid average z1 to 2 to aspartic acid 2
．． 0-z I KF was recovered (3m theoretical yield was 8
81G to 86H), the despartic acid thus produced was confirmed by elemental analysis for C, H, N, and OK, and the results were consistent with the 97-9911 theory. Raw +26 (C-1O in 2N HCl). This product was tested by the coupled enzyme test method and matched 9691 to an authentic aspartic acid standard.

Table I Culture medium volume versus aspartase activity. Culture material (
Effect of yeast extract concentration in ammonium fumarate (8-t-containing).

Yeast extract Aspartase LOQ22 L8 (L64 27 α90 10 L20 40 15G KG L76 [02,14 ?0 2.26 &O5L24 Table ■ Volume of culture medium vs. aspartase activity. Culture material (
Influence of ammonium heptalate concentration in yeast extract (containing 8g).

Fumaric acid aspartase Q LOO! 0 2.50 40 2.52 ao 048 Table ■ Extraction of aspartase from a cell population containing 4 mol of ammonium fumarate.

O- 470 778 1479 2480 4884 Table ■ Hollow fiber reactor Wt (CDAK 2.5 D) reacts with 184 L of ammonium heptalate using cell-free enzyme (extracted from approximately 5 grams of IL in the dry state). It was operated in recirculating patch mode with a 10 liter agitated reservoir containing 10 liters.

aOL81 α25 16g α6 L62 LOL8 0 L5 L12! O(198 26α82 aOα70 a6 058 Table Oa4g 10 082 to C21 ?Oα14 Continuation of 1st page oInt, C1,3 identification symbol Internal reference number (C
12N 9/88 C12R1/425) (C12N 9/88 C12R17185) (CI2 N 9/88 C12R1101) 0 Inventor Lois Jean Moser 1310 Apple Tree Court, Frederick, Maryland, USA 21701

Claims (1)

[Claims] 1. An aqueous cell suspension of microbial cells containing aspartase is prepared, and the aqueous cell suspension is subjected to aspartase-releasing conditions so that most of the intracellular aspartase is transferred to the extracellular cell suspension. Incubate for sufficient time to release into liquid. Remove the solids from the cell suspension to obtain a clear aspartase-containing solution. adding an aspartase stabilizing salt to the cell suspension or the clear aspartase-containing solution to render the clear aspartase-containing solution hypertonic with respect to the microbial cells; A method for producing an aqueous solution of aspartase free of wart cells. 2. The method according to claim 1, wherein said aqueous cell suspension is obtained by neutralizing and suspending a vacuoles of microorganisms in a hypertonic solution of said aspartase stabilizing salt. 8. The method according to claim 1 or 2, wherein the aspartase stabilizing salt comprises a water-soluble salt of 7-malic acid or aspartic acid. 4. The method according to claim 1 or 2, wherein the aspartase stabilizing salt comprises ammonium heptase malate or ammonium aspartate. 5. The method according to claim 2, wherein the aspartase stabilizing salt comprises ammonium fumarate. 6. The method of claim 6, wherein the concentration of ammonium fumarate in the hypertonic solution of ammonium fumarate is within a range from about α6 molar to a saturation concentration. 7. The method of claim 5, wherein the concentration of ammonium fumarate in said hypertonic solution of ammonium fumarate is within the range of about LO to about 14 molar. 8. The above aspartase release conditions are from about 2°C to about 70°C.
up to an incubation temperature of at least about 1
7. The method according to claim 6, wherein the method is incubated for 0 minutes. 9. The above aspartase release conditions range from about 25°C to about 60°C.
including culture temperatures up to about 12°C to about 6°C.
7. The method according to claim 6, wherein the method is cultured for 0 hours. 10. The method according to claim 9, wherein the culture temperature is about 87°C. ■ Bacterial cells are suspended in a sufficiently hypertonic solution of ammonium 7 malate in VCl2 to obtain a cell temperature of at least about 6 grams per liter of dry cell weight, and the aspartase release conditions described above are further maintained. Qp from 7 to about 9
10. The method of claim 9 comprising H@l. lλ Cells of the above bacteria are suspended in a sufficiently hypertonic solution of ammonium fumarate to form a warm solution containing from about 10 g2 to about 15 g cells per liter, by dry cell weight. 12. The method of claim 11, wherein the pH value is within the range of about 8 to about 9. 13. The above bacterial cells are Pseudomonas fluorescens, Pseudomonas aeroginosa, Bacillus subtilis, Noccilus magatherium, Proteus no (Rugaris, Upper 2cha marsetuscens, Escherichia col), and Asnocultase-producing varieties of Aerobacterium aerogenes A method as claimed in claim 1.5.6, 7.9 or 12. 14. The method according to claim 13, wherein the bacterial cell molecule is Nische IJ Shakori. 15. The above bacterial cells are of variety ATCC81976.
15. The method according to claim 14, which is Escherichia coli. 16. An aqueous solution of aspartase obtained by the method according to claim 1, which does not contain human cells. 17. An aqueous solution of aspartase obtained by the method according to claim 11, which contains mostly cells. 18. Claims: [An almost cell-free aqueous solution of aspartase obtained by the method according to claim 13. 19. A reactor comprising a first chamber, a second chamber, and a semipermeable membrane separating the first chamber from the second chamber,
The semipermeable membrane is completely permeable to aspartate and fumarate, but impermeable to aspartase, and the substantially cell-free aqueous aspartase as claimed in claim @16 or 17 A solution is directed into the first chamber into fluid contact with the semi-permeable membrane, and an ammonium fumarate solution is directed into the second chamber into fluid contact with the semi-permeable membrane. A method for producing and distributing aspartic acid, which comprises culturing under aspartic acid producing conditions and recovering aspartic acid from the second chamber solution. 20. An outer shell in which the reaction device can be sealed; a device for filling the shell with liquid; and a plurality of hollow, semi-permeable shells 11e which are passed through the shell so as to be sealed from the interior of the shell.
4. One end of each fiber opens into the inlet end manifold, and the other end opens into the outlet end manifold. The aqueous solution of aspartase free of wart cells is introduced into the shell, the ammonium fumarate is passed through the hollow semipermeable fiber, and aspartic acid is recovered from the ammonium fumarate solution. 20. The method according to claim 19, characterized in that: 21. The method of claim 4, wherein the temperature of ammonium heptalate in the ammonium fumarate solution ranges from about 1 mole to the saturation temperature. 22. The method of claim 21, wherein a substantially saturated solution of ammonium fumarate is used. The above aspartic acid production conditions are from about 2°C to about 70°C.
4. The method according to claim 4, which includes culturing temperatures up to. U, the above aspartic acid production conditions range from about 25°C to about 50°C.
5. A method according to claim 4, comprising culturing temperatures up to .degree. The reactor is operated in a recirculating batch mode in which the ammonium fumarate solution is passed through the reactor from a reservoir. 25. The method of claim 11, wherein substantially all of the ammonium chloride is recycled until it is converted into amino acids.