FR2490241A1 - PROCESS FOR THE PREPARATION OF L-ASPARTIC ACID, HYDROPHILIC POLYETHER POLYURETHANE FOAM FOR USE AND METHOD FOR THE PREPARATION THEREOF - Google Patents

Abstract

The invention relates to a hydrophilic polyether-polyurethane foam in which at least 50 mol% of the alkylene oxide units in the polyether sections of the polyurethanes consist of ethylene oxide. The foam contains immobilised aspartase-producing microorganisms. It also embraces the preparation of this foam and the use thereof for preparing L-aspartic acid from substrates containing fumarate ions.

Description

It is known that aspartase has the ability to convert ammonium fumarate to L-aspartic acid. Various methods of producing L-aspartic acid by the enzymatic reaction of aspartase with ammonium fumarate are known. For example L-aspartic acid can be prepared by culturing a microorganism producing aspartase in a nutrient medium containing fumaric acid or the fumarate ion. We can for example refer to patents
US No. 2,927,050; 2,971,899; 3,198,712; 3,214,345; 3,310,475; 3,999,586; 4,013,508; 4,048,018, to British Patent No. 880,234 and to Canadian Patent No. 697,310.
US No. 3,214,345 are used E. coli cells to produce L-aspartic acid from ammonium fumarate. Alternatively, L-aspartic acid can be prepared either by reacting whole cells containing aspartase activity with ammonium fumarate or by extracting the enzyme and heating with ammonium fumarate. However, these methods are disadvantageous because the resulting acid is contaminated with either the enzyme, microbial cells, food sources and the like. Therefore, in order to recover high purity L-aspartic acid, additional steps to remove the enzyme and other contaminants are required. Frequently, these methods destroy the enzyme and / or the microorganism, and these sources can therefore be used only once.

 To overcome the above drawbacks, it has been suggested to immobilize the aspartase by combining it with an adsorbent anion-exchange polysaccharide. Reference may be made to the publication of Japanese patent No. 6,870 / 1970. It is also known to encapsulate microorganisms in polymeric vehicles. Reference may be made to the publication of Japanese patent No. 17,587 / 1970 where polyacrylates are used as polymeric vehicles. Also refer to US Pat. Nos. 3,649,457, 3,791,926 and 3,898,128. The use of carroghenine as a polymeric vehicle is described by Sato in Biochemica and Biophysica Acta, 570, 179-186 (1979). In the Russian patent publications SU 423,976 and SU 451,483, the immobilization of E cells is described.

Coli in polyacrylamide cells and the production of L-aspartic acid from ammonium fumerate.

 Encapsulation of microorganisms in hydrophilic polyurethane foams has also been suggested.

Reference may be made to US Patent No. 3,905,923 (Klug) and to US Patent Application 362,488 (filed May 21, 1973) from -Louis
L. Wood et al., Entitled "Preparation and use of enzymes bound to polyurethane". The binding of enzymes to polyurethane is also described in US Patent No. 3,672,955 (Stanlev), in particular in column 5, lines 27-35. In the patent
US No. 4,000,040 describes the preparation of L-aspartic acid from substrates other than fumaric acid such as acetic acid, ethyl alcohol and carbohydrates. Hydrophilic polymers have also been used as vehicles for chemicals, such as bacteriostats and fungicides. Refer to U.S. Patent No. 3,975,350 (Hudgin). It is also known to immobilize the bacterial cells in hydrophobic polyurethanes. eg see example 4 of British Patent No. 953,414. The immobilization of a large number of enzymes in hydrophilic polyurethanes is also described in US patent application No. 849,999 (Hartdegen and others filed on November 9, 1977 and entitled "Immobilized Biological Material".

We can cite as other references of interest,
Sonomoto et al. (Agric. Biol. Chem. 44 (5), 1119-1126, 1980) where steroid conversions are accomplished using bacterial cells trapped in polyurethanes prepared from urethane prepolymers. Fukui et al. (Biochemistry, 1980, 62, 381-386) describe the use of immobilized enzymes in photo-crosslinkable prepolymers and urethane prepolymers to prepare acidic L-amincr. The immobilization of bacterial cells using urethane prepolymers is also described. At a recent "Gordon Research Conference" (11-15 August 1980), the immobilization of whole cells (ie E. coli genetically produced containing large amounts of penicillin amidase) using urethane prepolymers has been discussed. Biotransformation systems have also been mentioned as "undervaluation" for the preparation of L-sterine and L-tryptophan.

 The present invention relates to a hydrophilic polyether polyurethane foam in which at least 50 mole of the alkylene oxide units in the polyether segments of the polyurethane are ethylene oxide, a microorganism producing aspartase being immobilized there. The present invention also relates to a method of preparing the foam as well as a method of using the foam to produce aspartic acid. Preferably, the polyether segments of the foam contain at least 90 mole% of ethylene oxide units. Depending on the amount of the crosslinking agent used, the foam can be either rigid or flexible. Based on the dry weight of the microorganism used, the weight ratio of the polyurethane polymer to the microorganisms in the foam is of the order of 1:10 to 10: 1, and preferably of the order of 2 : 1 to 4: 1. Before mixing the culture with the prepolymer, the dry weight of the microorganism in the culture can be determined by evaporation of the culture to dryness at an appropriate temperature, such as 500C. Then, when mixing a similar culture with the prepolymer, it was found that 50 to 90% of the microorganisms could be immobilized in the foam. The term "immobilization" indicates that the microorganisms are retained in the foam rather than being leached therefrom upon contact with water or an aqueous solution of a substrate. It is believed that the microorganisms are encapsulated in the foam to accomplish immobilization. It is also likely that during the foaming process, a bond occurs between the isocyanate groups of the prepolymer and the groups on the surface of the microorganisms, i.e. the amino groups.

 Foams are prepared by mixing a culture of microorganisms producing aspartase directly with a urethane prepolymer in the presence of enough water to promote foaming. Traditionally, water is brought into the culture, in fact suitable cultures generally contain around 10 to 90% by weight of water, the water content of the particular culture being determined by evaporation of a sample of dry culture as described above. Thanks to the presence of water, the prepolymer will undergo foaming and simultaneously the microorganisms will be immobilized in the foam. To optimize immobilization, the pu of the aqueous culture is between approximately 4 and approximately 11 and preferably it is greater than 7. During immobilization, the prepolymer / water weight ratio is generally from 2: 1 to 1: 2 and preferably from 3: 2 to 2: 3, this water being supplied by the culture or by the combination of the culture and the water added before or during mixing. During mixing, the culture must be sufficiently dispersed in the water of the prepolymer so that no lumps or clumps visible to the eye are present. This mixture is easily obtained after contact of the culture with water according to the description above. Following mixing, the foaming reaction is generally completed in 5-10 minutes and the foam is cured to its rigid or flexible final shape in an additional 5-10 minutes. However, with large masses of foam, it is it is conceivable that the times for foaming and curing can be considerably extended.

Aspartase producing microorganisms are used in the context of the present invention. As an example of microorganisms producing aspartase, there may be mentioned appropriate strains of the following where the production capacity of aspartase can easily be determined by consulting a catalog of an agency such as ATCC.
Pseudomonas fluoresceus
Serratia marcescens
Bacterium succincum
Bacillus megaterium
Bacillus subtilisa
Bacillus natto
Micrococcus sp.

 Escherrichia coli.

 Aerobacter aerogenes.

 All the above microorganisms are publicly available at the culture sites indicated. However, it will be noted that the present invention is not limited to the use of these specific microorganisms but within its framework are incorporated all the microorganisms producing d 'aspartase.

 L-aspartic acid can be prepared by contacting the foam with ammonium fumarate or a mixture of fumaric acid or its salt and an inorganic ammonium salt. Mention may be made, as suitable examples of the fumaric acid salt, of its alkali metal salts such as sodium fumarate and potassium fumarate. Ammonium chloride, ammonium sulfate and ammonium phosphate are preferred as the inorganic ammonium salts. When a mixture of fumaric acid or its salt and an inorganic ammonium salt is. used in the enzymatic reaction, the preferred proportions of the inorganic ammonium salt in the mixture are of the order of 1 to 2 moles for one mole of fumaric acid or of the salt of fumaric acid. divalent should be added to the enzyme reaction solution (i.e. the substrate solution) to improve the enzyme activity and the stability of the immobilized microorganism, although this improvement in activity has been found to be was frequently unnecessary or for short periods of time. Mention may be made, as suitable examples, of divalent metal ions which can be used, calcium, magnesium, manganous and strontium ions.

If used, the concentration of the ion of a divalent metal in the substrate solution is from 0: 1 to 10 millimoles / liter.

 The concentration of the substrate used is not critical, in fact, ammonium fumarate or a mixture of fumaric acid or its salt and an inorganic ammonium salt is dissolved in water at any concentration. Then, the pH is adjusted to 8 to 10. The foam of the immobilized microorganism is brought into contact with the substrate, and the mixture is incubated at a temperature of 15 to 600C while stirring, until the reaction is finished. When the reaction is complete, the foam is separated and can be stored under refrigeration for subsequent use. L-aspartic acid is recovered from the substrate by traditional methods.

Alternatively, the enzymatic reaction can be accomplished by a column process, for example on a continuous basis.

For example, the foam is introduced into a column at a density sufficient to remain permeable through the substrate. The substrate solution, having a pH of 8 to 10 and containing the fumarate ion and the ammonium salt, passes to a temperature of 15 to 600C and at an appropriate flow rate. An aqueous solution containing L-aspartic acid is obtained as an effluent. This solution can be put back into circulation if desired. L-aspartic acid can be released from the solution by adjusting the pH to about 2.8-3, with concentrated sulfuric acid. L-aspartic acid is thus obtained in the form of a crystalline precipitate. The purity of the product can be improved by washing in ethanol if desired.

 Urethane prepolymers useful for the preparation of polyurethane foam are prepared by capping a polyoxyalkylene polyol with an excess of a polyisocyanate, such as toluene diisocyanate. Before styling it, the polyol must have a molecular weight of the order of 200 to approximately 20,000 and preferably of the order of 600 to approximately 6,000. The hydroxyl functionality of the polyol and the functionality of the corresponding isocyanate after styling is between 2 and about 8. If foams are formed of prepolymers having isocyanate functionality on the order of 2, the resulting product is essentially linear and does not have as much tensile strength as if it were cross-linked. Consequently, a hydroxyl content greater than 2 per molecule is desired. This can be obtained by using mixtures of diols with triols or other polyols of higher functionality or triols or other polyols of higher order, as such, can be styled. with di- or polyisocyanates.

 As examples of suitable polyols (to be used as polyisocyanates), there may be mentioned: (A) essentially linear polyols formed for example by reaction of ethylene oxide with ethylene glycol as initiator.

Mixtures of ethylene oxide with other alkylene oxides can be used as long as the molar percentage of ethylene oxide is at least 50%. If the linear polyethers are mixtures of ethylene oxide with, for example, propylene oxide, the polymer can be either random or block and the terminal units can be either oxyethylene or oxypropylene.

The second class of polyols (B) contains those having a hydroxy functionality of 3 or more. Such polyols are commonly formed by reacting a'Goylene oxides with a polyfunctional initiator such as trimethylolpropane, pentaerythritol, and the like. In forming polyol B, the alkylene oxide used can be ethylene oxide or mixtures of ethylene oxide with other alkylene oxides. Useful polyols can be further represented by (C) linear and branched polyfunctional polyols as indicated in A and B above with an initiator or a crosslinking agent. As a specific example of C, a mixture of polyethylene glycol (molecular weight of the order of 1,000) with trimethylolpropane, trimethylolethane or glycerol can be cited. This mixture can subsequently be reacted with excess polyisocyanate to produce a prepolymer useful in the present invention. Alternatively, the linear or branched polyols (such as polyethylene glycol) can be reacted separately with an excess polyisocyanate. The initiator, such as trimethylolpropane, can also be reacted separately with the polyisocyanate. Subsequently, the two capped materials can be combined to form the prepolymer.

 Suitable polyisocyants and initiators are indicated in US Patent No. 3,903,232 incorporated herein by reference. The initiators are generally cross-linking agents which are soluble in water or dispersible in water as described in US Pat. No. 3,903,232.

Preparation of prepolymer A
Prepolymer A is prepared by mixing two molar equivalents of polyethylene glycol having an average molecular weight of 1,000 (PEG - 1,000) and 0.66 molar equivalent of trimethylolpropane (TMOP). The mixture is dried at 100-1100C under a pressure of 666.5 - 2,000 N / m2 to remove the water. The resulting dried mixture is slowly added, over a period of about 1 hour, to a container containing 5.52 molar equivalents of toluene diisocyanate (TDI), while stirring the mixture of TDI and polyol.

The temperature is maintained at 600C while stirring for an additional 3 hours. Then, an additional quantity of 0.78 molar equivalent of TDI is added while stirring, over a period of the order of 1 hour while maintaining the temperature at 600C. The final reaction mixture contains a 5 percent molar excess of TDI. All the hydroxyl groups are capped with isocyanate and a certain chain extension occurs due to the crosslinking of the polyols with the TDI.

EXAMPLE 1
E. coli cells (ATCC, 11.303) suspended in 0.1 M phosphate buffer (pH 8.0) were centrifuged at 5,000 rpm for 30 minutes. The mass of the cells was placed under refrigeration and was drained overnight. The cells (11 g) were mixed with 11 g of prepolymer A. As a result of the water in the cell mass, a flexible and hydrophilic polyurethane foam formed which hardened in about 15 minutes. The cells were encapsulated in or linked to the polyurethane foam matrix.

EXAMPLE 2
The foam was placed under refrigeration and was then cut into small pieces (cubes of about 6.45 mm) for later use.

 A substrate-forming medium was prepared from the following recipe: 11.6 g of fumaric acid and 20.3 mg of MgCl2. 6 H2O, dissolved in 25% ammonia with pH adjustment to 8.5 using concentrated ammonia. Sufficient deionized water was added to bring the volume to 100 cc.

The temperature of the substrate was maintained at 370C and about 1.4 g (dry weight basis) of foam was placed in the solution of the substrate. After 30 minutes, analysis of the samples with thin layer chromatography indicated that considerable amounts of L-aspartic acid had been produced. Other test results indicated that 95% of the fumaric acid had been used (i.e. converted to L-aspartic acid).

EXAMPLE 3
The particulate foam prepared in Example 1 was introduced into a 75 cc column equipped with a water jacket. The temperature was maintained at 370C and the substrate was passed at various flow rates. The composition of the substrate was as follows: qD3cc of WH40H, 1200 of deionized water, 232 g of fumaric acid, 406 mg of Mec12.

6 H20, with the addition of deionized water to bring the volume to 2 liters (pH 9.0). (done in two lots). The volume of the liquid bed in the column (i.e. the portion of the column not occupied by the foam) was 31.25 cc.

About 3500 ml of the substrate was passed through the column at a constant rate of 0.6 ml / min at a pH of 9 and at 370C. Samples were taken periodically and analyzed for fumaric acid, by high pressure liquid chromatography. The percentage of conversion to L-aspartic acid relative to the number of hours the column had operated at the time of sampling is indicated in the table below and illustrates the stability of the column
Fumaric acid / L-aspartic conversion
Time (hours) (a O cc / mn)
,
5 71
46 69
150 61
218 61
EXAMPLE 4
After the column of Example 3 had operated for approximately 54 hours, the flow rate was established at 0.28 c / min and a 288 ml sample was collected over the following period of 16 hours and 50 minutes. L-aspartic acid was isolated from a 250 ml portion of the sample, and it was found that the actual rate of conversion of fumaric acid to L-aspartic acid (based on direct analysis of l (L-aspartic acid dried by gas-liquid chromatography) was of the order of 85%.

EXAMPLE 5
About 3,500 ml of the substrate which had passed through the column of Example 3 was collected at a flow rate of 0.60 ml / min. The sample was heated to 900 C and the pH was reduced to 2.8 (the isoelectric point for Laspartic acid) by adding 200 ml of 60% H2SO4. The acidified mixture was cooled to 150C to precipitate the acid
L-aspartic which was removed by filtration, dried and washed in 95% ethanol in water at 300C. The unreacted fumaric acid dissolved in ethanol was thrown into solution. The insoluble L-aspartic acid was removed and dried under vacuum. A sample of the powder was analyzed at 97% L-aspartic acid, which represents 89% of the theoretical production.

EXAMPLE 6
Foam was prepared as in Example 1 and 1 g of sample was placed in a flask and mixed with 100 cc of the substrate (126 mg / ml of fumaric acid) at about 370C and at pH 9 for 15 minutes. Then, the process was repeated three times using a fresh batch of the substrate each time. In the first batch (based on the analysis of residual fumaric acid), the conversion to L-aspartic acid was of the order of 57% in the first batch and of the order of 35% for each of the three batches following. It is believed that the difference between the first batch and the following batches is due to the unbound cells removed by washing.

EXAMPLE 7
A column was prepared and it was operated as in Example 3 (116 mg / cc of fumaric acid, pH 9.04) but at a temperature of 200C. The substrate was continuously recirculated through the column. Based on the reduction in fumaric acid levels, the conversion to L-aspartic acid was essentially complete after 120 hours. Achievement levels of 33 and 50% were achieved after approximately 30 and 50 hours respectively. The 50% level is an extrapolated value.

Claims (18)

 1. Process for the preparation of L-aspartic acid, characterized in that a substrate containing the fumarate ion is brought into contact withwe moEs of hydrophilic polyether polyurethane or at least 50 mol% of the alkylene oxide units in the segments polyether polyurethane are ethylene oxide, a microorganism producing aspartase being immobilized in said foam.  2. Method according to claim 1 characterized in that the substrate has a pH of 8 to 10.  3. Method according to claim 1, characterized in that the temperature of the substrate is from 15 to 600C.  4. Method according to claim 1, characterized in that the substrate contains ammonium fumarate.  5. Method according to claim 1 characterized in that the substrate contains the magnesium ion.  6. Method according to claim l, characterized in that it is carried out on a continuous basis.  7. Method according to claim 1, characterized in that it is carried out on a discontinuous basis.  8. Hydrophilic polyether polyurethane foam characterized in that at least 50 mol% of the alkylene oxide units in the polyether segments of the polyurethane are ethylene oxide, a microorganism producing aspartase being immobilized in said foam.  9. Foam according to claim 8, characterized in that at least 90% by mole of the alkylene oxide units are ethylene oxide.  10. Foam according to claim 8, characterized in that it is prepared by reaction of a hydrophilic polyether urethane prepolymer with water under foaming conditions, said water being mixed with a culture of a micro -organism producing aspartase, and in that at least 50 mol% of the units of alkoulene oxide in the polyether segment of the prepolymer are formed of ethylene oxide.  11. Foam according to claim 10 characterized in that the prepolymer is a mixture of polyoxyethylene glycol with a low molecular weight polyhydric alcohol, said mixture reacting with a molar excess of a polyisocyanate.  12. Foam according to claim 8, characterized in that it is rigid.  13. Foam according to claim 8, characterized in that it is flexible.  14. Foam according to claim 8, characterized in that the weight ratio, on an anhydrous basis, of the polyurethane polymer to the culture of the microorganism is between 1: 1 () and 10: 1.  15. A process for the preparation of a hydrophilic polyether polyurethane foam, characterized in that a culture of a microorganism producing aspartase is mixed with a hydrophilic polyether urethane prepolymer in which at least 50 mol% of the oxide units d The alkylene in the polyether segments of the prepolymer are ethylene oxide, sufficient water being present in the mixture to force the prepolymer to undergo foaming to trap and immobilize the microbial cells of the culture.  16. Method according to claim 15, characterized in that the water content of the culture is of the order of 10 to 90% by weight.  17. Method according to claim 15, characterized in that the pH of the mixture is greater than 7.  18. The method of claim 15, characterized in that on a weight basis, the prepolymer / culture weight ratio in the mixture is from 1:10 to 10: 1, the weight of said culture being calculated on a dry basis.