LU84273A1 - ASPARTASE PRODUCTION - Google Patents

Description

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The present invention relates to the production of aspartase. More particularly, the invention relates to a process for the preparation of an aqueous aspartase solution, free of cells and which can be used to transform ammonium fumarate into aspartic acid. L-aspartic acid is an amino acid. important who. is used in pharmaceutical applications and as a raw material for specialty chemicals such as L-alanine and aspartame * This amino acid is also used as an additive to food products for humans and animals. Aspartic acid was prepared by. chemical means but, as a general rule, chemical processes give a racemic mixture of the two forms D and L. 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, in U.S. Patent No. 3,214,345 to Chibata et al., There are described fermentation processes for the production of aspartic acid, processes in which the acid is used. fumaric (as its ammonium salt) as the main source of carbon. The process discussed in this reference involves a batch type process in which a microorganism is inoculated into a nutrient fermentation medium while after an incubation period the aspartic acid is recovered of this fermentation medium.

Mention has also been made of the production of aspartic acid using immobilized cell reactors. For example, in U.S. Patent 35,771,926 to the names of

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Chibata et al., Describes the preparation of an immobilized cell reactor by enclosing cells of draspartate producing microorganisms in a diacrylamide polymer. The aspartic acid is then prepared by bringing the immobilized cells into contact with ammonium fumarate. The reaction can be of the batch or continuous type. In continuous reactors, the immobilized cells are loaded into a column through which a solution of ammonium fumarate is continuously passed.

Immobilized cell reactors have several advantages over batch type fermentation processes. In batch-type processes, recovery of the product frequently involves complex processes which reduce the overall yield and in many cases result in a product which is contaminated with cells, metabolites and other materials present in the medium. nutrient. By using immobilized cells, many of these problems are eliminated; however, significant amounts of cellular impurities may still be present in the final product.

"The methods by which recovery and contamination problems posed by immobilized and continuous-type cell methods are avoided involve the isolation, purification and use of the essential enzyme (s) to carry out the reaction The enzyme involved in the transformation of ammonium fumarate into aspartic acid is aspartase. Attempts have been made to immobilize the aspartase in columns or on solid supports in order to directly transforming ammonium fumarate into aspartic acid, for example, in the introductory part of United States Patent 3,791,926, there are described 4 methods involving the attachment of aspartase to an anion exchange polysaccharide or entrapment of the aspartase in an acrylamide polymer, however, these methods have met with limited success owing to the fact that it is difficult to extract and purify the enzyme aspartase, without expect that there is a significant loss of enzyme activity during the purification, concentration and immobilization of the enzyme * 10 Reactors of the dialysis type have also been proposed in order to immobilize the enzymes. As it appears in this specification, the expression "dialysis" is used in its broad sense and it includes real dialysis (exchange of molé-1 $ cules of solutes between two liquids separated by a membrane), 1 * osmosis ( transport of a solvent through a membrane) and 1 * ultrafiltration (movement of a solvent or a solute caused by a differential pressure through a membrane), In reactors of the dialysis type, a solution the enzyme is effectively retained on one side of a semi-permeable membrane, the substrate intended for the enzymatic reaction is then brought into contact with the other side of this semi-permeable membrane, the semi-permeable membrane is designed to be permeable to the substrate and to the product, however, it must be impermeable towards the enzyme. Consequently, the substrate can migrate towards the face of the membrane which contains the enzyme, where it is transformed into a product, which 30 can migrate back to the subs solution trat from which it can be recovered.

Dialysis type reactors can have different shapes. For example, industrial units generally have the configuration of a filter press comprising numerous plates of * 5 * parallel membranes separated by chambers through which liquids can flow *

In "Biotechnology and Bioengineering", 20, 217—230 (197 ^) 9 Kan, JK and Shuler, M * L * describe the use of a hollow fiber kidney dialysis unit as a dialysis type reactor * When using such a unit, whole microbial cells are immobilized on the envelope side of the kidney dialysis unit. A solution of a substrate is circulated through the tube side of the hollow fibers of the dialyzer. The substrate diffuses through the fibers and up to the cells where reactions take place, while the products formed diffuse to return to the recycling stream. The particular system described involves the immobilization of a strain of fluorinated Pseudomonas. - cens for the enzymatic conversion of L-histidine to urocanic acid *

So far, attempts to use the immobilized aspartase enzyme in dialysis-type reactors for the production of aspartic acid have generally been unsuccessful (see, for example, United States patent * America No. 3 * 791 * 926 mentioned above) · It is believed that the reason for this failure is that known methods for isolating and purifying aspartase have resulted in significant losses of 1 * enzymatic activity *

According to the present invention, there is described a process for preparing an aqueous aspartase solution substantially free of cells, this process consisting in forming an aqueous suspension of microbial cells containing 1 ace per bunch - incubating this suspension aqueous cells in conditions which give rise to the release of aspartase pen- Λ.

"6 for a sufficient period of time for a substantial part of the intracellular aspartase to pass into the extracellular fluid; remove the solids from the suspension of cells to form a clear solution containing aspartase and add, to the aqueous cell suspension or the clarified solution containing aspartase, an aspartase stabilizing salt in sufficient quantity to make this solution hypertonic with respect to the microbial cells. In a preferred embodiment, the aqueous suspension of cells is formed with a hypertonic solution of the aspartase stabilizing salt.

This process is simple and efficient and generally more than 80% of the aspartase activity of the cells is recovered in the cell-free aspartase solution. In addition, it is found that the enzyme keeps its activity for long periods in a solution of this type. These solutions can be used very effectively in 20. dialysis-type reactors described above.

The aspartase enzyme is produced by a wide variety of microorganisms, and the present invention is by no means limited to the use of any one genus, species or strain.

For example, bacteria such as Pseudomonas fluorinated cens, Pseudomonas aeruginosa., Bacillus subtil is, Bacillus megatherium, Proteus vulgaris, Serratia mar-cescens, Escherichia coli and Aerobacter aerogenes have been described as bacteria suitable for the production of bacteria. aspartic acid. A particular organism from which aspartase has been recovered is a mutant of E, coli using glutamate (ATCC No. 31976).

As a source of aspartase, aspartase-producing cells can be grown on a nutrient medium designed for the production of the enzyme and rapid cell growth. Methods for preparing and maintaining cell cultures are well known and are not part of the present invention. ... · 5 As a rule, a nutritious medium containing essential mineral substances is used. and growth factors, as well as assimilable sources of carbon and nitrogen. According to an advantageous characteristic, the medium contains 10 - 1 fumaric acid and it also preferably contains a yeast extract as a source of nitrogen, vitamins and growth factors, the ammonium ion as an additional source of nitrogen, likewise as calcium and magnesium salts as essential mineral substances. Phosphate and carbonate salts of potassium or sodium can be used as buffers.

The inoculated medium is generally subjected to fermentation under growing conditions for a period sufficient to produce about 2 to about 6 g of solids per liter. Normally, fermentation carried out at about 37 ° 0 for about 12-14 hours is sufficient to obtain the desired concentration of cells.

After an appropriate growth period, the cells are advantageously separated from the extracellular portion of the fermentation medium (for example, by centrifugation or by filtration) and washed.

Aspaftase can be released from cells by suspending the cells in an aqueous medium and incubating them under conditions which result in the release of aspartase. The aqueous suspending medium can be water or any aqueous solution having no detrimental effect on the enzyme (eg, ordinary buffers). Preferred aqueous suspending media are hypertonic solutions of aspartase stabilizing salts (as will be described below) and particularly preferred media are hypertonic solutions of soluble salts of fumaric acid or aspartic acid. Ammonium fumarate solutions of a concentration of between about 1 molar and saturation, preferably about 1.4 molar, are particularly preferred. The cells are advantageously suspended in the aqueous medium at a concentration of *. at least about 5 g / liter (based on dry cell weight), preferably from about 10 to about 15 g / liter.

• 15 The cell suspension is generally incubated under conditions which result in the release of aspartase for a period of time sufficient for a large portion of the intracellular aspartase to pass into the fumarate solution * On, 20 generally employs a incubation temperature between about 2 ° C and about 70 ° C, preferably between about 25 ° C and about 50 ° C. A particularly preferred incubation temperature is about 37 ° C. Slower enzyme release rates and lower enzyme activities are associated with lower incubation temperatures, while higher incubation temperatures can have a detrimental effect on the enzyme.

The incubation time depends to some extent on the incubation temperature, but is generally at least about 10 minutes. Preferably, an incubation period of at least about 1 hour is adopted, - better - still, - d-1 about-J.2-to-about .00 hours.

After ~ i4 ± ncubation, - the solids are removed -------- 35 from the cell suspension, for example, by centri— 9 r ί *! 4 fugation or by filtration to form a clarified solution containing 1 * aspartase. Any clarification process which does not result in significant loss of aspartase activity can be adopted. 5 As a general rule, the solution obtained contains approximately 80% of the aspartase activity initially contained in the. cells. These solutions are relatively free of undesirable impurities and have been found to retain practically all of their aspartase activity for more than 40 days at 37 ° C. # To the clarified solution is added a stabilizing salt 1 * aspartase in an amount sufficient to render this hypertonic solution. In a preferred embodiment of the invention, the solution of the stabilizing salt 1 * aspartase is used as the aqueous medium for suspending the cells and, when this method is adopted, it is generally not necessary to * add a stabilizing sel 1 * aspartase to the clarified solution.

The aspartase stabilizing salt has been found to stabilize the enzyme against loss of activity over a period of time and also to stabilize it against thermal degradation. For example, comparisons of aspartase activity in 25 solutions stored at 65 ° C indicate that the enzyme extracted with a buffer virtually loses all activity after 30 minutes, while an enzyme extracted in 1.4M ammonium fumarate retains at least 80% of its stability after storage for two hours.

The preferred salts stabilizing the aspartase are soluble salts of fumaric acid or of aspartic acid. Since fumaric acid constitutes the enzymatic substrate for the production of aspartic acid, using salts of these Γ • · ι '* i - ίο acids, it is generally not necessary to subsequently separate the salt from aspartase, In addition, these salts, in particular, ammonium fumarate, have been found to be particularly effective in extracting and stabilizing the enzyme. Although the fumarate and aspartate salts are preferred, it will be understood that any water-soluble salt having a stabilizing effect on the enzyme can be used. These salts can be represented generally by the formula MX in which M represents a cation such as an alkali or alkaline earth metal, ammonium, a lower alkyl-ammonium compound, a lower alkyl-quaternary ammonium compound and the like, while X represents an anion such as a halide, a nitrate, carbonate, phosphate, sulfate, maleate, fumarate, aspartate and the like. The concentration of the as-9 partase stabilizing salt in the clarified solution is advantageously in the range of from about 0 0.5M at saturation, preferably between about 1M and about 1.4M.

The aspartase solutions prepared by this process are particularly useful in dialysis type reactors, in particular in hollow fiber reactors. The cell-free solution can be easily introduced into the shell side of this reactor by simply applying suction on the tube side of the latter. After the enzyme has been introduced into the reactor, an ammonium fumarate solution can be circulated through the tube side of the reactor. The substrate, namely ammonium fumarate and the product, aspartic acid, pass freely through the walls of the dialysis tubes, and the aspartic acid can be recovered from the circulating solution. .

| · U On the other hand, the enzyme aspartase cannot pass through the walls of the tubes; consequently, it is effectively retained in the reactor. L1 elimination of the dialysable impurities and of the colo-5 res bodies takes place immediately after the start of the reaction and, consequently, they do not contaminate the mass of the product stream.

The reaction conditions can be varied depending on the type of reactor used. As a rule, the substrate is a concentrated solution of ammonium fumarate, for example, of a concentration of about IM at saturation, preferably of about Ι, δΜ. The reaction media are incubated under conditions producing aspartic acid. The reactor is advantageously maintained at a high temperature in order to ensure efficient transformation of the substrate into the product. Temperatures are generally adopted from about 2 ° C to about 70 ° C, preferably from about 25 ° C to 50 ° C and more preferably from about 37 ° C. The enzymatic reaction is exothermic, so that in large reactors or in reactors in which low flow rates are adopted for the substrate, elements can be used to dissipate the excess heat. * 25 The desired flow rate of the solution of the substrate through the reactor depends on the particular reactor adopted, the concentration of the substrate, the configuration of the system (for example, in circulation or in a single pass), the enzymatic charge and the speed at which it is desired to dissipate the heat.

The hollow fiber reactor system can operate in a batch mode with recycling in which the reactor output stream returns to a reservoir. The solution from the substrate is continued to circulate until a substantial portion ϋ fs 12 carrying ammonium fumarate has been transformed into aspartic acid. Among other reaction modes, there are single pass systems in which several reactors can operate in series.

The present invention has been found to provide a simple and effective method for recovering aspartase from aspartase producing cells. The aspartase solution thus obtained and free from.

10 cells can be used in batch or immobilized modes and is particularly suitable for reactors of the hollow fiber dialysis type.

The aspartase solutions free from cel-15 Iules according to the present invention offer several advantages over whole cells in reactors of the dialysis type. In order to obtain a high degree of enzyme activity with whole cells, the cells must generally be in a concentrated form. The concentrated cell media are very dense and viscous, which poses at least two problems which are avoided by the cell-free system according to the present invention. First, impurities and color bodies from the cell slurry migrate through the dialysis membrane to enter the product stream. Rather than appearing for a short period at the initial stage of the reaction as is the case with cell-free solutions, impurities and color bodies tend to separate from whole cells by leaching during extended periods. A second advantage of the cell-free system relates to the diffusion problems posed by whole cell systems. Aspartase is believed to be an intracellular enzyme, so that the reagents and products must be e diffusing not only through the membrane of the reactor, but also through the extracellular fluid and the cellular constituents. In sequence, with these whole cell systems, less efficient transformation rates are observed. For example, with the same amount of enzyme activity, a cell free and hollow fiber reactor system has been found provided a transformation rate approximately 45% higher than that observed with the whole cell system. The invention is illustrated in more detail by the following examples which, however, are in no way limiting,

15 Example I

Several experiments were carried out in which nutrient media containing varying amounts of yeast extract were subjected to produce faspartase * Each 20 'fermentation medium contained, per liter: 24-60 gd * yeast extract, 30 gd * fumaric acid, 2 g potassium dibasic phosphate, 0.5 g sodium carbonate, 2 mM magnesium sulfate and 0.1 mM calcium chloride, while the pH was adjusted to about 7.2 25 with ammonium hydroxide, In each medium, 1 ml of a culture of E, coli (ATCC No. 31976) was inoculated, which was incubated for 12-16 hours at 37 ° C. peptone containing $ 0.5 monosodium glutamate. Each inoculated medium was incubated at 37 ° C for 12-14 hours, after which time the cells were collected. When harvested, the dry cell weight was 2-7 g per liter depending on the amount of yeast extract used.

Table I below gives the production rate ------- 35 of aspartase for each fermentation medium.

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A second group of experiments was carried out in which fermentation media were prepared containing different concentrations of ammonium fumarate and subjected to fermentation as described above. Each of these places contained $% yeast extract and the other ingredients in the same concentrations as those listed above. Table II shows the results of these experiments. The cell culture prepared from the fermentation medium containing 2.4% w / v yeast extract and 3% ammonium fumarate was centrifuged, decanted and the liquid removed. The cells were then washed with water, subjected to centrifugation again, decanted and resuspended at 1/20 of the original volume in tris buffer, HCl 6.05 d, pH 8.5 · Next, the slurry was diluted with a 1.8 m solution of ammonium fumarate, pH 8.5 d to form a suspension of cells 20 having 1/5 of the initial volume of fermentation. The suspension obtained was separated into six portions which were incubated at 37 ° C for the periods indicated in Table III, then subjected to centrifugation, The supernatants were decanted and removed the solids. The supernatants were subjected to an analysis in order to determine the activity of aspartase. The results obtained are shown in Table III,

Example II

A cell-free aspartase solution formed from 12.5 g (dry weight) of cells prepared according to the method of Example I (2.4% yeast extract, 3% fumarate) was introduced. ammonium) into the envelope side of the Cordis-Dow artificial kidney (CDAK 2.5D) by applying suction to the tube side of the artificial kidney. After introducing the enzyme into the reactor, the envelope side was sealed and a 1.8m solution of ammonium fumarate, pH $ 8.5, was passed through the tube side of the reactor at a rate of approximately 11- 15 liters / hour. A recycling mode was adopted in which the solution of the substrate was recycled to the reactor from a tank. The reactor was maintained at a temperature of 37 ° C. The contents of the tank were analyzed periodically to determine the residual fumarate. The results of the analyzes are shown in Table IV. The aspartic acid was precipitated to a pH of 2.8 by addition of H 2 SO 4, filtered, washed, and then dried. From an average quantity of 2.1 kg

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fumaric acid, 2-2.1 kg of aspartic acid was recovered (theoretical yield! 83 at & 6%). The aspartic acid thus produced was characterized by elementary analyzes of determination of the contents • 20 in C, H, N and 0 which agreed at a rate of 97—99% with the theory * One measured the specific rotation 2Ç ° of the product and it was as follows: [a] ^ = +26 (C = 10 in 2N HCl). The product was analyzed by an enzyme assay associated with a diagnosis and the result was 9% consistent with an authentic aspartic acid standard.

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Table I

Effect of the concentration of the yeast extract in the fermentation medium (containing 3% ammonium fu ~ marate) on the volumetric activity of as-5 partase of the broth.

Aspartase yeast extract (% w / v) millimoles h-ml _________________ _culture__ broth 1.0 0.22 10 1.8 0.64 2.7 0.90 3.0 1.20 4.0 1, 50 5.0 1.76 15 6.0 2.14 '7.0 2.26 8.0, 2.24

Table II

Effect of the concentration of ammonium fumarate in the fermentation medium (containing 8% yeast extract) on the volumetric activity of aspartase in the broth.

Fumaric acid Aspartase (% w / v) (millimoles / h-ml) 25 0 1.00 2.0 2.50 4.0 2.52 6.0 0.48, ψ * 17

Table III

Extraction of aspartase from cells with 1.4M # ammonium fumarate

Duration (hours ·) Extracted activity ($) 5 0 - 4 70%.

7 73% 14 79% 24 80% 10 48 84%

Table IV

Hollow fiber reactor (CDAK 2.5D) loaded with cell-free enzyme (extracted from about 12.5 g of cells, dry weight) $ used. In a batch recycling mode with a stirred tank of 10 liters of Ammonium fumarate 1, MS, Samples were taken from the tank and subjected to * analysis to determine residual fumarate * Duration (hours) Residual fumarate (m) 20 0.0 1.81 0 , 25 1.63 0.5 1.52 1.0 1.30 1.5 1.12 25 2.0 0.98 2.5 0.82 3.0 0.70 3.5 0.58 4.0 0.48 30 5.0 0, 32 6.0 0.21 7.0 0.14

Claims (15)

1. A process for the preparation of a substantially cell-free aqueous aspartase solution, this process comprising forming an aqueous suspension of microbial cells containing aspartase; incubate this aqueous suspension of cells under conditions giving rise to the release of aspartase for a period of time sufficient for a substantial part of the intracellular aspartase to pass into the extracellular fluid 3 to remove the solids from the suspension cells to form a clarified solution containing aspartase and add, to the aqueous cell suspension or to the clarified solution containing 1 aspartase, an aspartase stabilizing salt in an amount sufficient to render this solution hypertonic —To microbial cells *. 2 * Process according to claim 1, characterized in that * this aqueous suspension is prepared of cells by putting the microbial cells in suspension in a hypertonic solution of this salt stabilizing aspartase. r 3 · Process according to 1 * any one of claims 1 and 2, characterized in that the salt 25 stabilizing * aspartase is a water-soluble salt of 1 * fumaric acid or 1 * aspartic acid * 4 · A method according to any one of claims 1 and 2, characterized in that the stabilizing salt * aspartase is ammonium fumarate or 30 * ammonium aspartate. 5 · Method according to claim 2, characterized in that the stabilizing salt * aspartase is ammonium fumarate * _______ 6 * Process-following — claim ^, ca ---------- 35 acted as that the concentration of ammonium fumarate * in ammonium fumarate hypertonic solution is between about 0.5M and the saturation * 7 ·· A process according to claim 5 * ca-5 characterized in that the concentration of fumarate of ammonium in the hypertonic solution of ammonium fumarate is between approximately 1 and approximately 1.4M '8 * Process according to Claim 6, characterized in that the conditions for recovery of aspartase are as follows: temperature of incubation -tion: about 2 ° C to about 70 ° G; incubation time of cell suspension: at least about 10 minutes # 9 · A method according to claim 69 ca-15 characterized in that the recovery conditions-. aspartase are as follows: incubation temperature î approximately 25 ° C to approximately 50 ° C · 9 incubation time of cell suspension: approximately 12 to approximately 60 hours * 20 10 # Process according to claim 9j characterized in that the incubation temperature is around 37 ° C * * 11 * A method according to claim 93 characterized in that the 25-born bacterial cells are suspended in a hypertonic solution of ammonium fumarate in an amount sufficient to obtain a cell concentration of at least about 5 g / liter (based on dry cell weight), the conditions for recovery of aspartase 30 also encompassing a pH of about 7 to. about 9 · 12 * A method according to claim 11, characterized in that the bacterial cells are suspended in a hypertonic solution of ammonium fumarate in an amount sufficient to obtain a cell concentration of about 10 to t * »#. about 15 g / liter (based on dry cell weight), while the pH is between about 8 and about 9 · 13 · Method according to any one of claims 5, 6, 7, 9 and 12, characterized in that the bacterial cells are chosen from the group comprising strains producing aspartase from Pseudomonas fluoréscens, Pseudomonas aeruginosa, Bacillus subtilis, Bacillus megaterium3 Proteus vul— 10 garis »Serratia marcescensa Escherichia coli and Aero-bacter aerogenes · 14 · Method according to claim 13j characterized in that the bacterial cells are cells of E. coli 15 15 · Method according to claim 14> characterized in that the bacterial cells are cells of the strain * E · coli ATCC 31976 · 16. A substantially cell-free aqueous aspartase solution prepared by the method of claim 1 * 17 · Aqueous solution of aspartase practically free of cells, prepared by the process * according to claim 11 " 18. A practically cell-free aqueous solution of aspartase, prepared by the process according to claim 13. 19 · Process for the production of aspartic acid in a reactor comprising a first chamber and a second chamber, as well as a semi-permeable membrane separating the first chamber from the second, t this semi-permeable membrane being permeable to the as- part ate and fumarate, but impermeable to 1 * aspartase, characterized in that it consists in depositing - the queasy solution ~ dJ aspartase practically cell-free -------- 35 according to claim 16 or 17 in this first r φ. c * chamber in fluid communication with the semi-permeable membrane and place a solution comprising ammonium fumarate in the second chamber in fluid communication with the semi-permeable membrane 5 5 incubate these solutions under conditions producing aspartic acid and recover the aspartic acid from the solution in the second chamber. 20. The method of claim 19 * characterized in that the reactor comprises a sealed envelope which includes elements for filling this envelope with liquid, as well as several hollow fibers forming the semi-permeable membrane and passing through this envelope, so that the lumens of these fibers are sealed from the interior of the envelope, each fiber having an inlet end opening into an inlet manifold having a tube inlet orifice, as well as an outlet end opening into an outlet manifold having a tube outlet orifice, the aqueous solution of aspartase practically free of cells being deposited in this envelope, while the ammonium fumarate passes through the hollow fibers of the semipermeable membrane and that the aspartic acid is recovered from the ammonium fumarate solution. 21. The method of claim 20, characterized in that the concentration of ammonium fumarate in the ammonium fumarate solution is between about 1M and saturation. 22. Method according to claim 21, characterized in that a practically saturated solution of ammonium fumarate is used. 23 · Process according to claim 20, characterized in that the conditions for producing aspartic acid include an incubation temperature of between approximately 2 ° C and approximately 70 ° C1 24 · Process according to claim 20, · Characterized in that the conditions for producing aspartic acid include an incubation temperature between about 25 ° C and about 50 ° C1 25 · Process according to claim 24, characterized in that the reactor is put into service * in a discontinuous recycling mode in which the ammonium fumarate solution is recycled from a tank through the reactor until a large quantity of the ammonium fumarate is transformed into aspartic acid1, t