NL8202854A - METHOD FOR PREPARING ASPARTASE. - Google Patents

Description

r λ i I VO 3557

Method for preparing aspartase.

The invention relates to a process for preparing aspartase, more particularly a process for preparing an aqueous, cell-free solution of aspartase, which are used for the conversion of ammonium fumarate to aspartic acid.

5 L-aspartic acid is an important amino acid, which is used in pharmaceutical preparations and as a raw material for special chemicals, such as L-alanine and aspartam. This amino acid is also used as an additive for human and animal foods. Asparagine Neighbor has also been prepared by chemical methods, but chemical work tools usually provide a racemic mixture of as many of the D and L forms. Only the L-form of aspartic acid is biologically assimilable. Preferred methods for the production of aspartic acid therefore include culturing aspartate-producing microorganisms on appropriate culture media. For example, U.S. Pat. No. 3,231U.3 ^ 5 describes a fermentative method of preparing aspartic acid using fumaric acid in the form of the ammonium salt as the main carbon source. A batch-wise working precede is used, in which a culture medium is seeded with a micro-organism, and aspartic acid is recovered from the medium after incubation for some time.

The production of aspartic acid using immobilized cell reactors is also known. For example, U.S. Patent 3,791,926 describes the preparation of aspartic acid with immobilized cells, wherein the aspartic acid-producing cells are enclosed in an acrylamide polymer. Aspartic acid is then prepared by contacting these immobilized cells with ammonium fumarate. The reaction can proceed in a batch or continuous manner. In continuous reactors, the immobilized cells are placed in a column through which an ammonium fumarate solution is continuously passed.

Reactors with immobilized cells have several advantages over low-fermentation fermentation processes. In an operating mode of operation, recovering the product often involves complex operations which reduce the overall yield while in many cases results in a product resulting with cells, metabolites and others.

8202854 2 »Λ components of the culture medium are contaminated. The use of immobilized cells overcomes many of these problems, but nevertheless significant amounts of cellular contaminants may be present in the final product.

Methods in which the recovery and impurity problems which have been overcome in batch-free and immobilized cell processes include isolation, purification and use of the enzyme or enzymes essential to carry out the desired reaction. The enzyme that handles the conversion of ammonium fumarate to aspartic acid is aspartase. Attempts have been made to immobilize this aspartase on columns or solid supports for a direct conversion of ammonium fumarate to aspartic acid. For example, the introduction to U.S.P. 3,791,926 methods, in which aspartase is bound to an anion exchange polysaccharide or entrapped aspartase in an acrylamide polymer. These compounds have proved to be only partially successful, because the extraction and purification of aspartase enzyme is difficult and significant losses of enzyme activity occur during the purification, concentration and immobilization of the enzyme: ...

Dialysis-type reactors have also been proposed for immobilizing enzymes. The term "dialysis" is used in a broad sense. It includes both actual dialysis (exchange of dissolved molecules between two liquids through a membrane), osmosis (solvent transport through a membrane) and ultrafiltration of solvent or dissolved material through a membrane under the influence of a pressure difference.) 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 mixed with the other side of this dull-permeable membrane. Brane contacted The semipermeable membrane is made to be permeable to the substrate and product but impermeable to the enzyme, thus allowing the substrate to migrate to the enzyme side of the membrane where it is converted into the product after which this product can migrate back to the substrate solution, from which it can be recovered · 35 Reactors of the dialys The type can have a wide variety of shapes. For example, industrial equipment 8202854 ...............

Usually in the form of a filter press, in which there are a large number of parallel membrane plates, separated by chambers through which the liquids can flow.

Kan and Shuler, in Biotechnology and Bioengi-Nation 20, 217-230 (1978), describe the use of a hollow fiber kidney dialysis unit as a dialysis type reactor. When using such a unit, whole microbe cells were immobilized on the sleeve side of the kidney dialysis unit. A solution of the substrate is then passed through the tube side of the hollow fibers into the dialyzer.

The substrate diffuses through the fibers to the cells, where the reaction takes place, after which the formed products diffuse back into the circulating stream. This particular system involves the immobilization of a stain Pseudomonas fluorescens for an enzymatic conversion of L-histidine to urocanic acid.

So far, attempts to use immobilized aspartase in dialysis-type reactors to produce aspartic acid have been unsuccessful - (see U.S.P-3 * 791 * 926). The cause of this failure is sought in the significant losses of enzyme activity in the isolation and purification of aspartase.

The invention now relates to a process for preparing an aqueous, substantially cell-free solution of aspartase, whereby an aqueous cell suspension of aspartase-containing microbe cells is formed, this aqueous cell suspension is incubated under asparfcase-releasing conditions for a sufficient period of time. to transfer a substantial portion of the intracellular aspartase to the extracellular fluid; the solids are removed from the cell suspension to yield a clear aspartase-containing solution, and a sufficient amount of an aspartase-stabilizing salt is added to this aqueous cell suspension or the clarified aspartase-containing solution to cause this solution to be hypertonic to the microbe cells * In a preferred embodiment, the aqueous cell suspension is formed using a hypertonic solution of the aspartase stabilizing salt.

This method is simple and effective, and more than 80% of the aspartase activity of the cells is usually recovered in the cell-free aspartase solution. * In addition, the enzyme was thus shown to be active for a long time in maintain such a solution. These solutions can be used very effectively in dialysis type reactors,

The enzyme aspartase is produced by a wide variety of microorganisms and the invention is therefore not limited to the use of any special genus, species or strain. For example, bacteria such as Pseudomonas fluorescens, Pseudomonas aeruginosa., Bacillus subtilus, Bacillus megatherium, Proteus vulgaris, Serratia marcescens, Escherichia coli and Aerobacter aerogenes have been described as suitable for producing aspartic acid. A specific organism from which asparase is derived is a glutamate-consuming mutant of E. coli (ATCC 319Τβ)

As a source of aspartase, aspartase-producing cells can be cultured on a culture medium intended for the production of this enzyme in rapid growth of the 3rd cells. Methods of preparation and. parts of this type of cell culture are known and do not form part of the invention.

A culture medium containing the essential minerals and growth factors as well as assimilable carbon and nitrogen sources is usually used. Most preferably, the medium should also contain fumaric acid and. preferably also a yeast extract as a source of nitrogen, vitamin. and growth factors, further ammonium ions as a further nitrogen source, calcium and magnesium salts as minerals, while phosphate and carbonate salts of potassium or sodium can be used as buffers.

The grafting medium is usually fermented under suitable conditions for a sufficiently long time until two to six grams of solid per liter are formed. Usually, a 12 - 1 hour hour fermentation at about 37 ° C is sufficient to obtain the desired cell concentration.

For an appropriate growth period, the cells are preferably separated from the extracellular part of the culture medium, for example by centrifugation or filtration. and then washed. Aspartase can be released from the cells by suspending them in an aqueous medium and incubating under aspartase releasing conditions. The aqueous suspension medium can be water or an aqueous solution which does not adversely affect the enzyme (such as conventional buffers). Preferred aqueous suspension media are hypertonic solutions of aspartase stabilizing salts (discussed in more detail below) and particularly preferred media are hypertonic solutions of soluble salts of fumaric or aspartic acid. Ammonium phosphate solutions with concentrations between about 7.0 mol to saturation, most preferably about 1.1 mol, are particularly preferred.

The. cells are preferably suspended in the aqueous medium at a concentration of at least about 5 grams per liter (dry cell weight), and most preferably in an amount of 70 to 15 grams dry cell weight per liter.

The cell suspension is usually incubated under aspartase-releasing conditions for a period of time sufficient to pass a major portion of the intracellular aspartase into the fumarate solution. An incubation temperature of about 2 to T0 ° Cs.

Preferably 25-50 ° C is usually used. A particularly favorable incubation temperature is about 3T ° C. Slower enzyme release and lower enzyme activity is associated with lower incubation temperatures, while higher incubation temperatures may have a decomposing effect on the enzyme. The incubation period depends to some extent on the incubation temperature, but usually it is at least 10 minutes. Preferably, an incubation period of at least about 1 hour applies, most preferably 12 to 6θ hours are incubated.

After this incubation, the solids of the cell suspension are removed, for example, by centrifugation or filtration, thereby forming a clarified aspartase-containing solution. Any clarification method can be used as long as it does not, at least, involve significant losses in aspartase activity. The resulting solution usually contains about 10% of the aspartase activity originally present in the cells. These solutions are virtually free of unwanted contaminants and have been found to substantially retain their aspartase activity for 37 days at 37 ° C. -

An adequate amount of an aspartase stabilizing salt is added to the clarified solution to make this solution hypertonic. In a preferred embodiment of the invention, an aspartase stabilizing saline is used as an aqueous cell suspension medium and when this embodiment is used ~ 82 0-2 8T4 .............. "" .......................................

6, addition of an aspartase stabilizing salt to the clarified solution is usually unnecessary.

The aspartase stabilizing salt was found to stabilize the enzyme against loss of activity for a long time, while the enzyme was also protected against thermal decomposition. For example, comparisons of aspartase activity in solutions stored at 65 ° C indicate that buffer-extracted enzyme free-sheet has lost all its activity after 30 min, t-file enzyme extracted with 1.1 mole of axonium fumate at least 80% of its stability after 2 hours. -10 stroke still beats.

The preferred aspartase stabilizing salts are soluble salts of fumaric or aspartic acid. Since fumaric acid is the enzyme substrate for the production of aspartic acid, the use of salts of these acids usually avoids the need to later separate this salt from the aspartase. In addition, these salts, especially ammonium fumarate, have been found to be particularly effective in extracting and stabilizing the enzyme. How much preferred fumarate and aspartate salts can be any further more soluble salt that has a stabilizing effect. applied to the enzyme. Such salts 20 correspond conventionally to the formula MX, where M is a cation such as an alkali or alkaline earth metal, ammonium, alkyl ammonium, alkyl quaternary ammonium and the like X is an anion, for example a halide, nitrate, carbonate, phosphate, sulfate, maleate, fumarate, aspartate and the like. The concentration of the aspartase stabilizing salt in the clarified solution preferably ranges from 0.5 mole to saturation, most preferably from 1.0 mole to about 1 mole.

The aspartase solutions thus prepared are particularly useful in dialysis type reactors, especially in hollow fiber reactors. The cell-free solution can easily be introduced to the bulk side of such a reactor by suction from the tube side of the reactor. After the enzyme has been introduced into the reactor, an ammonium fumarate solution can be circulated through the tube side of the reactor. The ammonium fumarate substrate and the aspartic acid product can pass freely through the fins of the dialysis tubes and the aspartic acid can be easily recovered from the circulating solution. On the other hand, the aspartase enzyme cannot migrate through the vents of the tubes, so it is effectively retained in the reactor. Removal of the dialysable impurities and colored substances occurs soon after the start of the reaction, so that the vast majority of the product stream is not disarinated.

The reaction conditions may vary depending on the type of reactor used. Usually, the substrate is a concentrated solution of ammonium fumarate, i.e., from about 1 mole to saturation, most preferably about 1.8 moles. The reaction media are incubated under da-aspartic acid-producing conditions. The reactor is preferably kept at an elevated temperature, whereby a good conversion of substrate into product is achieved. Temperatures between about 2 and about 70 ° C, preferably between 25 and 50 ° C, most preferably about 37 ° C are used for this. The enzyme reaction is exothermic, so that in. large reactors, or reactors with low substrate flow rates, means for dissipating the developing heat can be provided. The desired flow rate of the substrate solution through the reactor depends on the specific reactor used, the concentration of the substrate, the configuration of the system (e.g., circulating or single flow), the degree of enzyme loading and the desired amount of heat dissipation.

The hollow fiber reactor system can be operated by a circulating batchwise method, whereby the effluent from the reactor is returned to a reservoir. The circulation of the substrate solution is continued until an important part of the ammonium fumarate has been converted into aspartic acid. Other ways in which the reaction can be carried out include single flow systems, where a large number of reactors can be arranged in series.

The invention provides a simple and effective method for the recovery of aspartase from aspartase-producing cells. The resulting cell-free aspartase solution can be used in batch or immobilized cell methods and is particularly suitable for hollow dialysis type reactors.

The cell-free aspartase solutions of the invention have several advantages over whole cells in dialysis type reactors. In order to achieve a high level of whole cell enzyme activity, the cells must usually be used in a concentrated form. Concentrated cell media are particularly thick and viscous, causing at least two problems which do not arise with the present cell-free system by the invention. First, impurities and dyes from a cell suspension migrate through a dialysis membrane to the product stream. This is not the case with cell-free solutions alone. The beginning of the reaction is the case, because impurities and dyes tend to diffuse away from the cells for quite a long time. A second advantage of the cell-free system concerns the diffusion problems that occur with gel cell systems. Aspartase is probably an intracellular enzyme so that reactants and products must diffuse not only through the reactor membrane but also through the extracellular fluid and cellular components. Thus, less high conversions are obtained with this type of cell system. For example, at approximately the same amount of enzyme activity, a cell-free convex fiber reactor system is approximately b5% more effective in conversion rate than a conventional cell system.

The invention is further illustrated by the following examples, which should not be construed as limiting.

Example I

Various experiments and verden were conducted, in which nutrient media with varying amounts of yeast extract were fermented to produce aspartase. Each culture medium contained per liter 2k - 60 g yeast extract, 30 g fumaric acid, 2 g dipotassium phosphate, 0.5 g sodium carbonate, 2 mM magnesium sulfate and 0.1 ami calcium chloride tervxjl 5 25 pH with ammonia. . Each medium was seeded with 1 cm of a culture of E. coli (ATCC 31976) incubated in a peptone medium with 0.5% monosodium glutamate for 12-16 hours at 37 ° C. Elk inoculate medium for 12 - incubated at 37 ° C for hours, then cells were found. The dry cell density was then 2 - 7 g per 1, depending on the amount of yeast extract used. The production rate of aspartase for each medium is shown in Table A.

A second group of experiments was conducted, where culture media with various concentrations of ammonium fumarate were prepared and incubated as borons. Each of these media contained 8% yeast extract and the same concentrations of the further ingredients as described above. The results of these experiments are presented in Table B. The cell culture obtained from a culture medium containing 2, 8% 8202854 .................. ~ 9 yeast extract and 3.0 Ammonium fumarate was centrifuged and the liquid scrubbed and discarded. The cells were then washed with water, re-centrifuged, scrubbed and resuspended in 1/20 of the original volume of a 0.05 tris-HCl buffer with pH · 8.5 *. This suspension was then diluted with 1.8 II solution ammonium fumarate with pH 8.5, giving the cell suspension 1/5 of the original fermentation volume. The resulting slurry was divided into 6 portions which were incubated at 3T ° C for the time periods shown in Table C and then centrifuged. The supernatants were decanted and the solids discarded. The supernatants were analyzed for his aspartase activity and the results are shown in Table C.

Example II

Fast cell-free solution of aspartase, prepared from 12.5 g (dry weight) of cells obtained according to Example I (2.1 yeast extract, 3 ammonium fumarate), was applied to the sleeve side of an artificial kidney of Cordis-Dow (CDAK 2.5D) by aspirating from the tube side of the artificial kidney after the enzyme had been added to the reactor. The tube side was closed and a 1.8 mol solution of ammonium fumarate with pH 8.5 was passed through the tube side of the reactor at a flow rate of about 11 - 15 l per hour. A recirculation method was used in which the substrate solution several times the reactor was introduced from a reservoir. The reactor was kept at 3T ° C. The reservoir contents were periodically analyzed for residue fumarate with the results of these analyzes shown in Table B. The aspartic acid was precipitated at pH 2.8 by the addition of sulfuric acid, filtered off, washed and dried. From an average of 2.1 kg of fumaric acid, 2.0-2.1 kg of aspartic acid were recovered (83 to 86% of the tbeo-retisc yield). The aspartic acid thus obtained was characterized by an elemental analysis on C, H, IT and O, which were found to be the same for 97-99%. The specific rotation of the product was: [α] D = + 26 ° (C = 10 in 2H HCl). This product was analyzed according to a diagnostic coupled enzyme assay and found to be $ 96 in accordance with an authentic aspartic acid-35 standard.

820 2 8 54 ........

10

T A B E L A

Effect of & yeast yeast extract concentration (with 3% ammonium fumarate) on the aspartase activity of the fermented liquid.

w / v% Aspartase

Yeast extract mmol u-cm3 fermentation broth 5 1.0 0.22 1.8 0.6k 2, T 0.90 3.0 1.20 k, 0 1.50 10 5.0 1.76 6.0 2.1k 7.0 2 , 26 8.0 2.2k

TABLE B

Effect of the ammonium fumarate concentration in the feedstock (with 8 $ 15 yeast extract) on the volume-aspartase activity of the fermentation broth.

w / v% Aspartase

Fumaarznur mmol / h-cm ^ 0 1.00 20 2.0 2.50 M 2.52 6.0 0.08

IABEL C

Extraction of aspartase from cells with 1 mol ammonium fumarate

Time in hours Percentage of activity extracted 25 0 '---

It · 10% 7 13% 1k 19% 2k Q0% 30 * k8 Sh% 8202854 “--------------- I" »11 '

T A B E L D

Hollow fiber reactor (CME 2.5D) loaded with callous-free enzyme (extracted from about 12.5 g of cell dry matter); applied In a recirculation method with a 10 1 stirred reservoir with 1.8 mol ammonium fumarate. Samples were collected from the reservoir and analyzed for residual anti-fumarate.

5 Time Antifumarate residual _ (M) 0.0 h 1.81 0.25 t, 63 0.5 1.52 10 .1.0 1.30 1.5 1.12 2.0 0.98 2, 5 0.82 3.0 0.70 15 3.5 0.5 * 8 if., 0 0, ½ 5.0 0.32 6.0 0.21

7.0 0.1U

8202854

Claims (18)

2. A method according to claim 1, characterized in that the vascular cell suspension is prepared by suspending microbe cells in a bypertonic solution of said aspartase stabilizing salt. 3. Operate according to conelusile or 2, characterized in that the aspartase stabilizing salt is a more soluble salt of fumaric or aspartic acid. Method according to claim 1 or 2, characterized in that the aspartase-stabilizing salt is ammonium fumarate or ammonium aspartate. Method according to claim 2, characterized in that the aspartase-stabilizing salt is ammonium fumarate. Award method according to claim 5, characterized in that the concentration of ammonium fumarate in the bypertonic ammonium fumarate. solution is between 0.5 mole and the saturation concentration. 7. Method according to claim 5, characterized in that the concentration of ammonium fumarate in the hypertonic ammonium fumarate solution is between 1.0 and 1.¼ Method according to claim 6, characterized in that the aspartase-finning conditions comprise an incubation temperature between 2 and 70 ° C, the cell suspension is incubated for at least about 10 minutes. 8 2 0 2 8 5 4 ..... ....... ft t Method according to claim 6, characterized in that the aspartase-winning conditions comprise an incubation temperature between 25 and 50 ° C and the cell suspension is incubated for 12-60 hours. 10. A method according to claim 9, characterized in that the incubation temperature is 3T ° C. 11. A method according to claim 9, characterized in that the bacterial cells are suspended * in an adequate amount of a hypertonic ammonium fumarate solution to obtain a cell concentration of at least 5 g of dry matter per 1 and the aspartase recovery conditions. a pH between T and 9. Method according to claim 11, characterized in that the bacterial cells are suspended in a sufficient amount of hypertonic ammonium fumarate solution. obtain a cell concentration of 10 to 15 g dry cell weight per 1 and the pH is between 8 and 9. Method according to claim 1, 5, 8, 7 * 9 or 12, characterized in that the bacterial cells are selected from aspartase-propagating β-primers of Pseudomonas fluorescens. Pseudomonas aerigonosa, Bacillus subtilis, Bacillus megatherium ,. Proteus vulgaris, Serratia marcescens, Escherichia coli and Aerobacter aerogedes ♦ Method according to claim 13, characterized in that the bactericells originate from E. coli. Method according to claim 1U, characterized in that the bacterial cells originate from E. coli strain ATCC 31976- Aqueous substantially cell-free solution of aspartase prepared according to claim 1. 1T. Aqueous, substantially cell-free aspartase solution prepared according to claim T1. An aqueous, at least substantially cell-free aspartase solution obtained according to claim 13. 30. Method for preparing aspartic acid in a reactor comprising a first space and a second space as well as a semipermeable membrane comprising the first separates from the second space, each semipermeable membrane permeable to aspartate and fumarate, to impermeable front aspartase, characterized in that an aqueous substantially cell-free aspartase solution according to claim 16 or 17 is in the first space in fluid communication with the semi-permeable membrane ..... 82 0 2 8 54 ........... Λ * 1¾. and a solution containing ammonium fumarate in the second space in liquid communication with the semipermeable membrane, incubating both solutions under aspartic acid-producing conditions and in aspartic acid from the solution. the second space wins. Method according to claim 19, characterized in that the reactor comprises a closable space with means for filling this space with a. liquid, as well as a number of hollow semi-permeable membrane fibers which pass through this space, such that the lumens of the fibers are closed off from the inside of the sleeve and each fiber has an inlet and an outlet, wherein the aqueous, at least virtually cell-free aspartase solution is housed in space and the ammonium fumarate solution is passed through the hollow semi-permeable membrane fibers and aspartic acid is recovered from the ammonium fumarate solution. A method according to claim 20, characterized in that the concentration of ammonium fumarate in the ammonium fumarate solution is between 1 molar and saturation. The method of claim 21, wherein a substantially saturated ammonium fumarate solution is used. 23- A method according to claim 20, wherein the aspartic acid-producing conditions comprise an incubation temperature between 2 and about 0 ° C. 2k. The method of claim 20, wherein the aspartic acid-producing conditions comprise an incubation temperature between 25 and 50 ° C. 25. Process according to claim 2k, characterized in that • the reactor is operated according to the batchwise recirculation method, wherein the ammonium fumarate solution from a reservoir is recycled through the reactor until a large part of the ammonium fumrate has been converted into aspartic acid. . 820 2 854 - -..........................................