CH651066A5 - METHOD FOR PRODUCING L-alanine AND MEANS FOR ITS IMPLEMENTATION. - Google Patents

Description

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PATENT CLAIMS

1. A process for the preparation of L-alanine, characterized by the contacting of a hydrophilic polyether polyurethane foam in which at least 50 mol% of the alkylene oxide units ia the polyether segments of the polyurethane are ethylene oxide and which foam contains a microorganism immobilized, which L- Aspara-ginsäure-ß-decarboxylase is able to form with a substrate containing L-aspartic acid.

2. The method according to claim 1, characterized in that the substrate is an aqueous solution with pH 3 to 11 and that it has a temperature of 15 to 60 ° C.

3. The method according to claim 1, characterized in that the microorganism is Pseudomonas dacunhae and that the substrate contains pyridoxal-5-phosphate.

4. The method according to claim 1, characterized in that the microorganism is Alcaligenes fae-calis.

5. The method according to claim 1, characterized in that it is carried out continuously or batchwise.

6. Hydrophilic polyether polyurethane foam as an agent for carrying out the method according to claim 1, characterized in that it contains at least 50 mol% of the alkylene oxide units in the polyether segments of the polyurethane ethylene oxide and contains a microorganism immobilized which L-aspartic acid-ß-decarburized -oxylase is able to form.

7. Foam according to claim 6, characterized in that at least 90 mol% of the alkylene oxide units therein are ethylene oxide.

8. Foam according to claim 6, characterized in that the foam is in rigid form.

9. Foam according to claim 6, characterized in that the foam is in a flexible shape.

10. Foam according to claim 6, characterized in that in it the ratio of the dry weights of polyurethane polymer to the microorganism is between 10: 1 and 1:10.

11. Foam according to claim 6, characterized in that Pseudomonas dacunhae or Alcaligenes faecalis is present in it as a microorganism.

The invention described here relates to a process for the preparation of L-alanine using an immobilized microorganism which produces aspartic acid decarboxylase, and to an agent for carrying out this process.

In the following description, aspartic acid decarboxylase stands for L-aspartic acid-β-decarboxylase according to enzyme classification No. 4-1-1-12).

Various methods are known for producing L-alanine from L-aspartic acid or its salts by means of an enzymatic reaction of aspartic acid decarboxylase on the starting compounds mentioned. For example, L-alanine can be obtained by cultivating a microorganism capable of producing aspartic acid carboxylase. The cultivation takes place in a culture broth, which is then reacted with the L-aspartic acid (published JP patent application No. 7560/1971). On the other hand, L-alanine can be obtained by reacting whole cells which have asparagine carboxylase activity with L-aspartic acid salts or esters. However, the enzymes can also be extracted for the time being and only then brought into contact with the starting compounds (Biochimica et Biophisica Acta, volume 67, 1963). However, the L-alanine obtained in the above-described processes inherently contains enzymes, enzyme residues, cells, cell components, components from the culture broth and other impurities such as proteins. Accordingly, the above manufacturing process steps must be followed by purification steps to obtain a high purity L-alanine. Likewise, in the above-mentioned reactions, it often happens that the reaction solution has to be boiled or fired after the reaction has ended in order to precipitate the enzyme or the microorganism. The precipitated solid is then filtered off. This means that the enzyme or microorganism can then only be used once.

In order to eliminate the disadvantages mentioned, the immobilization of microorganisms which are capable of forming L-asparagine decarboxylase in polymeric carriers has been proposed. For example, U.S. Patent 3,989,128 describes this immobilization in semi-permeable polyacrylamide membranes. US Pat. No. 3,458,400, on the other hand, describes the enzymatic conversion of L-aspartic acid to L-alanine using Pseudomonas dacunhae or Achromobacter pestifer as microorganisms which are capable of forming decar-boxylase aspartic acid.

The immobilization of microorganisms in hydrophilic polyurethane foams has also been proposed. U.S. Patent 3,905,923 (inventor: Klug) and U.S. Patent Application Serial No. 362,488 dated May 21, 1973 (inventor Louis L. Wood et al) entitled "Preparation and Use of Enzymes Bound to Polyurethane) an example of such a publication. The immobilization of enzymes on polyurethanes is also described in US Pat. No. 3,672,955 (inventor Stanley), especially in column 5, lines 27 to 35. Hydrophilic polymers have also been proposed as carriers for other chemicals, for example bacteriostats and fungicides; see US Pat. No. 3,975,350 (inventor: Hudgin). It is also known to immobilize bacterial cells in hydrophobic polyurethanes; see Example 4 of UK Patent 953,414. Immobilization of a large number of enzymes in hydrophilic polyurethanes is also described in U.S. Patent Application Serial No. 849,999 (inventor Hartdegen et al) dated November 9, 1977, entitled "Immobilized Biological Material »taught.

Other relevant references include:

Sonomoto et al; Agric. Biol. Chem., 44 (5), pages 1119 to 1126, 1980. In this work steroid conversions are carried out using bacterial cells which are enclosed in polyurethanes. Polyurethanes are obtained from urethane prepolymers.

Fukui et al, Biochimie, 1980, 62, pages 381 to 386. This work describes the use of enzymes which are immobilized in photocrosslinkable prepolymers and urethane prepolymers and are suitable for the preparation of L-amino acids. The immobilization of bacterial cells using urethane prepolymers is also described.

A recent Gordon Research Conference in August 1980 discussed the immobilization of whole cells using urethane prepolymers. Examples of such cells are genetically manipulated E. coli that contain large amounts of penicil linamidase. At the same conference

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also reported biotransformation systems for the production of L-serine and L-tryptophan.

The inventive method for the production of L-alanine is characterized in the preceding claim 1.

The polyether segments of the polymethane preferably contain at least 90 mol% of ethylene oxide units. Depending on the amount of crosslinking agent used, the foam can be present as a rigid body or as a flexible agent. Based on the dry weight of the microorganism used, the weight ratio of the polyurethane polymer to the microorganism in the foam is normally between 1:10 and 10: 1, preferably between 2: 1 and 4: 1. Before adding the culture to the prepolymer, the dry weight of the microorganism in the culture can be determined by evaporating the liquid phase of the culture. This evaporation takes place at a suitable temperature, for example at 50 ° C. Then, when such a culture is foamed with the prepolymer, 10 to 90% of the microorganism is enclosed in the foam and immobilized.

In freshly prepared foams of this type, washout rates of enzyme cells of normally less than 25% by weight of the culture used have been observed. In general, this wash-out rate increases with the degree of loading of the foam. Even if lumps remain in the mass during the admixture, i.e. if the degree of mixing is insufficient, the subsequent degree of washout increases. In this document, the term “immobilization” means the fact that the microorganisms are retained in the foam and are not rinsed out of the foam when it comes into contact with an aqueous medium. It is assumed that the microorganisms are enclosed in the foam in such a way that they are practically immobilized there. It is also believed that during the foaming process, connections are made between the isocyanate groups of the prepolymer and between groups on the surface of the microorganism, for example amino groups.

The foams can be obtained by mixing the cultures with the microorganisms capable of producing L-aspartic decarboxylase directly with a urethane prepolymer in the presence of enough water under conditions that cause foaming. Usually the water of the organism culture is sufficient; this is the case, for example, if the cultures mentioned contain 10 to 90% by weight of water. Due to the presence of water, the prepolymer will foam and at the same time enclose and immobilize the microorganism in the foam. In order to optimize the immobilization, the pH of the aqueous phase of the culture should be between 3 and 11, preferably it is above 7. During the immobilization, the weight ratio between prepolymer and water is generally 2: 1 to 1: 2, preferably 3: 2 to 2: 3. If the water is not sufficient for the culture, water is added before or during mixing.

After the culture has been mixed in, the foaming reaction generally proceeds within 5 to 10 minutes. The foam is then cured to its final shape, which can be both rigid and flexible. This curing takes another 5 to 10 minutes. If the foam masses are very large, both the foaming times and the curing times can be significantly longer.

Those microorganisms which are able to form aspartic acid decarboxylase and therefore in the invention

methods according to the invention can include strains of:

Alcaligenes faecalis Pseudomonas dacunhae Clostridium perfrigines Nocaradia globurela Desulfovibrio desulfuricans Achormobacter pestifer.

The list represents examples of the microorganisms to be used according to the invention.

All of the microorganisms listed in the list above are freely available from culture collection points. The strains derived from which L-aspartic acid decarboxylase are capable of being formed can be determined by consulting the corresponding descriptions of the collections; for example the ATCC catalog. However, it should be noted that the present invention described is not limited to the above specific microorganisms; rather, the invention encompasses all L-asparagine decarboxylase-producing microorganisms.

According to the invention, L-alanine can now be obtained by contacting the prepared foam with L-aspartic acid. The L-aspartic acid is normally used in the form of a 0.1 to 1.5 molar aqueous solution with a pH between 3 and 7. Ammonium hydroxide, phosphoric acid and other compounds can be used to adjust the appropriate pH.

The foam with the immobilized microorganism is brought into contact with the substrate solution and the mixture is incubated at temperatures between 15 and 60 ° C. with stirring until the reaction is complete. After completion of the reaction, the foam is separated off and can be stored under cooling until the next use. The L-alanine is obtained from the substrate using conventional methods. Alternatively, the enzymatic reaction can be carried out in a column, i.e. the process is ongoing. For example, the foam is packed into a column in such a density to allow the substrate to flow through. The substrate solution, again with a pH between 3 and 7 and containing the L-aspartic acid, is passed through the column at a temperature of 15 to 60 ° C. and under a suitable flow rate. A solution containing L-alanine is obtained as the outflow. If necessary, this solution can be pumped around. The L-alanine can be obtained therefrom according to the method of Example 4 below.

The urethane prepolymers to be used to produce the polyurethane foam are obtained, for example, as follows: a polyoxyalkylene polyol is reacted with an excess of polyisocyanate, for example toluenediiso cyanate. Before the reaction, the polyol should have an average number molecular weight between 200 and 20,000, preferably between 600 and 6,000. The hydroxyl functionality of the polyol and the corresponding isocyanate functionality after the reaction is between 2 and 8. If foams are formed from prepolymers with an isocyanate functionality of approximately 2, the product obtained is essentially linear and does not have the tensile strength of a corresponding cross-linked material. Therefore, a hydroxyl group content of more than 2 per molecule is aimed for. This can be achieved by mixing mixtures of diols and triols or higher

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neller polyols are implemented with di- or polyisocyanates.

Examples of usable polyols (for the reaction with the polyisocyanates) include: (A) essentially linear polyols, for example obtained by reacting ethylene oxide with ethylene glycol as an initiator. Mixtures of ethylene oxide with other alkylene oxides can also be used; however, the molar proportion of ethylene oxide must be at least 50 mol%. If the linear polyethers are mixtures of ethylene oxides with, for example, propylene oxide, the polymer can be either a random or a block polymer. The end groups can then be either oxyethylene or oxypropylene.

A second group of polyols (B) include those with a hydroxy functionality of 3 or more. Such polyols are normally obtained by reacting alkylene oxides with polyfunctional initiators such as trimethylolpropane, pentaerythritol, etc. When the group B polyols are obtained, the alkylene oxides used are either ethylene oxide or mixtures of ethylene oxide with other alkylene oxides.

Another exemplary group of the polyols (C) to be used comprises the linear, branched polyfunctional polyols as in groups R and B above, together with an initiator or branching agent. A specific example from group C is a mixture of polyethylene glycol with an average molecular weight of 1,000 with trimethylolpropane, trimethylolethane or glycerin. This mixture can then be reacted with an excess of polyisocyanate in order to obtain a prepolymer which can be used according to the invention. On the other hand, linear or branched polyols (e.g. polyethylene glycol) can also be reacted separately with an excess of polyisocyanate. The initiator, for example trimethylolpropane, can also be reacted separately with the polyisocyanate. The two pretreated materials can then be combined to form the prepolymer.

Suitable polyisocyanates and initiators are, for example, those described in U.S. Patent 3,903,232. The initiators are usually water-soluble or water-dispersible crosslinking agents as described in the same US patent.

To prepare prepolymer A, two molar equivalents of polyethylene glycol with an average number molecular weight of 1000 and 0.66 molar equivalents of trimethylolpropane are mixed. The mixture is now dried at 100 to 110 ° C and at a pressure of 5 to 15 torr to remove the water. The resulting dried mixture is slowly added to a stirred container containing 5.52 molar equivalents of toluene diisocyanate for about 1 hour. The temperature is maintained at 60 ° C during the addition; after the addition is complete, stirring is continued for 3 hours. A further 0.78 molar equivalents of toluene diisocyanate are then added, with stirring again. Even after this addition, stirring is continued at 60 ° C. for 1 hour. The reaction mixture now contains a 5% molar excess of toluenediisocyanate. All hydroxyl groups react with isocyanate and chain extensions occur because of the crosslinking of the polyols with the toluenediisocyanate.

example 1

Alcaligenes faecalis cells (ATCC No. 25 094) were mixed with 5 g of prepolymer A, the preparation of which was described above. 8 g of cell culture were used. The dry weight of the cells in the culture was 1 g. The water in the culture created a flexible, hydrophilic polyurethane foam that cured in about 15 minutes. The culture cells were included or connected in the foam itself or on the surface thereof.

Example 2

The foam from Example 1 was placed in a column and covered with a Millipore filter at the top and bottom. At the bottom of the column, a container with 50 g of L-aspartic acid was attached in contact with the column. Now 4 l of a solution with 40 ml of NH4OH and 10 ml of Tween 80 with a pH of 10 (adjusted with 50% H3P04) were pressed through the 11-aspartic acid and then through the column. The solution temperature was about 25 ° C and the flow rate was about 0.67 ml per minute. During the first 40 hours of flow, there was 0.90 mg / ml L-alanine in the outflow, which corresponds to a production rate of 0.60 mg / min. The flow rate was then reduced to 0.34 ml / min. reduced, which increased the conversion rate to 1.62 mg / ml. At a flow rate of 0.17 ml per minute, the conversion rose to 2.66 mg / ml.

The L-alanine content determination was carried out by means of the Technicon auto-analyzer using L-amino acid oxidase. The L-alanine is converted to the corresponding a-keto acid, ammonia and H202. The peroxide formed is reacted with 2,4-dichlorophenol and 4-aminotanipyrene in the presence of the corresponding peroxidase. The resulting reaction product, a chi-noneimine dye, was determined colorimetrically at 505 nm.

Example 3

Foam as produced in example 1,

was crushed and then packed into a column. The column had a double jacket. The temperature was set by passing water through the double jacket mentioned. The substrate was passed at a flow rate of 0.67 ml per minute. It consisted of 500 ml of deionized water, which contained 10 ml of a concentrated NH4OH solution and 2.5 ml of Tween 80. The substrate, before entering the packed column, passed through a container containing 53 g solid L-aspartic acid at room temperature. When passing through the column, the substrate was warmed to 27 ° C. and then, before returning to the substrate container, cooled again to room temperature. The solid L-aspartic acid was present in the container mentioned. Through the contact of the two substances, a solution weight was formed in the template. The substrate was continuously passed through the column. The process was repeated at temperatures of 37 and 50 ° C. The run at 27 ° C lasted 20 hours, the one at 37 ° C 72 and the last at 50 ° C for about 24 hours. The rate of conversion for the formation of L-alanine was determined in the same way as in Example 2; it was 112.5 mg / h at 27 ° C, 334 mg / h at 37 ° C and 0 mg / h at 50 ° C.

Example 4

26 g cells of Pseudomonas dacunhae (ATCC number 21 192) were mixed with 20 g prepolymer A according to Example 1; the mixture was then foamed and cured. The foam was then cut into cubes approximately 1 cm3 in size and placed in a 100 cm3 column. A substrate of approximately 0.5 m

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L-Aspartic acid was passed through the column. The pH of the substrate was adjusted to 5.3 by adding H2S04; it also contained 10 mg / lt of pyridoxal-5-phosphate and 2.5 g / lt of Tween 80. The flow rate was 0.65 ml / min, which led to a conversion of L-aspartic acid to L-alanine (analyzed as in the example 2) from 14 to 17 mg / ml. The substrate was not circulated, i.e. it only flowed through the column once. After 700 cm 3 had flowed through the column, the L-alanine was isolated from the exit solution. For this purpose, the substrate was heated to 85 ° C., the pH of the latter was brought to 5.5, and finally the solution was cooled to 15 ° C. Both the L-aspartic acid and the L-alanine failed. The separated solids were taken up in deionized water, with L-alanine going into solution. The undissolved L-aspartic acid was filtered off. The water was then evaporated from the solution under vacuum and crystals of L-alanine were obtained. The yield of crude product was 151.4 g.

Example 5

The process according to Example 4 was carried out for approximately 2 weeks. Another substrate was then reacted with the same column. This new substrate was 1 molar with respect to L-aspartic acid and additionally contained 125 cm3 NH4OH / lt. The pH of the new substrate was brought to 5.3 by adding 60% H2S04

set. The flow rate was 0.65 ml per minute. The initial rate of reaction was approximately 14 mg / ml. Now a third substrate was used, one that was only 0.05 molar in relation to L-aspartic acid per liter. The conversion rate decreased to 4 to 5 mg / ml.

Other new approaches with substrates that were 1 molar with respect to L-aspartic acid gave conversion rates of 27 to 32 mg / ml. When the io liquid was circulated, conversions of 45 to 50 mg / ml were achieved. By adding pyridoxal-5-phosphate (12.5 mg / lt), average conversions of approximately 36 mg / ml were obtained. Subsequent batches without pyrid oxal-5-phosphate but with circulation gave 45 mg / ml 15 L-alanine. After the same column had been in operation for about 45 days, the degree of conversion fell to about 16 ml / ml. During this time, however, the work was carried out mainly without the addition of pyridoxal-5-phosphate, and, when the compound (12.5 mg / ml) mentioned was added again, the reaction rate rose again to 40 mg / ml. When this new substrate was circulated for 24 days, the pH of the substrate rose to approximately 8.7, which corresponds to a conversion rate of over 90%. The content of L-Ala-25 nin in the solution was determined according to the procedure from Example 2. In the end, the conversion of L-aspartic acid to L-alanine was 94%.

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