GB2108128A

GB2108128A - Production of aspartase

Info

Publication number: GB2108128A
Application number: GB08220598A
Authority: GB
Inventors: David Charles Sternberg; Lois Jean Moser
Original assignee: Genex Corp
Current assignee: Genex Corp
Priority date: 1981-10-15
Filing date: 1982-07-15
Publication date: 1983-05-11
Also published as: PL238624A1; AU8592482A; IT8267896A0; DE3226532A1; ES514018A0; FR2514782A1; LU84273A1; DK307582A; GR78398B; IL66262A0; DD207219A5; NL8202854A; ES8400142A1; BR8204099A; JPS5867184A; SE8204306L; FI823502L; SE8204306D0; BE893838A; FI823502A0

Abstract

Aspartic acid is enzymatically produced from ammonium fumarate in the presence of the enzyme, aspartase. A cell-free aspartase solution is prepared by forming an aqueous cell suspension of aspartase- containing microbial cells; incubating the cell suspension, centrifuging the cell suspension to form a clarified solution that contains a major portion of the aspartase activity originally present in the cells, a sufficient amount of an aspartase-stabilizing salt being included in the cell suspension or to the clarified solution, to make the clarified solution hypertonic. Aspartase recovered in this manner is particularly useful in immobilized enzyme reactors for the production of aspartic acid.

Description

Summary of the invention In accordance with the present invention, a method is disclosed for preparing an aqueous, substantially cell-free solution of aspartase, which comprises forming an aqueous cell suspension of aspartase-containing microbial cells; incubating the aqueous cell suspension under aspartasereleasing conditions for a time sufficient for a substantial portion of the intracellular aspartase to pass into the extracellularfluid and removing the solids from the cell suspension to form a clarified, aspartase-containing solution, a sufficient amount of an aspartase-stabilizing salt being included in the aqueous cell suspension or the clarified, aspartase-containing solution to make said solution hypertonic with respect to the microbial cells. In a preferred embodiment, the aqueous cell suspension is formed with a hypertonic solution of the aspartase-stabilizing salt.

The procedure is simple and efficient, and more than 80% of the aspartase activity of the cells is generally recovered in the cell-free aspartase solution. Moreover, the enzyme has been found to maintain its activity for extended periods of time in such a solution. The solutions can be very effectively employed in dialysis-type reactors, as discussed above.

Detailed description The enzyme, aspartase, is produced by a wide variety of microorganisms, and the present invention is not limited to the use of any particular genus, species or strain. For instance, bacteria such as Pseudomonas fluorescens, Pseudomonas aeroginosa, Bacillus subtilus, Bacillus mega therim, Proteus vulgaris, Serratia marcescens, Escherichia colt and Aerobactor aerogenes have been described as suitable for the production of aspartic acid. A particular organism from which aspartase has been recovered is a glutamate-utilizing mutant of E. coli (ATCC No.

31976).

As a source of aspartase, aspartase-producing cells may be cultured on a nutrient medium designed for production of the enzyme and rapid growth of the cells. Methods for preparing and maintaining cell cultures are well known and do not constitute a part of the present invention.

Generally, a nutrient medium containing essential minerals and growth factors, and assimilable sources of carbon and nitrogen is employed. Advantageously, the medium contains fumaric acid, and also preferably contains a yeast extract as a source of nitrogen, vitamins and growth factors, ammonium ion as an additional nitrogen source, and calcium and magnesium salts as essential minerals. Phosphate and carbonate salts of potassium or sodium may be employed as buffers.

The inoculated medium is generally fermented under growth conditions for a sufficient period of time to produce from about two to about six grams per liter of solids. Normally a fermentation at about 37 C for about 12-14 hours is sufficient to produce the desired cell concentration.

After a suitable period of growth, the cells are advantageously separated from the extracellular portion of the fermentation medium (e.g., by centrifugation or filtration), and washed.

Aspartase can be released from the cells by suspending them in an aqueous medium and incubating them under aspartase-releasing conditions. The aqueous suspending medium may be water or any aqueous solution that does not adversely affect the enzyme (such as common buffers). Preferred aqueous suspending media are hypertonic solutions of aspartase-stabilizing salts (as hereinafter discussed), and particular preferred media are hypertonic solutions of soluble salts of fumaric or aspartic acid. Ammonium fumarate solutions, ranging in concentration from about 1.0 molar to saturation, preferably about 1.4 molar are especially preferred.

The cells are advantageously suspended in the aqueous medium at a concentration of at least about 5 grams per liter (dry cell weight basis) and preferably from about 10 to about 1 5 grams per liter.

The cell suspension is generally incubated under aspartase-releasing conditions for a time sufficient for a substantial portion of the intracellular aspartase to pass into the fumarate solution. An incubation temperature of from about 20C to about 700C. preferably from about 250C.

to about 500C. is generally employed. A particularly preferred incubation temperature is about 37 CC. Slower enzyme release rates and lower enzyme activities are associated with lower incubation temperatures, whereas higher incubation temperatures can deleteriously affect the enzyme. The incubation time is, to some extent, dependent on the incubation temperature, but generally is at least about 10 minutes.

Preferably, an incubation period of at least about 1 hours, most preferably, from about 12 to about 60 hours is employed.

After incubation, the solids are removed from the cell suspension, e.g., by centrifugation or filtration to form a clarified aspartase-containing solution. Any clarification procedure may be used that does not result in substantial losses of aspartase activity. The resulting solution generally contains about 80% of the aspartase activity originally contained in the cells. These solutions are relatively free of undesirable contaminants, and have been found to maintain substantially all of their aspartase activity for over 40 days at 370C.

A sufficient amount of an aspartase-stabilizing salt is added to the clarified solution to make that solution hyperton. In a preferred embodiment of the invention, the aspartase-stabilizing salt solution is used as the aqueous cell suspension medium, and when that procedure is used, the addition of an aspartase-stabilizing salt to the clarified solution is generally not necessary.

The aspartase-stabilizing salt has been found to stabilize the enzyme against loss of activity SPECIFICATION Production of aspartase Background of the invention The present invention relates to the production of aspartase. More particularly, the invention relates to a method for preparing an aqueous, cell-free solution of aspartase, which can be used for the conversion of ammonium fumarate to aspartic acid.

L-aspartic acid is an important amino acid that is used in pharmaceutical applications and as a raw material for specialty chemicals, such as Lalanine and aspartame. This amino acid is also used as an additive for animal and human foods.

Aspartic acid has been produced by chemical means, but chemical processes generally produce a racemic mixture of both the D- and L-forms.

Only the L-form of aspartic acid is biologically assimilable. Accordingly, preferred methods for the production of aspartic acid involve the cultivation of aspartate-producing microorganisms on suitable nutrient media. For example, Chibata, et al., U.S. Patent 3,214,345 describe fermentative procedures for the production of aspartic acid in whichfumaric acid (as the ammonium salt) is used as the major carbon source. The referenced procedure involves a batch-type process, wherein a nutrient fermentation medium is inoculated with a microorganism, and after a period of incubation, aspartic acid is recovered from the fermentation medium.

The production of aspartic acid using immobilized cell reactors has also been reported.

For example, Chibata, et al., U.S. Patent 3,791,926 describe the preparation of an immobilized cell reactor by entrapping aspartateproducing microorganism cells in an acrylamide polymer. Aspartic acid is then prepared by contacting the immobilized cells with ammonium fumarate. The reaction may be of a batch-type or a continuous-type. In continuous reactors the immobilized cells are packed in a column, through which an ammonium fumarate solution is continuously passed.

Immobilized cell reactors have several advantages over batch-type fermentation processes. In the batch type methods, recovery of the product frequently involves complex procedures which reduce the overall yield, and in many cases, result in a product that is contaminated with cells, metabolites and other materials present in the nutrient medium. The use of immobilized cells eliminates many of these problems; however, substantial amounts of cellular impurities may nevertheless be present in the final product.

Procedures by which the recovery and contamination problems associated with batchtype and immobilized cell processes have been overcome have included the isolation, purification and use of the enzyme or enzymes essential for carrying out the desired reaction. The enzyme involved in the conversion of ammonium fumarate to aspartic acid is aspartase. Attempts have been made to immobiiize aspartase on columns or solid supports for direction conversion of ammonium fumarate to aspartic acid. For example, the introductory portion of U.S. patent 3,791,926 describes procedures involving the binding of aspartase to an anion exchange polysaccharide or entrapping aspartase in an acrylamide polymer.These methods have, however, met with limited success, in that extraction and purification of the aspartase enzyme is difficult, and substantial loss of enzymatic activity has occurred during the purification, concentration and immobilization of the enzyme.

Dialysis-type reactors have also been proposed for the immobilization of enzymes. As used herein the term "dialysis" is used in the broad sense to include true dialysis (exchange of solute molecules between two liquids separated by a membrane), osmosis (solvent transport across a membrane), and ultrafiltration (solvent or solute movement brought about by a transmembrane pressure differential). In dialysis-type reactors, an enzyme solution is effectively retained on one side of a semipermeable membrane. The substrate for the enzymatic reaction is then contacted with the other side of the semipermeable membrane. The semipermeable membrane is designed to be permeable to the substrate and product but impermeable to the enzyme.Thus, substrate can migrate to the enzyme side of the membrane where it is converted to product, and the product can migrate back into the substrate solution, from which it can be recovered.

Dialysis-type reactors can take a variety of forms. For example, industrial units are generally of the filter-press configuration, which comprise numerous parallel membrane plates, separated by chambers through which liquids may flow.

Kan, J. K. and Shuler, M. L., Biotechnology and Bioengineering, 20, 217-230 (1978) describe the use of a hollow fiber kidney dialysis unit as a dialysis-type reactor. In using such a unit, microbial whole cells are immobilized on the shell side of the kidney dialysis unit. A solution of a substrate is circulated through the tube side of the hollow fibers in the dialyzer. The substrate diffuses through the fibers to the cells, where reactions take place, and formed products diffuse back into the recirculating stream. The particular system described involves the immobilization of a strain of Pseudomonas fluorescens, for the enzymatic conversion of L-histidine to urocanic acid.

Heretofore, attempts to use immobilized aspartase enzyme in dialysis-type reactors for the production of aspartic acid have generally been unsuccessful (e.g., see U.S. Patent 3,791,926, supra). The reason for this lack of success is thought to be that known procedures for isolating and purifying aspartase result in substantial losses of enzyme activity.

over a period of time and also to stabilise it against thermal degradation. For example, comparisons of aspartase activity in solutions stored at 650C indicate that buffer-extracted enzyme loses virtually all of its activity after thirty minutes, whereas enzyme extracted into 1.4 M ammonium fumarate maintains at least 80% of its stability after two hours of storage.

The preferred aspartase-stabiiizing salts are soluble salts of fumaric or aspartic acid. Inasmuch as fumaric acid is the enzyme substrate for the production aspartic acid, the use of salts of these acids generally obviates the need for later separating the salt from the aspartase. Moreover, such salts, especially ammonium fumarate, have been found particularly effective for the extraction and stabilization of the enzyme. Although fumarate and aspartate salts are preferred, it will be appreciated that any water-soluble salt that has a stabilizing effect on the enzyme may be used.Such salts may be generally represented by the formula, MX, wherein M represents a cation, such as an alkali or alkaline earth metal, ammonium, lower alkylammonium, lower alkyl quaternary ammonium, and the like, and X represents an anion, such as halide, nitrate, carbonate, phosphate, sulphate, maleate, fumarate, aspartate, and the like. The concentration of the aspartase-stabilizing salt in the clarified solution advantageously ranges from about 0.5 molar to saturation, preferably from about 1.0 molar to about 1.4 molar.

The aspartase solutions prepared by this method are particular useful in dialysis-type reactors, especially in hollow fiber reactors. The cell-free solution may easily be introduced into the shell side of such a reactor simply by applying suction to the tube side of the reactor. After the enzyme has been introduced into the reactor, an ammonium fumarate solution may be circulated through the tube side of the reactor. The ammonium fumarate substrate and aspartic acid product freely pass through the walls of the dialysis tubes, and aspartic acid may be recovered from the circulating solution. On the other hand the aspartase enzyme cannot pass through the walls of the tubes, therefore, it is effectively retained in the reactor. Removal of the dialyzable impurities and color bodies occurs soon after the reaction is started, and therefore do not contaminate the bulk of the product stream.

The conditions for reaction may be varied depending on the type of reactor used. Generally, the substrate is a concentrated solution of ammonium fumarate, e.g. from about 1 molar to saturation, preferably about 1.8 molar. The reaction media are incubated under aspartic acidproducing conditions. The reactor is advantageously maintained at an elevated temperature to provide efficient conversion of substrate to product. Temperatures of from about 2 OC to about 70"C, preferably from about 25 OC to 50or, most preferably about 370C are generally employed. The enzymatic reaction is exothermic, therefore, in large reactors or reactors in which low substrate flow rates are employed, means for removing excess heat may be employed.The desired flow rate of the substrate solution through the reactor depends upon the particular reactor employed, the concentration of the substrate, the configuration of the system, (e.g., circulating or a single pass), the level of enzyme loading, and the desired rate of heat dissipation.

The hollow fiber reactor system can be operated in a recirculating batch mode, in which the exit stream of the reactor returns to a reservoir. Circulation of the substrate solution is continued until a major portion of the ammonium fumarate has been converted to aspartic acid.

Alternative modes of reaction involve single pass systems in which a plurality of reactors may be operated in series.

The present invention has been found to provide a simple and efficient procedure for recovering aspartase from aspartase-producing cells. The resultant cell free aspartase solution can be used in batch or immobilized modes, and is particularly suited to hollow dialysis type reactors.

The cell-free aspartase solutions of this invention have several advantages over whole cells in dialysis-type reactors. To obtain a high level of enzymatic activity with whole cells, the cells must generally be in a concentrated form.

Concentrated cell media are very dense and viscous, which leads to at least two problems that are alleviated by the cell-free system of the present invention. First, impurities and color bodies from the cell slurry migrate through the dialysis membrane into the product stream.

Rather than occurring during a short period of time at the initial stage of the reaction, as is the case with the cell-free solutions, impurities and color bodies tend to leach out of whole cells over extended periods of time. A second advantage of the cell-free system relates to diffusional problems associated with whole-cell systems.

Aspartase is thought to be an intracellular enzyme, therefore, reactants and products must diffuse not only through the reactor membrane, but also through the extraceliular fluid and cellular constituents. Accordingly, less efficient rates of conversion are observed with such whole-cell systems. For example, with approximately the same amount of enzyme activity, a cell-free, hollow fiber reactor system has been shown to have about 45% greater conversion rate than is observed with the whole-cell system.

The invention is further illustrated by the following examples which are not intended to be limiting.

Example I Several experiments were conducted in which nutrient media containing varying amounts of yeast extract were fermented to produce aspartase. Each fermentation medium contained, per liter: 24-60 grams of yeast extract, 30 grams of fumaric acid, 2 grams of dibasic potassium phosphate, 0.5 grams of sodium carbonate, 2 mM magnesium sulfate, and 0.1 mM calcium chloride, and the pH was adjusted to about 7.2 with ammonium hydroxide. Each medium was innoculated with 1 mt of a culture of E. coli (ATCC No. 31976) that had been incubated for 12-16 hours at 370C inna peptone medium containing 0.5 percent monosodium glutamate.

Each innoculated medium was incubated at 370C for 12-14 hours and at that time, the cells were harvested. The dry cell weight at harvest was about 2-7 grams per liter, depending upon the amount of yeast extract used. The rate of production of aspartase for each fermentation media is shown in Table I.

A second group of experiments was conducted, in which fermentation media containing various concentrations of ammonium fumarate were prepared and fermented as described above. Each of these media contained 8% yeast extract and the same concentrations of other ingredients as described above. The results of these experiments are shown in Table II. The cell culture prepared from the fermentation medium containing 2.4 w/v% yeast extract and 3.0% ammonium fumarate was centrifuged, and the liquid decanted and discarded. The cells were then washed with water, recentrifuged, decanted and resuspended to 1/20 the original volume in 0.05 tris.HCI buffer, pH 8.5. This slurry was then diluted with 1.8 M solution of ammonium fumarate, pH 8.5, to make a cell suspension having 1/5 of the original fermentation volume.

The resulting suspension was separated into six portions which were incubated at 370C forth time periods shown in Table III and then centrifuged. The supernatants were decanted, and the solids were discarded. The supernatants were analyzed for aspartase activity, and the results are shown in Table III.

Example II A cell-free solution of aspartase, from 12.5 g (dry weight) of cells prepared in accordance with the procedure of Example 1(2.4% yeast extract, 3% ammonium fumarate), was introduced into the shell side of Cordis-Dow artificial kidney (CDAK 2.5D) by applying suction to the tube side of the articificial kidney. After the enzyme was introduced into the reactor, the shell side was sealed and a 1;8 M solution of ammonium fumarate, pH 8.5, was passed through the tube side of the reactor at a flow rate of about 2 11-1 5 liters per hour. A recirculating mode was employed, in which substrate solution was recirculated through the reactor from a reservoir.

The reactor was maintained at a temperature of 370C. The reservoir contents were periodically analyzed for residual fumarate, and the results of the analyses are shown in Table IV. The aspartic acid was precipitated at pH 2.8 by addition of H2SO4, filtered, washed and dried. From an average of 2.1 Kgfumaric acid, 2.0--2.1 Kg aspartic acid was recovered (83% to 86% theoretical yield). The aspartic acid thus produced was characterized by elemental analyses for C, H, N and 0, which were in 9799% agreement with theory The specit tatioR u tte product \ r' measured and was as follows: [1o25=+26 (C=1 0 in 2N HCI).The product was analyzed by a diagnostic coupled enzyme assay and was in 96% agreement with an authenic aspartic acid standard.

Table I Effect of yeast extract concentration in fermentation medium (containing 3% ammonium fumarate) on volumetric aspartase activity of broth.

aspartase w/v /0 mmols h-ml of yeast extract culture broth 1.0 0.22 1.8 0.64 2.7 0.90 3.0 1.20 4.0 1.50 5.0 1.76 6.0 2.14 7.0 2.26 8.0 2.24 Table II Effect of ammonium fumarate concentration in fermentation medium (containing 8% yeast extract) on volumetric aspartase activity of broth.

w/v /0 aspartase fumaric acid mmols/h-ml 0 1.00 2.0 2.50 4.0 2.52 6.0 0.48 Table III Extraction of Aspartase from Cells with 1.4 M Ammonium Fumarate.

%Activity Time (hrs.) Extracted 0 4 70% 7 73% 14 79% 24 80% 48 84% Table IV Hollow fiber reactor (CDAK 2.5D) loaded with cell free enzyme (extracted from approximately 12.5 g. cells-dry weight); used in recirculating batch mode with a 10 liter stirred reservoir of 1.8 M ammonium fumarate. Samples removed from reservoir and analyzed for residual fumarate.

Residual Time fumarate (M) 0.0 h 1.81 0.25 1.63 0.5 1.52 1.0 1.30 1.5 1.12 2.0 0.98 2.5 0.82 3.0 0.70 3.5 0.58 4.0 0.48 5.0 0.32 6.0 0.21 7.0 0.14

Claims

1. A method for preparing an aqueous, substantially cell-free solution of aspartase, which comprises forming an aqueous cell suspension of aspartase-containing microbial cells; incubating the aqueous cell suspension under aspartasereleasing conditions for a time sufficient for a substantial portion of the intracellular aspartase to pass into the extracellularfluid and removing the solids from the cell suspension to form a ciarified, aspartase-containing solution, a sufficient amount of an aspartase-stabilizing salt being included in said cell suspension or said clarified aspartase-containing solution to make said clarified aspartase-containing solution hypertonic with respect to the microbial cells.

2. The method of claim 1, said aqueous cell suspension is prepared by suspending the microbial cells in a hypertonic solution of said aspartase stabilizing salt.

3. The method of claim 1 or 2, said aspartasestabilizing salt is a water-soluble salt of fumaric or aspartic acid.

4. The method of claims 1 or 2, wherein said aspartase-stabilizing salt is ammonium fumarate or ammonium aspartate.

5. The method of claim 2, wherein said aspartase-stabilizing salt is ammonium fumarate.

6. The method of claim 5, wherein the concentration of ammonium fumarate in the hypertonic ammonium fumarate solution is from about 0.5 molar to saturation.

7. The method of claim 5, wherein the concentration of ammonium fumarate in the hypertonic ammonium fumarate solution is from about 1.0 to about 1.4 molar.

8. The method of claim 6, wherein the aspartase-recovering conditions include an incubation temperature from about 20C to about 700 C, and the cell suspension is incubated for at least about 10 minutes.

9. The method of claim 6, when the aspartase-recovering conditions includes an incubation temperature from about 250C to about 500C, and the cell suspension is incubated for about 12 to about 60 hours.

10. The method of claim 9, wherein the incubation temperature is about 370C.

11. The method of claim 9, wherein the bacterial cells are suspended in sufficient hypertonic ammonium fumarate solution to provide a cell concentration of at least about 5 g/liter, on a dry cell weight basis, and the aspartase-recovering conditions further include a pH of from about 7 to about 9.

12. The method of claim 11, wherein the bacterial cells are suspended in sufficient hypertonic ammonium fumarate solution to provide a cell concentration of from about 10 g/liter to about 1 5 liter on a dry cell weight basis, and the pH is from about 8 to about 9.

13. The method of claim 1,5,6,7,9or 12, wherein the bacterial cells are selected from the group consisting of aspartase-producting strains of Pseudomonas fluorescens, Pseudomonas aeroginosa, Bacillus subotilis, Bacillus megatherium, Proteus vulgaris, Serratla marcescens, Escherichia coli and Aerobacter aerogenes.

14. The method of claim 13, wherein the bacterial cells areE. coli.

1 5. The method of claim 14 wherein the bacterial cells are E. coli strain ATCC 31976.

1 6. An aqueous, substantially cell-free solution of aspartase prepared by the method of claim 1.

1 7. An aqueous, substantially cell-free solution of aspartase prepared by the method of claim 11.

1 8. An aqueous, substantially cell-free solution of aspartase prepared by the method of claim 13.

1 9. A method for producing aspartic acid in a reactor which comprises a first chamber and a second chamber and a semi-permeable membrane which separates the first chamber from the second chamber, said semipermeable membrane being permeable to asparate and fumarate, but impermeable to aspartase, which method comprises placing the aqueous, substantially cell-free aspartase solution of claim 1 6 or 1 7 in said first chamber in fluid communication with said semipermeable membrane and placing a solution comprising ammonium fumarate in said second chamber in fluid communication with said semipermeable membrane; incubating said solutions under aspartic acid-producing conditions, and recovering aspartic acid from the solution in said second chamber.

20. The method of claim 19, wherein said reactor comprises a sealable shell having means for filling said shell within a liquid, and a plurality of hollow semipermeable membrane fibers that pass through said shell, such that the lumens of said fibers are sealed from the interior of said shell, and each fiber has an inlet end which opens into an inlet manifold having a tube inlet port, and an outlet end which opens into an outlet manifold having a tube outlet port, wherein said aqueous, substantially cell-free aspartase solution is placed in said shell, and said ammonium fumarate is passed through said hollow semipermeable membrane fibers, and aspartic acid is recovered from said ammonium fumarate solution.

21. The method of claim 20, wherein the concentration of ammonium fumarate in the ammonium fumarate solution is from about 1 molar to saturation.

22. The method of claim 21, wherein a substantially saturated ammonium fumarate solution is employed.

23. The method of claim 20, wherein the aspartic acid-producing conditions include an incubation temperature of from about 20C to about 7O0C.

24. The method of claim 20, wherein the aspertic acid-producing conditions include an incubation temperature of from about 250C to about 500 C.

25. The method of claim 24, wherein the reactor is operated in a recirculating batch mode, in which the ammonium fumarate solution is recycled from a reservoir through the reactor until a substantial quantity of the ammonium fumarate is converted to aspartic acid.

26. A method as claimed in claim 1 substantially as herein described with reference to Example I.

27. A method as claimed in claim 19 substantially as herein described with reference to Example II.