CS213127B1 - Process for the enzymatic production of L-aspartic acid - Google Patents

Abstract

The invention relates to a method for the enzymatic production of L-aspartic acid from ammonium fumarate in a discontinuous, semi-continuous or continuous manner using specially developed cell catalysts based on cross-linked, permeabilized, or activated biomass, or based on chemically bonded microbial producers of the aspartase enzyme.

Description

The invention relates to a method for the enzymatic production of L-aspartic acid, an important amino acid used increasingly in the pharmaceutical and food industries; in this context, there is therefore increasing interest in its economic large-scale production.

Aspartic acid can in principle be produced by chemical or biological means. Due to the fact that only the L-stereoisomer of aspartic acid is usable for the desired purposes, the biological method is gaining industrial importance, which allows for the selective production of the desired isomer, thus eliminating the complex and expensive separation of the D,L-form, which occurs by chemical synthesis.

There are several biological routes for the preparation of L-aspartic acid: a/fermentation of suitable microorganisms secreting the acid into a culture medium, from which it is then isolated, b/single-shot submerged cultivation of cells of microorganisms with aspartase activity is used to grow them, which are either suspended with the culture medium or after separation from the culture medium in a solution of the substrate, ammonium fumarate, which is converted to L-aspartic acid after a certain period of time under stirring at a suitable temperature. The latter is isolated after separation of the cells.

c/use of isolated aspartase enzyme bound to a suitable carrier, d/use of bound cells with aspartase activity.

The methods mentioned under a/ and b/ require that new cells be grown in large-capacity fermentation tanks for each production batch, which is a matter that is very demanding in terms of raw materials and energy, not to mention that the L-aspartic acid produced in this way is not very pure, since it contains autolysis products of native cells of production microorganisms, arising during the conversion, and various impurities originating from the remains of the fermented nutrient medium.

In an effort to economize the production of L-aspartic acid, a number of procedures have been developed in which the enzyme and cells with aspartase activity are bound either to a suitable carrier or to each other, as mentioned under c/ and d/. These procedures allow multiple continuous use of the enzyme or cells and the isolated L-aspartic acid is very pure /P. Sveo et al., Chemické listy 74, 82, 1980/. It is obvious that the use of bound cells has a number of advantages over the bound enzyme, especially of an economic nature, reflected in lower production costs for the preparation of the bound biocatalyst and in higher operational stability. So far, the most successful results have been achieved by the method of capturing native nutrient cells in a gel matrix, either by direct incorporation into polymers soluble in water or organic solvents or into the network of the resulting polymer, especially in the radical copolymerization of acrylamide with methylene-bis-acrylamide /USA/ patent no. 3 791 926; SU patent no. 659 611 /, when up to 70%> of the activity yield of microbial cells bound in the aforementioned way was achieved, especially E. coli. However, the above methods of binding native cells with aspartase activity also have their disadvantages, in particular the fact that with multiple or continuous use, native cells in the gel matrix domain autolyze, which is manifested by a temporary increase in their activity and then a gradual decrease in the activity of the catalyst as a result of leaching from the gel matrix, which results in a shorter operational half-life of the material / T. Tosa se sp., Appl. Microbiol. 27, 886, 1974 /.

The cited work /Chemické listy 74,82,1980/ also presents other methods of binding cells with aspartase activity, for example the use of polyfunctional crosslinking agents, especially 2,4-toluene diisocyanate and glutaraldehyde, with the aim of covalently crosslinking the intracellular content of individual microbial cells with aspartase activity and thus achieving fixation of this enzyme in cells with the possibility of their repeated use for the preparation of L-aspartic acid. In the case of application of 2,4-toluene diisocyanate, the activity of crosslinked cells was zero, in the case of glutaraldehyde, the activity of crosslinked cells was in the optimal case 34% of the initial activity. For the reasons indicated, these methods of binding cells did not reach the stage of industrial use.

It was found that under selective reaction conditions of crosslinking the intracellular content of cells of a production microorganism with aspartase activity using dialdehydes, especially glutaraldehyde, a product can be obtained which, after subsequent permeabilization of the crosslinked cells by physical or physicochemical influences, or by a combination, exhibits up to 70% activity.

213 127 aspartase in cross-linked and permeabilized cells. By selective activation of the aspartase enzyme during further treatment of these cells, up to 100% of the original activity can be achieved. The cells modified in this way represent a catalyst that is usable for the enzymatic production of L-aspartic acid and that is so stable that it allows long-term discontinuous, semi-continuous or continuous operation at a high initial substrate concentration, i.e. ammonium fumarate, which is at the limit of its solubility (1.5 mol) liter/. The isolated L-aspartic acid is very pure and usable for common purposes, especially for the preparation of salts serving as drugs. The yield of pure acid is around 95% of theory. The efficiency of enzyme conversion in the range of initial ammonium fumarate concentrations of 0.5 to 1.5 mol/liter is 100%, provided that optimal conditions are provided, i.e. the amount of catalyst, the initial substrate concentration and the reaction time.

Based on the above findings, a method for enzymatic production of L-aspartic acid according to the invention was developed, the essence of which consists in that an aqueous solution of ammonium fumarate in a concentration of 0.05 to 1.5 mol/liter, preferably 1.35 mol/liter, with a pH value of 6.5 to 9.5, preferably 8.5, with the addition of magnesium cations and optionally with the addition of a nonionic surfactant, is brought into contact at a temperature of 10 to 50 °C, preferably 35 to 38 °C, discontinuously, semi-continuously or continuously with a catalyst based on cross-linked and permeabilized, optionally activated biomass of a microbial aspartase producer or on the basis of chemically bonded cells of this producer, after which L-aspartic acid is isolated from the separated liquid portion after the conversion is completed.

In addition to the advantageous possibility of repeated use of the catalyst based on bound cells with aspartase activity for the enzymatic production of L-aspartic acid, the advantage of the method according to the invention is its speed, since it is not necessary to ensure complete separation of the cell mass into a paste form and the catalyst does not need to be washed. The residual volume of the reaction mixture after biotransformation, which remains in the thickened sediment, is immediately returned to the process at the start of the second and subsequent enzyme transformation cycles, the one-time procedure can thus be changed to a semi-continuous procedure, where after 1 to 2 hours the cycle can be repeated. The supernatant obtained after separation of the catalyst is immediately processed by heating to 90 -°C and adjusting the pH with 60% sulfuric acid to a value of 2.8, after about 2 hours of standing of the solution at a temperature of 10 to 15 °C, the freely crystallized L-aspartic acid is filtered off, washed with cold water and dried, or further processed as needed. Ammonium sulfate can be advantageously regenerated from mother liquors, which, as is known, has many uses, e.g. as a fertilizer or component of fertilizers or as a component of nutrient media for various fermentation technologies.

Another advantage of the method according to the invention is the possibility of continuous enzymatic production of L-aspartic acid, in principle, for example, by suspending the catalyst in the substrate solution in a reaction vessel, the bottom of which is separated by a semipermeable material, preferably a semipermeable membrane of suitable porosity, for example from cellulose acetate with an average pore size distribution of 0.5 to 1.0 microns, after which, after achieving a quantitative concentration of the substrate to the product with continuous stirring or liquid flow, the inflow of fresh substrate is regulated so that the reaction liquid taken from under the membrane is quantitatively reacted. To prevent the pores of the semipermeable membrane from being clogged with the sedimenting catalyst, this can be adjusted, for example, by placing the reactor horizontally with side suction under intensive stirring of the reaction mixture or by placing the stirring blade just above the membrane surface. Of course, other structural adjustments are also possible.

The catalyst based on cross-linked and permeabilized cells is stable for at least 6 months, especially when stored in the form of an aqueous suspension in the presence of 1 M ammonium fumarate at a temperature of 1 to 15 °C.

For the continuous enzymatic production of L-aspartic acid, it is particularly advantageous to use a catalyst based on chemically bound cells with aspartase activity (aspartate-ammonia-lyase activity), prepared in a known manner (Czech Republic, Author's Certificate No. PV 5145-79). This catalyst has advantageous sedimentation and hydrodynamic properties and is usable in a column in the form of a fixed or fluidized bed through which an aqueous solution of ammonium fumarate flows, while the degree of conversion can be regulated by the amount of catalyst charge and the flow rate of the substrate. For continuous production, a continuous stirred reactor or a cascade of continuous stirred reactors connected side by side can also be used.

For the method of enzymatic production of L-aspartic acid according to the invention, various microbial species and strains synthesizing the enzyme aspartase /aspartate ammonia lyase/, for example various strains from the genera Alcaligenes, Escherichia and Corynebacterium, or high-production mutants of various microorganisms, can be used.

For the production of microbial cells forming the starting material for a catalyst with aspartase activity, hydrolysates of plant or animal matter, especially hydrolysates of peanut or soybean flour, or pancreatic residues, which can replace expensive raw materials such as peptone, beef extract, broth, etc., are preferred as the main components of nutrient media. Plant hydrolysates stimulate aspartase biosynthesis during submerged cultivation of production microorganisms to the same extent as expensive raw materials of animal origin.

The following examples of embodiments of the method according to the invention only illustrate, but are not in any way limiting, the materials, equipment, process or conditions set forth therein.

Example 1 a/ The production strain Alcaligenes metalcaligenes was inoculated from fresh slant agar into ten 500 ml flasks containing 100 ml of sterile nutrient medium of the following composition/weight/volume/ » 4% technical peptone, 0.1% yeast extract, 0.5% primary potassium phosphate, 0.05% crystalline magnesium sulfate, pH 7.2. The vegetative inoculum was cultivated on a rotary shaker for 16 hours at a temperature of 28 °C. Then the inoculum was sterilely decanted from the flasks and prepared for inoculation of the fermentation tank.

b/ The production strain Corynebacterium sp. was inoculated from fresh slant agar into ten 500 ml boiling flasks containing 100 ml of sterile nutrient medium of the same composition as above, the conditions and the cultivation procedure were also the same. The obtained inoculum was also sterilely decanted and prepared for inoculation of the fermentation tank.

o/ The production strain of Escherichia coli was inoculated, as above, into ten 500 ml boiling flasks containing 100 ml of sterile nutrient medium of the same composition as above. The cultivation conditions were the same as before, with the difference that the cultivation temperature was 37 °C. The obtained inoculum was prepared for inoculation of the fermentation tank after decantation.

The obtained inocula were inoculated into 150 liters of sterile nutrient medium in 2001-liter fermentation tanks, which had the following composition /weight/volume/ with 4% technical peptone, 0.1% yeast extract, 0.5% primary potassium phosphate, 0.05% crystalline magnesium sulfate. The pH value of the medium was adjusted to 7.1 with ammonia water before sterilization. A sterile solution of ammonium salt of glutamic acid with an initial concentration of 0.5% weight/volume was added to the tank inoculated with the E.coli inocula.

Cultivation of production microorganisms was carried out with stirring at 300 rpm and aeration at 40 liters of air/min, at a temperature of 28 °C for 9 hours. Cultivation of E. coli was carried out at a temperature of 37 °C. After completion of cultivation, native cells were centrifuged, the obtained pastes were weighed, and their dry matter and enzyme activity were determined.

Production strain Alcaligenes metalcaligenes Corynebacterium sp. Escherichia coli Wet mass of nat. cells / g/ 2680 2240 2400 Specific activity /+/ nat.cells /j /g wet mass/ 496 232 360

213 127

Production strain Alcaligenes rne t al oal ige no s Corynebacterium sp. Escherichia coli Specific activity A/ nat .cells 2000 1160 1800 /j/g dry matter /

/+/ The unit of enzyme activity of aspartase is the amount of enzyme that, at a temperature of 37°C, pH 8.5 and an initial substrate concentration of 1 M, produces 1 µmol of L-aspartic acid per minute from ammonium fumarate under zero-order reaction kinetics. The concentration of L-aspartic acid was determined by thin-layer chromatography on silica gel, quantitatively by densitometric evaluation and manometrically, using a technical preparation of L-aspartic acid decarboxylase obtained from Mycobacterium sp. cells.

The obtained wet cell paste of each of the microbial aspartase producers used was mixed separately in 3.3 liters of water with the addition of 0.5 mg/ml of crystalline magnesium sulfate, so that homogeneous suspensions with a cell content of 0.3 kg/liter were formed. The pH of the suspensions was adjusted to 7.0 with 20% sodium hydroxide solution while stirring, and a 25% aqueous solution of glutaraldehyde was added so that its final concentration in the suspensions was 0.5% vol. The reaction mixtures were continuously stirred for 60 minutes at room temperature, then they were diluted by adding drinking water to a volume of about 35 liters, mixed, and the cross-linked cells were centrifuged separately into the form of wet pastes.

The obtained pastes were separately suspended in 3 to 4 liters of drinking water to form homogeneous suspensions, to which was added crystal magnesium sulfate to a concentration of 1 mg/ml, ammonium sulfate to a concentration of 0.5 ml/liter and nonionic surfactant to a mass concentration of 0.1%# The pH of the suspensions was adjusted with 25% ammonia water to a value of 8.5 while stirring and the suspensions were heated to 30 °C in a water bath and stirred at this temperature for 3 hours. After this time, the suspensions were diluted with a tenfold volume of drinking water, mixed and the cross-linked, permeabilized and activated cells were centrifuged to form wet cell pastes.

100 g of the obtained cell paste of each microbial producer was suspended in a liter of 1 M aqueous solution of ammonium fumarate with the addition of magnesium ions, containing 0.1% by volume of non-ionic surfactant, with a pH value of 8.5 (ammonia water). The suspensions were stirred at a temperature of 37 °C until 100% conversion of the substrate to the product (theory 133.1 mg/ml L-aspartic acid) occurred in 2 hours for A. metalcaligenes, 2.5 hours for E. coli and 3 hours for Corynebacterium sp. After these reaction times, the individual reaction mixtures were centrifuged, the sediments were suspended in the same volume of substrate as in the previous cycle (1 liter) and under the same reaction conditions, the conversions were repeated a total of 13 times with the same catalyst charge, with the average conversion efficiency being 99.4% of theory. L-aspartic acid was isolated from the supernatants by heating to . 90 °C, adjusting the pH with 60% sulfuric acid to 2.8 and cooling. The total yield of L-aspartic acid was 1643 g, i.e. 95% of theory. In a control run of repeated enzymatic conversion of the substrate to the product using native cells of the same aspartase producers under the same reaction conditions, 75% conversion was achieved in the second cycle and less than 50% in the third cycle, with the cells in the third cycle already being strongly autolyzed.

Example 2

As in example 1, an inoculum of the A. metalcaligenes strain was prepared, intended for inoculation of soil in a fermenter with a volume of 1 m , the fermenter was filled with 800 liters of soil of this composition and 240 liters of acid hydrolysate of peanut flour with the addition of 0.5% by weight of primary potassium phosphate, diluted with drinking water and the pH adjusted to 7.2 with ammonia water.

After sterilization / 120 °C, 30 minutes / the nutrient medium was cooled to 28 °C, a solution of 400 g of crystalline magnesium sulfate in 2 liters of water was added aseptically and the medium was inoculated with the prepared vegetative inoculum of A. metalcaligenes. Cultivation was carried out for 16 hours at a temperature of 20 °C,

213 127 with stirring at 300 rpm and aeration at 300 liters/min. After the end of the cultivation, the cells were centrifuged into a wet paste. 10 kg of wet paste of native cells/ 2.5 kg of dry matter were obtained /with specific activity of 400 U/min/g of wet mass, respectively 1800 U/min/g of dry matter of cells.’ kg of wet cell paste was homogeneously suspended in 33 liters of drinking water, the pH was adjusted to 7.0 with 20% sodium hydroxide solution, a 25% aqueous solution of glutaraldehyde was added with stirring to a concentration of 0.5% /weight/volume/. Crosslinking of the cells took 60 minutes at room temperature with constant stirring. Then the suspension was diluted with 350 liters of drinking water and the cells were isolated by centrifugation. 9.6 kg of wet mass of cross-linked cells with a specific activity of 136 U/min/g of wet mass were obtained.

The entire amount of cross-linked cells was suspended in a 1.35 M aqueous solution of ammonium fumarate to a total volume of 30 liters, the pH was adjusted to 8.5, the magnesium sulfate content to 1 mg/ml and a non-ionic surfactant was added in an amount of 0.1%/ weight/volume/. The cross-linked cell suspension was permeabilized and activated for 48 hours at a temperature of 30 °C. The cells were then centrifuged to form a wet paste. 7.1 kg of wet cell paste of cross-linked, permeabilized and activated cells with a specific activity of 350 U/min/g bulk mass was obtained.

L-aspartic acid was isolated from the supernatant in a yield of 85% of theory. Since the product was not pure, it was recrystallized and 79.6% of theory of pure L-aspartic acid was obtained.

The obtained cell paste was further used for semi-continuous production of L-aspartic acid.

95 liters of 1.35 M aqueous fumarate solution with pH 8.5, containing 1 mg/ml magnesium sulfate, were introduced into a 100-liter reactor. The substrate solution was heated to 37 °C with stirring and 5 kg of wet mass of the cell catalyst was added. After 3 hours of reaction with stirring, ammonium fumarate was quantitatively converted to ammonium aspartate (theoretical yield 179.7 mg/ml L-aspartic acid). After the first completed conversion, the catalyst was separated by centrifugation and reused for the enzymatic transformation of ammonium fumarate, a total of 20 times, with 0.5 kg of catalyst paste being added after each fifth cycle to compensate for handling losses. L-aspartic acid was isolated from the supernatant by the procedure according to Example 1 with a total yield of 90% of theory and an active substance content of 99.7% by weight.

Example 3

290 g of wet mass of chemically bound cells with aspartase activity prepared according to Czechoslovak author's certificate No. /PV 5145-79/, was filled into a glass column of size x 20 cm, equipped with a tempered jacket. The conversion of the substrate to the product was carried out continuously. A 1 M aqueous solution of ammonium fumarate with pH 8»5, containing 1 mg/ml magnesium sulfate, flowed through the column at a rate of 150 ml/h. The liquid flowing into the column with a fixed catalyst bed/flow from top to bottom/and the column itself were tempered to 37 °C. After flow through the column, quantitative conversion of ammonium fumarate to ammonium aspartate/133.1 mg/ml L-aspartic acid/was achieved under the specified conditions, and the yield of isolated L-aspartic acid was 88% of theory, with an active substance content of 99.8% by weight. During 110 hours /5 days/ of continuous operation/ 16.5 liters of substrate flowed through the column, which was converted to product with 100% efficiency/ no decrease in the specific activity of the catalyst was observed.

Claims (1)

SUBJECT OF THE INVENTION Process for the enzymatic production of L-aspartic acid, characterized in that an aqueous solution of ammonium fumarate having a concentration of 0.05 to 1.5 mol / liter, preferably 1.35 mol / liter, of pH 6.5 to 9.5, preferably 8.5, with the addition of magnesium cations and optionally with the addition of a non-ionic surfactant, contacted at a temperature of 10 to 50 ° C, preferably 35 to 38 ° C, discontinuously, semi-continuously or continuously with a catalyst based on a cross-linked and permaabilized or activated bipmas of the microbial aspartase producer or on the basis of the chemically coupled cells of this producer, after which L-aspartic acid is isolated from the separated liquid fraction after the conversion.