NL8102156A - PROCESS FOR THE PREPARATION OF L-ALANINE BY CAPTURED MICROORGANISMS, AND FOAM TO BE USED THEREIN - Google Patents

Description

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Process for the preparation of L-alanine by established microorganisms, and foam to be used therewith.

In this specification, "aspartate decarboxylase" is intended to mean the enzyme which cleaves the β-carboxyl group from L-aspartic acid; in International Enzyme Classification No. 4.1.1.12.

Various methods of forming L-alanine through the action of aspartate decarboxylase on L-aspartic acid or salts thereof are known. For example, L-alanine can be prepared by culturing an aspartate decarboxylase-forming microorganism in a nutrient medium and reacting the culture fluid with L-aspartic acid; see Japanese Patent Publication No. 7560/1971. Alternatively, L-alanine can be prepared either by acting on resting L-cells with aspartate decarboxyl lase activity on L-aspartic acid or by extracting the enzyme (see, for example, Biochimica et Biophisica 15 Acta, part 67 from 1963) and let it act on the L-aspartate. However, these methods have the drawback that the resulting product is contaminated either with enzyme or with the microbe cells and the food sources, etc. In order to recover high purity L-alanine, additional operations are required to remove the enzyme and the other admixtures. Often, after the reaction is complete, the reaction mixture is boiled and / or acidified to precipitate the enzyme or microorganisms (which are then filtered off) so that enzyme or microorganisms can be used only once.

To overcome these drawbacks, it has been proposed to capture aspartate decarboxylase in polymeric supports; see U.S. Patent 3,898,128, which describes encapsulation in semi-permeable polyacrylamide membranes. See also U.S. Patent 3,458,400, which describes the enzymatic conversion of L-aspartic acid to L-alanine using Pseudomonas dacunhae or Achromobacter pestifer.

Encapsulation of microorganisms in hydrophilic polyurethane foams has also been proposed previously, see U.S. Patent 3,905,923. Binding of enzymes to polyurethanes is also described in U.S. Pat. No. 3,672,955, see column 5, lines 27-35. Hydrophilic polymers have also been used as carriers for chemicals, including bacterio-states and fungicides; see U.S. Patent 3,975,350.

Another pertinent reference is to the article by Sonomoto et al in Agric. Biol. Chem. 44 10 (1980) 1119-1126, according to which steroid conversions are achieved with bacterial cells enclosed in polyurethanes prepared from prepolymers. Fukui et al, in Biochemistry 62 (1980) 381-386, describe the use of enzymes captured in photo-crosslinkable prepolymers and urethane prepolymers to prepare L-amino acids. Capturing bacterial cells using urethane prepolymers has also been described. In a recent Gordon Research Conference of August 11-15, 1980, the capturing of whole cells (especially of genetically engineered E. coli which made a lot of penicillin amidase 20) into urethane prepolymers was discussed; biological systems for preparing L-serine and L-tryptophan were also "still under evaluation".

According to the invention, a hydrophilic polyether-polyurethane foam is used in which at least 50 mol% of the alkylene oxide units of the polyurethane are ethylene oxide, with an immobilized aspartate decarboxylase-forming microorganism in said foam. The invention also relates to a process for preparing the foam and its use for preparing L-alanine.

Preferably, the polyether segments consist of at least 90% ethylene oxide units. Depending on the amount of crosslinker used, the foam can be both stiff and flexible. The weight ratio between polyurethane and microorganism (based on dry weight) should be between 1:10 and 10: 1, preferably between 2: 1 and 4: 1. Before mixing the culture with the prepolymer, the dry weight of microorganisms of the culture can be determined by evaporating a sample at suitable temperature (e.g. 50 ° C). Then 50 to 90% of the microorganisms can be captured in the foam.

Some rinsing of the bacterial cells has been observed in freshly made foams, but usually less than 25% of the starting one. Generally, the more flushed out as the bacteria / foam ratio increases. More can also be rinsed out if lumps or clumps of the bacteria are present during mixing. By "attached" here is meant that the microorganisms remain in the foam on contact with water or an aqueous solution and cannot be washed out. It is believed that this capture is achieved by encapsulating the microorganisms in the foam. It is also likely that during foaming bonding occurs by reaction of the isocyanate groups of the polymer with certain groups (eg, amino groups) on the surface of the microorganisms.

The foams are prepared by directly mixing a culture of the aspartate decarboxylase-forming microorganism with a urethane prepolymer in the presence of enough water to promote foaming. Generally, the water comes along with the microbes culture, i.e. these cultures usually contain 10 to 90% by weight of water (which can be easily determined by evaporating a sample thereof). Due to the presence of the water, the prepolymer will foam and at the same time the microorganisms will be captured in it. To do this as best as possible, the pH of the culture should be between 3 and 11, preferably above 7. During capture, the prepolymer / water weight ratio is generally between 2: 1 # 3Q and 1: 2, preferably between 3: 2 and 2: 3, which water is provided by the culture or by a combination of the culture with water added before or during mixing.

After mixing, foaming is generally completed in 5-10 minutes and the foam cures to its final state in another 5-10 minutes. But with large amounts of foam, it is conceivable that you need a little more time.

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Examples of microorganisms that form aspartic decarboxylase and are useful in this invention include:

Alcaligenes faecalis 5 Pseudomonas dacunhae

Clostridium perfringens Nocardia globurela Desulfovibrio desulfuricans Achromobacter pestifer.

All these microorganisms are publicly available from the aforementioned official tribal collections. However, it should be noted that the present invention is not limited to the use of the named species but includes the use of all aspartate decarboxylase-forming microorganisms.

L-alanine can be prepared by contacting that foam with L-aspartic acid. In general, this L-aspartic acid is used in the form of a 0.1 to 1.5 M aqueous solution with a pH between 3 and 7. Ammonia and / or phosphoric acid and other substances can be used for 20 adjusting the correct pH.

The encapsulated microorganism foam is contacted with the substrate solution and the whole is kept at a temperature between 15 ° and 60 ° C with stirring until the reaction is complete. The foam is then separated and can be stored in the refrigerator for a subsequent use.

The L-alanine can be separated from the solution in the usual manner.

Otherwise, the enzymatic reaction can be continuous, using a column. For example, the foam is packed into a column of such density that the water can continue to flow through it. The substrate solution with a pH between 3 and 7 is passed through the column at an appropriate flow rate at a temperature between 15 ° and 60 ° C. An aqueous solution of L-alanine is obtained as an effluent. The L-alanine can be released from this solution as will be described in the forthcoming Example IV.

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Prepolymers suitable for preparing the polyurethane foam are obtained by reacting a polyoxyalkylene polyol with an excess of polyisocyanate such as toluene diisocyanate. That polyol must have a molecular weight between 200 and 20,000, preferably between 600 and 6000, and 2 to 8 hydroxy groups per molecule. When making foams from prepolymers with a functionality of about 2, the end products are essentially unbranched and have less tensile strength than if they were cross-linked. It is therefore preferable to start from a polyol with more than two functional groups per molecule; these can be obtained by starting from mixtures of diols with triols or polyols with even higher functionality.

Examples of suitable polyols are 15 (A) essentially unbranched polyols created by reacting ethylene oxide with an initiator such as glycol. Mixtures of ethylene oxide with other alkylene oxides can also be used as long as ethylene oxide thereof is at least 50 mole percent. If the unbranched polyethers are mixtures of ethylene oxide with, for example, propylene oxide, the order within the polymer chains can be either random or blockwise, and the end groups can be both hydroxyethoxy and hydroxypropoxy. A second class of poly (B) are those with three or more hydroxy groups per molecule; these are usually formed by reacting alkylene oxides with a polyvalent initiator such as trimethylolpropane, pentaerythritol, etc. In forming a polyol B, the alkylene oxide used may be ethylene oxide or a mixture thereof with other alkylene oxides. Still other suitable polyols are (C) branched or unbranched polyols as listed at A and B 30 together with an initiator or crosslinker; an example of this is a mixture of polyethylene glycol (MG ca 1000) with trimethylolpropane, trimethylolethane or glycerol. This mixture can be reacted with excess polyisocyanate to obtain a prepolymer useful for the invention, or the branched or unbranched polyols can be reacted separately with excess polyisocyanate. The initiator, for example trimethylol 8102156-6-propane, can also be reacted separately with the polyisocyanate. Finally, the separately prepared products are combined with each other to form the prepolymer.

Suitable polyisocyanates and initiators are described in U.S. Pat. No. 3,903,232. The initiators are generally water-soluble or dispersible crosslinkers described in that patent. Prepolymer A is prepared by combining two equivalents of polyethylene glycol with a molecular weight of about 1000 with 0.6 equivalents of trimethylol propane. To remove water, the mixture is dried at 100-110 ° C at a pressure of 5-15 torr. This dry mixture is then slowly added with stirring over an hour to a vessel containing 5.52 moles of toluene diisocyanate. Stirring is then continued for 3 hours, keeping the temperature at 60 ° C. Finally, an additional 0.78 mol of toluene diisocyanate is added over an hour at a temperature of 60 ° C with stirring. An excess of 5% isocyanate is ultimately used and all end groups are now isocyanate, not hydroxyl; the chains have now also become a bit longer.

Example I

Alcaligenes faecalis (ATCC No. 25094) was mixed 8 g (in which 1 g of dry matter) with 5 g of prepolymer A. The water in the cell mass produced a flexible hydrophilic polyurethane foam which cured in about 15 minutes. The 25 cells were encapsulated within or bonded to the polyurethane matrix.

Example II

The foam of Example I was placed in a column which was closed at both ends with a Millipore filter 30. Under the column, 50 g of L-aspartic acid was placed in a vessel that contacted the column. Then a solution of 40 ml of ammonia and 10 ml of Tween 80 in 4 liters of water, the pH of which was adjusted to 10 with 50% H 2 PO 2, was passed upward through the column via the aspartic acid supply. The temperature was about 25 ° and the flow rate about 0.67 ml / min. During the first 40 hours of the operation, the column gave about 8102156-7-0.90 mg / ml, i.e. 0.60 mg / min. of L-alanine. Thereafter, at a flow rate of 0.34 ml / min. the conversion 1.62 mg / ml, and at a flow rate of 0.17 ml / min. the conversion became 2.66 mg / ml.

The L-alanine was determined with a Technicon Auto Analyzer with L-amino acid oxidase, with which the L-alanine is converted into the corresponding α-keto acid, and in which the resulting 1 ^ 2 under the influence of peroxidase from black radish with 2,4-dichlorophenol and 4-aminoantipyrene is converted into a quinonimine dye, which is measured colorimetrically at 505 nm.

Example III

The particulate foam of Example 1 was packed into a column in a water jacketed tube.

The temperature was adjusted to various values, and the substrate was applied at a flow rate of 0.67 ml / min. forwarded. The base liquid was a solution of 10 ml of concentrated ammonia and 2.5 ml of Tween 80 in 500 ml of deionized water. This base liquid was contacted with 53 g of solid L-aspartic acid at room temperature, but before entering the column it was warmed to 27 ° C, and after leaving the column it was cooled to room temperature before being was returned to the storage vessel with the solid L-aspartic acid.

An equilibrium concentration therefore prevailed in this storage vessel.

The base liquid was continuously circulated through storage vessel, column and cooling coil. This happened not only at 27 ° G but also at 37 ° and 50 ° C. The test at 27 ° C was continued for 20 hours, that at 37 ° C for 72 hours and that at 50 ° C for about 24 hours. The conversion rates in L-alanine determined as described in Example II were 112.5 mg / h (at 27 ° C), 334 mg / h (at 37 ° C) and 30 mg / h (at 50 ° C) C).

Example IV

26 g of freshly harvested Pseudomonas dacunhae (ATGC no. 21192) was mixed with 20 g of prepolymer, followed by foam formation, as in Example 1. This foam was cut into cubes about 10 mm in length before use, and these were stacked into a 100 ml column. An approximately 8102156 - - 8 - 0.5 M L-aspartic acid solution, which also contains 10 mg / l pyridoxal-5-. phosphate and 2.5 g / l Tween 80, the pH of which was adjusted to 5.3 with H 2 SO 4 was run at a flow rate of 0.65 ml / min. passed through the column. This gave a conversion to L-alanine of 14-17 mg / ml. However, the substrate was not recycled and passed through the column only once. After 700 ml of effluent was collected, the L-alanine was isolated therefrom by concentrating the 85 ° C solution, adjusting the pH to 5.5 and then cooling to 15 ° C. Here, L-aspartic acid and L-alanine crystallized out, and the product was taken up in deionized water, the L-alanine dissolved, and the L-aspartic acid was filtered off. The filtrate was evaporated to dryness under vacuum to obtain crystalline L-alanine; the yield t was 151.4 g.

Example V

After the column of Example IV had been in operation for about 2 weeks, the substrate was replaced with a solution containing 1.0 M of L-aspartic acid and containing 125 ml of strong ammonia per liter; the pH was adjusted to 5.3 at 60%. The flow rate was again 0.65 ml / min. Initially, the conversion was about 14 mg / ml, but after replacing this substrate with one containing only 0.05 M L-aspartic acid, the conversion dropped to 4-5 mg / ml.

Subsequent tests with a 1 M solution gave conversions from 27 to 32 mg / ml. This increased to 45-50 mg / ml during recirculation. When 12.5 mg / l of pyridoxal-5-phosphate was added, the conversion increased to about 36 mg / ml. Subsequent experiments without pyridoxal-5-phosphate but with recirculation gave a conversion of about 45 mg / ml. After the column had been in continuous operation for about 45 days, the conversion had dropped to about 16 mg / ml, but most of the time there had been no pyridoxal-5-phosphate, and then 12.5 mg / ml ml was added, the conversion rose again to 40 mg / ml.

The same liquid was then passed through the column for 24 days, the pH rising to 8.72, 810 2 15 6-9 - which meant a conversion of above 90%. L-alanine was now determined in the circulating liquid (by the method of Example II), and it was found that 94% of the L-aspartic acid had been converted to L-alanine.

8102156

Claims (22)

1. A process for preparing L-alanine in which a solution of L-aspartic acid is contacted with a water-insoluble source of L-aspartate decarboxylase, characterized in that this source of aspartate decarboxylase is a hydrophilic polyester polyurethane foam in which at least 50 mol. Z of the alkylene oxide units is ethylene oxide. 2. A method according to claim 1, characterized in that the L-aspartic acid solution has a pH between 3 and 11. Process according to claim 1 or 2, characterized in that the reaction takes place at a temperature between 15 ° and 60 ° C. Method according to any one of the preceding claims, characterized in that the microorganism is Pseudomonas dacunhae. Process according to claim 4, characterized in that the L-aspartate solution also contains pyridoxal-5-phosphate. 20 contains. Method according to any one of claims 1 to 3, characterized in that the microorganism is Alcaligenes faecalis. A method according to any one of claims 25 to 6, characterized in that it is carried out continuously. Method according to any one of claims 1 to 6, characterized in that it is carried out in portions. 9. A hydrophilic polyether polyurethane foam in which at least 50 mol. Z of the alkylene oxide units is ethylene oxide, characterized in that this foam contains encapsulated cells of an aspartate decarboxylase-forming microorganism. Foam according to claim 9, characterized in that the at least 90 mol% of the alkylene oxide units is ethylene oxide. Foam according to claim 9 or 10, characterized in that the prepolymer was prepared by reacting a polyisocyanate 8102156-11 with a mixture of polyoxyethylene glycol and a polyhydric alcohol of low molecular weight. Foam according to any one of claims 9 to 12, characterized in that it is rigid. Foam according to any one of claims 9 to 12, characterized in that it is flexible. Foam according to any one of claims 9 to 14, characterized in that the weight ratio polyurethane / microorganism (based on dry matter) therein is between 1:10 and 10: 1. Foam according to any one of claims 9 to 15, characterized in that the microorganism is Pseudomonas dacunhae. Foam according to any one of claims 9 to 15, characterized in that the microorganism is Alcaligenes faecalis. 18. A process for preparing a hydrophilic polyether-polyurethane foam in which a culture of an aspartate decarboxylase-forming microorganism is mixed with a hydrophilic polyether-urethane prepolymer in which at least 50 mol% of the alkylene oxide units are ethylene oxide, and with enough water in the mixture to foam the prepolymer to encapsulate the culture microbe cells in the resulting foam. A method according to claim 18, characterized in that the water content of the culture is between 10 and 90% by weight. A method according to claim 18 or 19, characterized in that the pH of the mixture is above 7. 21. Method according to claim 18, 19 or 20, characterized in that the weight ratio of prepolymer / microbe cells (based on dry matter) is between 1:10 and 10: 1. A method according to any one of claims 35 to 21, characterized in that the microorganism is Pseudomonas dacunhae. 8102 156 V ï 'V - 12 - The method according to any one of claims 18 to 21, characterized in that the microorganism is Alcaligenes faecalis. 24. Method mainly according to description and / or examples. 8102156 i - i - .. _______