GB2084155A - Process for production of L- amino acids using immobilized microorganisms - Google Patents

Abstract

A hydrophilic polyether polyurethane foam wherein at least 50 mole % of the alkylene oxide units in the polyether segments of the polyurethane are ethylene oxide, said foam having an L-amino acid- producing microorganism immobilized therein. Also disclosed is a method for preparing the foam and utilizing the foam to prepare L-amino acids from the corresponding substrates

Description

SPECIFICATION Process for production of L-amino acids using immobilized microorganisms This invention relates to the production of L-amino acids.

Various methods for producing L-amino acids by enzymatic reactions with the appropriate substrate (i.e. precursor) are known. For example, L-alanine can be prepared by cultivating an Laspartic acid decarboxylase-producing microorganism in a nutrient medium and reacting the cultivation broth with L-aspartic acid (Japanese Patent Publication No. 7560/1971). Alternatively, L-alanine can be prepared by extracting aspartate decarboxylase from a microorganism, and reacting the enzyme with L-aspartate (e.g. Biochimica et Biophysica Acta, volume 67 (1963)). L-aspartic acid can be produced by similar procedures. US Patent 3,214,345 discloses the use of E coli cells to produce L-aspartic acid from ammonium fumarate. However, L-amino acids produced according to these methods are inevitably contaminated with the enzyme, microbial cells, nutrient sources of the medium and/or proteins.Accordingly, in order to prepare L-amino acids of high purity, additional steps for removing the enzyme and other contaminants from the reaction are required. Frequently after the reaction is completed, the reaction solution is boiled and/or acidified to precipitate the enzyme or microorganisms and the precipitate is filtered off. Thus, the enzyme or microorganism can be used only once and must be discarded.

To overcome the above disadvantages encapsulation of microorganisms in polymeric carriers has been suggested. Thus US Patent 3,898,128 described encapsulation in semipermeable polyacrylamide membranes. US Patent 3,458,400, describes the enzymatic conversion of Laspartic acid to L-alanine using Pseudomonas dacunhae or Achromobacter pestifer, as sources of aspartate decarboxylase. Japanese Patent Publication No 6870/1970 describes binding aspartase to an anion exchange polysaccharide adsorbent. In Japanese Patent Publication No 1 7 587/1 970 polyacrylates are used to encapsulate aspartase-producing microorganisms for the production of L-aspartic acid. The use of carrageenan as a carrier is described by Sato in Biochemica et Biophysica Acta, 570, 1 79-186 (1979).Russian Patent Publications SU 423,976 and SU 451,483 describe immobilization of Ecolicells in polyacrylamide gels, and production of L-aspartic acid from ammonium fumarate.

Encapsulation of microorgaisms in hydrophilic polyurethane foams has also been suggested (see e.g. US Patent 3,905,923). The binding of enzymes to polyurethane is also described in US Patent 3,672955. Hydrophilic polymers have also been used as carriers for chemicals, including bacteriostats and fungicides (see US Patent 3,975,350). It is also known to immobilize bacterial cells in hydrophobic polyurethanes (see Example 4 of UK Patent 953,414).

Other references of interest include Sonomoto et al (Agric Biol Chem, 44(5), 1119-1126, 1980) wherein steroid conversions are accomplished using bacterial cells entrapped in polyurethanes prepared from urethane prepolymers. Fukui et al (Biochimie, 1980, 62, 381-386) describe the use of enzymes immobilized in photocrosslinkable prepolymers and urethane prepolymers to prepare L-amino acids. Immobilization of bacterial cells using urethane prepolymers has also been described. At a recent Gordon Research Conference (August 11 - 15 1980) immobilization of whole cells (e.g. genetically engineered E coli containing large amounts of penicillin amidase) using urethane prepolymers was discussed.

The present invention provides a hydrophilic polyether polyurethane foam wherein at least 50 mole % of the alkylene oxide units in the polyether segments of the polyurethane are ethylene oxide units, said foam having an L-amino acid-producing microorganism immobilized therein.

The term "L-amino acid-producing microorganism" is intended to designate microorganisms which, when contacted with a substrate, convert the substrate to an L-amino acid. The invention applied to organisms which produce the appropriate enzymes intracellularly and/or extracellularly. A specific example of the invention is conversion of L-aspartic acid (a substrate here rather than a product) to L-alanine using an aspartate decarboxylase (i.e. L-aspartic acid P-decarboxy- lase (enzyme Classification No 4-1-1-12))-producing microorganism. Another specific example is the production of L-aspartic acid from fumarate using an aspartase-producing microorganism.

The present invention also provides a process for preparing the foam as well as a process for utilizing the foam to make L-amino acids. Preferably the polyether segments of the foam contain at least 90 mole % of ethylene oxide units. Depending upon the amount of crosslinking agent employed the foam can be either rigid or flexible. Based on the dry weight of the microorganism employed, the weight ratio of the polyurethane polymer to microorganisms in the foam is generally from 1:10 to 10: 1, and preferably from 2:1 to 4:1.Prior to mixture of the culture with the prepolymer, the dry weight of microorganisms in the culture can be determined by evaporating the culture to dryness at a suitable temperature, e.g. 50on. Thereafter on mixing a similar culture with the prepolymer, it has been found that 10-90% (e.g. 50-90%) by weight of the microorganisms can be immobilized in the foam.

In freshly-prepared foams some wash-out of cells has been observed, generally less than about 25 or 15% by weight of the culture. Generally wash-out increases as the loading of culture is increased. Also during mixing, if "lumps" are present in the mixture because of incomplete mixing, the amount of wash-out increases. By the term "immobilization" is meant that the microorganisms are retained in the foam rather than being leached therefrom upon contact with water or an aqueous substrate solution. It is believed that the microorganisms are encapsulated within the foam to accomplish the immobilization. It is also likely that during the foaming process binding occurs between the isocyanate groups of the prepolymer and groups on the surface of the microorganisms, e.g. amino groups.

The foams can be prepared by mixing a culture of the L-amino acid-producing microorganisms directly with a urethane prepolymer in the presence of sufficient water to promote foaming.

Conventionally the water is carried in the culture; thus suitable cultures generally comprise from 10 to 90 weight % of water; the water content of the particular culture can be determined by evaporating a sample of the culture to dryness as described above. Due to the presence of water, the prepolymer will undergo foaming and simultaneously the microorganisms are immobilized in the foam. To optimise immobilization, the pH of the aqueous culture is desirably from 4 to 11 (3 to 11 in the case of aspartate-decarboxylase-producing microorganism) and preferably in excess of 7. During immobilization, the prepolymer/water weight ratio is generally from 2:1 to 1:2 and preferably from 3:2 to 2:3, said water being provided by the culture or by combination of the culture and water added prior to or during mixture.

Following mixture the foaming reaction is generally completed within 5-10 minutes; the foam can be cured to its final rigid or flexible form in an additional, say, 5-10 minutes. However with large foam masses, it is conceivable that the times for foam formation and curing can be considerably extended.

L-amino acids which can be produced utilizing the foams of the invention, together with microorganisms to be immobilized and the corresponding substrate are set forth in the following Table. The term "corresponding substrate" is intended to designate a chemical compound which is fermented by an immobilized microorganism to produce the desired L-amino acid.

TABLE L-amino acid Microorganism Substrate L-alanine Pseudomonas dacunhae L-aspartic acid (ATCC 21192) Alcaligenes faecalis L-aspartic acid (ATCC 25094) Clostridium perfrigines L-aspartic acid Nocavadia globurela L-aspartic acid Desulfovitrio desulfuricans L-aspartic acid Achromobacter pestifer L-aspartic acid L-histidine Brevibacterium thiogenitalis imidazole (ATCC 19240) pyruvic acid Corynebacterium glutamicum glucose (ATCC 21604 and ATCC 21607) (1) Brevibacterium flavum(1) glucose (ATCC 21319) (2) Glutamic acid producer (2) histidinol e.g. Cornebacterium or Brevibacterium, e.g.

Brevibacterium flavum, ATCC 14067 L-isoleucine Serratia marcescens glucose (ATCC 21740) L-isoleucine Serratia marcescens glucose (ATCC 21741) L-isoleucine Serratia marcescens glucose (ATCC 21810) L-isoleucine Brevibacterium hydroxybutyric thiogenitalis acid (ATCC 19240) L-isoleucine Brevibacterium a-keto-ss-methyl- thiogenitalis n-valeric acid (ATCC 19240) L-leucine Brevibacterium a-keto-isocaproic thiogenitalis acid (ATCC 19240) L-leucine Brevibacterium glucose thiogenitalis (ATCC 19240) L-serine Microbacterium species glycine/ (ATCC 21376) formaldehyde L-serine Norcardiabutanica glycine/ (ATCC 21197) formaldehyde L-serine Corynebacterium glycine/ glycinophilum formaldehyde (ATCC 21341) L-theonine Brevibacterium flavum glucose (ATCC 21269) L-theonine corynebacterium glucose acetoacidophilum (ATCC 21270) L-theonine E. coli ATCC 13070) glucose L-proline Cornebacterium glucose glutamicum (ATCC 19223) L-proline Microbacterium glucose ammoniaphilum (ATCC 15354) L-proline Brevibacterium glucose ammonia genes (ATCC 13746) TABLE (cont.) L-amino acid Microorganism Substrate L-arginine Bacillus subtalis glucose (ATCC 21742) L-arginine Brevibacterium flavum glucose (ATCC 21742) L-arginine Corynebacterium glucose glutamicum (ATCC 21659) L-tryptophan Brevibacterium indolepyruvic thiogenitalis acid (ATCC 19240) L-tryptophan proteuo rettgerii indole + pyruvic (Ajinomoto) acid (or serine) L-tryptophan E. coli (ATTC 27553) indole + pyruvic acid (or serine) L-tryptophan E. coli(ATTC 8724) indole + pyruvic acid (or serine) L-phenylalamine Brevibacterium phenyl pyruvic thiogenitalis acid (ATTC 19240) L-phenylalamine Rhodosporidium trans-cinnamic toruloides acid (ATTC 19240) L-tyrosine Brevibacterium hydrosyphenyl thiogenitalis pyruvic acid (ATTC 19240) L-tyrosine Erwinia herbicola phenol + pyruvic (ATTC 21434) acid L-tyrosine Erwinia herbicola phenol + pyruvic (ATTC 21433) acid L-lysine Brevibacterium flavum glucose (ATCC 21127-21129) L-lysine Corynebacterium glucose glutamicum (Atcc 13826) L-lysine Corynebacterium glucose glutamicum (ATCC 13827) L-lysine Bacillus megaterium glucose (ATCC 21209) L-aspartic acid Pseudomonas fluoresceus fumarate Serratia marcescens fumarate Bacterium succincum fumarate Bacillus megaterium fumarate Bacillus subtilis fumarate Aerobacter aerogenes fumarate Bacillus natto fumarate Micrococcus sp fumarate Escherrichia coli fumarate All of the above microorganisms are publically available from the culture collections indicated.

To prepare the L-amino acid, the immobilized microorganism foam is contacted with a generally aqueous substrate solution, and the mixture is incubated at a temperature of, say, 15"C to 60"C with stirring, until the reaction is complete. When the reaction is completed the foam can be separated and stored under refrigeration for subsequent use. The amino acid can be recovered by conventional methods. Alternatively, the enzymatic reaction can be performed by a column method, i.e. on a continuous basis. For example the foam is packed into a column at a sufficient density to remain permeable to flow-through of the substrate. The substrate solution, generally having a pH of 3 to 11, is passed through at a temperature of from, say, 1 5"C to 60"C and at a suitable flow rate.An aqueous solution containing the desired L-amino acid is obtained as the effluent. This solution can be recirculated as desired.

L-aspartic acid can be prepared by contacting the foam with ammonium fumarate or a mixture of fumaric acid or its salt and inorganic ammonium salt. Suitable examples of the salt of fumaric -acid include alkali metal salts such as sodium fumarate and potassium fumarate. Ammonium chloride, ammonium sulfate and ammonium phosphate are preferred as the inorganic ammon ium salts. When a mixture of fumaric acid or its salt and an inorganic ammonium salt is employed in the enzymatic reaction, the preferred proportions of inorganic ammonium salt in the mixture is 1 to 2 moles to one mole of fumaric acid or fumaric acid salt.A divalent metal ion may be added to the enzymatic reaction solution (i.e. the substrate solution) to enhance the enzymatic activity and stability of the immobilized microorganism, although it has been discovered that such enhancement of activity is frequently unnecessary for short periods of time.

Suitable examples of the diva lent metal ions which can be employed include calcium, magnesium, manganous and strontium ions. If employed, the concentration of divalent metal ion in the-substrate solution is from 0.1 to 10 millimoles/liter.

The concentration of substrate employed is not critical, e.g. ammonium fumarate or a mixture of fumaric acid or its salt and an inorganic ammonium salt is dissolved in water in any concentration. Thereafter the pH is desirably adjusted to 8 to 1 0. If the enzymatic reaction is performed by a column method an aqueous solution containing L-aspartic acid is obtained as the effluent. L-aspartic acid can be liberated from the solution by adjusting the pH to, say, 2.8-3, with concentrated sulfuric acid. L-aspartic acid is thereby obtained as a crystalline precipitate.

The purity of the product can be improved by washing in ethanol if desired.

When L-alanine is prepared from L-aspartic acid, generally the L-aspartic acid is employed in the form of a 0.1 to 1.5 molar aqueous solution having a pH of from 3 to 7. Ammonium hydroxide/phosphoric acid and other materials can be employed as necessary to obtain the proper pH.

L-alanine can be liberated from the solution as described in Example 4.

Urethane prepolymers useful in preparing the polyurethane foam are prepared by capping a palyoxyalkylene polyol with an exess of polyisocyanate, e.g., toluene diisocyanate. Prior to capping the polyol should generally have a molecular weight of from 200 to 20,000 and preferably from about 600 to about 6,000. The hydroxyl functionality of the polyol and the corresponding isocyanate functionality following capping is usually from 2 to about 8. If foams are formed from prepolymers with an isocyanate functionality of about 2, the resulting product is essentially linear and does not have as much tensile strength as if it were crosslinked.

Accordingly, a hydroxyl content greater than two per molecule is desired. This can be obtained by using mixtures of diols with triols or other higher functionality polyols, or triols or other higher order polyols can be capped with di- or polyisocyanates.

Examples of suitable polyols (to be capped with polyisocyanates) include: (A) essentially linear polyols formed for example by reaction of ethylene oxide with ethylene glycol as an initiator.

Mixtures of ethylene oxide with other alkylene oxides can be employed so long as the mole percent of ethylene oxide is at least 50 percent. Where the linear polyethers are mixtures of ethylene oxide with, e.g. propylene oxide, the polymer can be either a random or a block copolymer and the terminal units can be either oxyethylene or oxypropylene. A second class of polyol (B) includes those with a hydroxy functionality of 3 or more. Such polyols are commonly formed by reacting alkylene oxides with a polyfunctional initiator such as trimethylolpropane or pentaerythritol. In forming the polyol B, the alkylene oxide used can be ethylene oxide or a mixture of ethylene oxide with other alkylene oxide. Thus a blend of a polyoxyethylene glycol with a low molecular weight polyhydric alcohol can be used.By "low molecular weight" is meant up to about 1,000, typically 90 to 1,000, preferably from 90 to 500. Such alcohols generally contain 3 to 20, especially 3 to 8, carbon atoms and 3 to 8, especially 3 to 6, hydroxyl groups. Useful polyols can be further exemplified by (C) linear or branched polyfunctional polyols as exemplified in A and B above together with an initiator or crosslinker. A specific example of C is a mixture of polyethylene glycol (m w about 1,000) with trimethylolpropane, trimethylolethane or glycerine. This mixture can subsequently be reacted with excess polyisocy anate to provide a prepolymer useful in the invention. Alternatively, the linear or branched polyols (e.g. polyethylene glycol) can be reacted separately with excess polyisocyanate. The initiator, e.g. trimethylolpropane, can also be separately reacted with polyisocyanate. Subse quently the two capped materials can be combined to form the prepolymer.

Suitable polyisocyanates and initiators are set forth in US Patent No. 3,903,232. The initiators are generally water-soluble or water-dispersible crosslinking agents, as described in US Patent No 3,903,232.

The following Examples further illustrate the present invention.

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) with 0.66 molar eqivalents of trimethylolpro pane (TMOP). The mixture is dried at 100-110 C under a pressure of 5-1 5 Torr to remove water. The resulting dried mixture is slowly added over a period of about one hour to a vessel containing 5.7 molar equivalents of toluene diisocyanate (TDI) while stirring the TDI and polyol mixture. The temperature is maintained at 60"C with stirring for three additional hours. Then an additional 0.92 molar equivalent of TDI is added with stirring over a period of about one hour while maintaining the temperature at 60"C.The final reaction mixture contains a 5 percent molar excess of TDI. All hydroxyl groups are capped with isocyanate and some chain extension occurs because of crosslinking of the polyols with TDI.

Example 1 Alcaligenes faecalis cells (ATCC 25094) were admixed with 5 gms of Prepolymer A. In forming the admixture eight grams of the harvested culture were employed. On a dry weight basis the culture contained 1 gram of cells. Due to the water in the culture, a flexible hydrophilic polyurethane foam was formed which cured in about 1 5 minutes. The cells were encapsulated within, or bound to, the polyurethane foam matrix.

Example 2 The foam prepared in Example 1 was inserted into a column capped at each end with a Millipore filter. At the bottom of the column 50 gms. of L-aspartic acid was placed in a container in contact with the column. Thereafter a solution (4 I.) of NH4OH (40 ml.) and Tween 80 (10 ml.) having a pH adjusted to 10 with 50% H3PO4 was passed into contact with the aspartic acid and thereafter upwardly through the column. The solution temperature was about 25", and the flow rate was about 0.67 ml./min. For the first 40 hours of operation the column yielded about 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 rate was 1.62 mg./ml. and at 0.17 ml./min. the conversion rate was 2.66 mg.ml.

Analysis for L-alanine was carried out on a Technicon Auto analyzer using L-amino acid oxidase to convert the L-alanine to the corresponding a-keto acid, NH3 and H202. thereafter the peroxide was reacted with 2,4-dichlorophenol and 4-aminoantipyrene in the presence of horseradish peroxidase. The resultant reaction product, a quinoneimine dye, was read colorimetrically at 505 nm.

Example 3 Particulate foam prepared in Example 1 was packed into a column equipped with a water jacket. The temperature was varied and the substrate was passed through at 0.67 ml./min. The substrate consisted of 500 ml. of deionized water containing 10 ml. of concentrated NH4OH, 0.5 ml. of Tween 80 and 53 g. of solid L-aspartic acid at room temperature. In passing through the column the substrate was heated to 27"C and was thereafter cooled to room temperature prior to return to the substrate collection vessel which contained the solid L-aspartic acid in equilibrium with the substrate solution. The substrate was continuously recirculated from the collection vessel, through the column and cooling coil and back to the collection vessel.The above procedure was repeated at temperature of 37"C and 50"C. The 27"C run was continued for 20 hours; the 37"C run for 72 hours; and the 50"C run for about 24 hours. The conversion rates for L-alanine, analyzed as in Example 2, were 11 2.5 mg/hr. (at 27"C); 334 mg./hr. (at 37"C); and 0 mg./hr. (at 50"C).

Example 4 Twenty-six grams of harvested Pseudomonas dacunhae cells (ATCC 21192) were admixed with 20 grams of prepolymer A followed by foam formation as in Example 1. The foam was cut into cubes approximately 1 cc in size, and packed into a 100 cc. column. A substrate containing about 0.5 molar L-aspartic acid was passed through the column. The pH of the substrate was 5.3 (adjusted with H2SO4), and 10 mg./l. of pyridoxal-5-phosphate was also present along with 2.5 g./l. of Tween 80. The column was operated at a flow rate of 0.65 ml./min. giving a conversion to L-alanine (analyzed as in Example 2) of 14 to 17 mg./ml. The substrate was not recirculated, i.e., the run was conducted on a "one-pass" basis.After 700 cc. of substrate had been collected, the L-alanine was isolated by heating the substrate solution to 85"C, adjusting the pH to 5.5, cooling to 1 5"C to precipitate L-aspartic acid and L-alanine, followed by dissolving the precipitate in deionized water to form a solution of L-alanine with L-aspartic acid being filtered off as an insoluble precipitate. The water was removed by vacuum from the filtrate to yield crystals of L-alanine. The yield of crude product was 151.4 grams.

Example 5 After about 2 weeks of operation, the substrate used with the column of Example 4 was changed to a 1 molar solution of L-aspartic acid containing 125 cc. of NK4OH/liter. The pH of the new substrate was adjusted to 5.3 using 60% H2SO4. The flow rate tunas 0.65 ml./min. The initial conversion rate was about 14 mg./ml. Upon substituting a substrate containing only 0.05 moles of L-aspartic acid/liter the conversion level was reduced to 4 to 5 mg./ml.

Subsequent runs with 1 molar substrate gave conversion levels of 27 to 32 mg./ml. Upon recycle the conversion levels reached 45 and 50 mg./ml. Upon addition of pyridoxal-5phosphate ("P-5-P") (12.5 mg./l.) the conversion levels averaged about 36 mg./ml.

Subsequent runs without the pyridoxal-5-phosphate, but with circulation, caused the conversion level to reach 45 mg./ml. After the column had been continuously in operation for about 45 days the conversion level fell to about 16 mg./ml. However much of the operating time was without P-5-P, and it was discovered that by adding P-5-P (12.5 mg./ml.) the conversion level increased to about 40 mg./ml. Subsequently, upon recycling the same batch of substrate for a period of 24 days, the substrate pH increased to about 8.72 indicating a conversion level in excess of 90%. The effluent solution was assayed for L-alanine using the automated procedure in Example 2. it was found that 94% of the L-aspartic acid had been converted to L- alanine.

Example 6 E. coli cells (ATCC 11303) suspended in 0.1 M phosphate buffer (pH 8.0) were spun down at 5000 rpm for 30 minutes. The cell mass was placed under refrigeration and drained overnight.

The cells (11 gms) were admixed with 11 gms of Prepolymer A. Due to the water in the cell mass, a flexible hydrophilic polyurethane foam was formed which cured in about 15 minutes.

The cells were encapsulated within, or bound to, the polyurethane foam matrix.

Example 7 Foam from Example 6 was placed under refrigeration and was thereafter cut into small pieces (approximately 1/4 inch cubes) for later use. A substrate media was prepared from the following recipe: 11.6 gms fumaric acid and 20.3 mgs MgCI2.6 H20, dissolved in 25% ammonium hydroxide with pH adjustment to 8.5 with concentrated ammonium hydroxide.

Sufficient deionized water was added to bring the volume to 100 cc. The temperature of the substrate was maintained at 37"C and about 1.4 gm (dry weight) of foam was placed in the substrate solution. After 30 minutes analysis of samples with thin layer chromotography indicated that considerable amounts of L-aspartic acid had been produced. Further analytical results indicated that 95% of the fumaric acid had been utilized converted to L-aspartic acid.

Example 8 Particulate foam prepared in Example 6 was packed into a 75 cc column equipped with a water jacket. The temperature was maintained at 37"C and substrate was passed through at various flow rates. The substrate composition was as follows: 400 cc NH4OH, 1200 cc deionized water, 232 gms fumaric acid; 406 mg MgCl2 . 6 H20, and add deionized water to bring volume to 2 liters (pH 9.0) (made in two batches). The liquid bed volume in the column (i.e., portion of the column not occupied by the foam) was 31.25 cc.

About 3500 ml. of substrate was passed through the column at a constant rate of 0.6 ml/min at pH 9. Samples were taken periodically and analyzed for fumaric acid by high pressure liquid chromatography. The % conversion to L-aspartic acid in relation to the number of hours the column had been in operation at the time of sampling is set forth in the following table: Stability Study (37 "C) Fumaric/L-Aspartic Con version Time (hours) % Conversion (at 0.6 cc/min) 5 71 46 69 150 61 218 61 Example 9 After the column of Example 8 had been in operation for about 54 hours the flow rate was set at 0.28 cc/minutes and a sample of 288 ml was collected over the succeeding period of 16 hours and 50 minutes. The L-aspartic acid was isolated from a 250 ml portion of the sample and it was found that the actual conversion rate of fumaric acid to L-aspartic acid (based on direct analysis of dried L-aspartic acid by gas-liquid chromatography) was about 85%.

Example 10 About 3500 ml of the substrate passed through the column of Example 8 at a flow rate of 0.60 ml/min was collected. The sample was heated to 90"C and the pH was reduced to 2.8 (the isolectric point for L-aspartic acid) by adding 200 ml of 60% H2SO4. The acidified mixture was cooled to 15"C to precipitate L-aspartic acid which was filtered off, dried and washed in 95% ethanol and water at 30"C. Unreacted fumaric acid dissolved in the ethanol was discarded in solution. The insoluble L-aspartic acid was removed and vacuum dried. A sample of the powder was analyzed at 97% L-aspartic acid for an 89% yield of purified powder.

Example ii Foam was prepared as in Example 6 and a 1 gram sample was placed in a flask and admixed with 100 cc of substrate (126 mg/ml of fumaric acid) at about 37"C and pH 9 for 15 minutes.

Thereafter the process was repeated three times using a fresh batch of substrate each time. In the first batch (based on analysis of residual fumaric acid) the conversion to L-aspartic acid was about 57% in the first batch and about 35% for each of the succeeding three batches. The difference between the first batch and subsequent batches is believed to be due to unbound cell wash-out.

Example 12 A column was prepared and run as in Example 8 (116 mg/cc of fumaric acid, pH 9.04) but at a temperature of 50"C. 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. Completion levels of 33% and 10% were obtained after about 30 and 50 hours respectively. The 50% level is an extrapolated value.

Example 13 Five grams of harvested Brevibacterium thiogenitalis (AHV resistant) cells were admixed with 5 grams of prepolymer A containing 2 wt. % Pluronic L-62, followed by foam formation as in Example 1. The foam was cut into cubes approximately 2 cc in size, and weighed portions (corresponding to 0.42 gm wet weight cells) were added to 25 cc of substrate solution (substrate: 1.64 MM a-ketoisocaproic acid, 1.70 MM L-glutamic acid, 5 x 10-5 M pyridoxal-5phosphate, pH adjusted to 8 or 9 with 2 N NH4OH). With constant mixing (magnetic stir bar) and at ambient temperature, samples were withdrawn at intervals during a day and assayed for leucine production. Typically, foam was in contact with substrate for 8 hours/day, and stored overnight in 0.05 M K phosphate buffer at 4-10"C, and the cycle repeated for 4 days.Analysis for leucine was done by semi-quantitative thin-layer chromatographic and quantitative highpressure liquid chromatographic methods. Foam/cell stability at ambient temperatures was determined at two pH's, 8.0 and 9.) (pH 7.0 was determined to be detrimental to activity).

Stability Study at pH 9.0 a-Keto Acid/Leucine Conversion Hours M Moles leucine/gm wet Days (Sample Time) weight bound cells 1 4 27.22 5 27.22 6 40.83 2 4 31.76 6 45.36 7 45.36 3 3 27.22 4 45.36 5 49.91 4 4 45.36 (After 72 6 72.13 hours 19 258.61 storage 22 285.85 in buffer at 4-10"C) The immobilized cells retained transaminase activity over the 4-day period at ambient temperatures. The gradual increase in rate of leucine production for each day is believed to be the result of "activation", i.e., the immobilized cells are believed to belying, thereby allowing easier substrate product diffusion to and from the transaminase active site. this apparent "activation" was more dramatic for the pH 8.0 study. In the first two days, the foam/cells showed very little activity. However, by day 3 the activity was dramatically increased as shown from the following data: Stability study at pH 8.0 -Keto acid/Leucine Conversion M moles leucine/gm wet Hours weight bound (70-85% Days (Sample Time) water cells 1 4-6 ND 2 4-6 ND 3' 4 186.01 5 245.02 6 249.50 4 4 163.32 (After 72 hours 5 294.88 storage in buffer 6 267.64 at 4-10 C)

Claims (41)

1. A process for preparing an L-amino acid which comprises contacting a substrate with a hydrophilic polyether polyurethane foam wherein at least 50 mole % of the alkylene oxide units in the polyether segments of the polyurethane are ethylene oxide units, said foam comprising an immobilized microorganism capable of converting the substrate to said L-amino acid.

2. A process according to claim 1 wherein the substrate is an aqueous solution having a pH of from 3 to 11.

3. A process according to claim 1 or 2 wherein the substrate is at a temperature of from 1 S'C to 60"C.

4. A process according to any one of claims 1 to 3 which is carried out on a continuous basis.

5. A process according to any one of claims 1 to 3 which is carried out on a batch basis.

6. A process according to any one of the preceding claims wherein at least 90 mol % of the alkylene oxide units in the foam are ethylene oxide units.

7. A process according to any one of the preceding claims wherein the foam is one obtained by reacting a hydrophilic polyether urethane polymer with water containing a culture of the said microorganism.

8. A process according to any one of the preceding claims wherein the prepolymer is a blend of a polyoxyethylene glycol and a low molecular weight (as hereinbefore defined) polyhydric alcohol, said blend being reacted with an excess of a polyisocyanate.

9. A process according to any one of the preceding claims wherein the ratio by weight on an anhydrous basis of the polyurethane polymer to the microorganism is from 1:10 to 10:1.

10. A process according to any one of the preceding claims for preparing L-aspartic acid wherein the substrate comprises fumarate ion and the foam comprises an immobilized aspartaseproducing microorganism.

11. A process according to claim 10 wherein the substrate has a pH of from 8 to 10.

1 2. A process according to claim 10 or 11 wherein the substrate comprises ammonium fumarate.

1 3. A process according to any one of claims 10 to 1 2 wherein the substrate comprises magnesium ion.

1 4. A process according to any one of claims 1 to 9 for preparing L-alanine wherein the substrate comprises L-aspartic acid and the foam comprises an immobilized aspartate decarboxylase-producing microorganism.

15. A process according to claim 1 4 wherein the microorganism is Pseudomonas dacunhae.

1 6. A process according to claim 15 wherein the substrate includes pyridoxal-5-phosphate.

1 7. A process according to claim 14 wherein the microorganism is Alcaligenes faecalis.

1 8. A process according to claim 1 substantially as hereinbefore described.

1 9. An L-amino acid whenever prepared by a process as claimed in any one of the preceding claims.

20. L-aspartic acid whenever prepared by a process as claimed in any one of claims 1 to 1 3 and 18.

21. LAlanine whenever prepared by a process as claimed in any one of claims 1 to 9 and 14to 18.

22. A hydrophilic polyether polyurethane foam wherein at least 50 mole % of the alkylene oxide units in the polyether segments of the polyurethane are ethylene oxide units, said foam comprising an immobilized L-amino acid-producing microorganism.

23. A foam according to claim 22 wherein at least 90 mole % of the alkylene oxide units are ethylene oxide units.

24. A foam according to claim 22 or 23 which has been prepared by reacting a hydrophilic polyether urethane prepolymer with water under foaming conditions, said water containing a culture of a said microorganism.

25. A foam according to any one of claims 22 to 24 wherein the prepolymer is a blend of a polyoxyethylene glycol with a low molecular weight (as hereinbefore defined) polyhydric alcohol, said blend having been reacted with a molar excess of a polyisocyanate.

26. A foam according to any one of claims 22 to25 which is rigid.

27. A foam according to any one of claims 22 to 25 which is flexible.

28. A foam according to any one of claims 22 to 27 wherein the ratio by weight on an anhydrous basis of the polyurethane polymer to the microorganism culture is from 10:1 to 1:1 O.

29. A foam according to any one of claims 22 to 28 wherein the microorganism is an aspartase-producing microorganism.

30. A foam according to any one of claims 22 to 28 wherein the microorganism is an aspartase decarboxylase-producing microoganism.

31. A foam according to claim 22 substantially as hereinbefore described.

32. A process for preparing a hydrophilic poiyether polyurethane foam wherein a culture of an L-amino acid-producing microorganism is mixed with a hydrophilic polyether urethane prepolymer wherein at least 50 mole % of the alkylene oxide units in the polyether segment of the prepolymer are ethylene oxide units, sufficient water being present to cause the prepolymer to foam and thereby entrap and immobilize the microbial cells of the culture.

33. A process according to claim 32 wherein the water content of the culture is from 10 to 90% by weight.

34. A process according to claim 32 or 33 wherein the pH of the mixture is greater than 7.0.

35. A process according to any one of claims 32 to 34 wherein on a weight basis, the prepolymer/culture weight ratio in the mixture is from 1 0:1 to 1:10, the weight of said culture being calculated on a dry weight basis.

36. A process according to any one of claims 32 to 35 wherein the microorganism is an aspartase-producing microorganism.

37. A process according to any one of claims 32 to 35 wherein the microorganism is an aspartase decarboxylate-producing microorganism.

38. A process according to claim 37 wherein the microorganism is Pseudomonas dacunhae or Alcaligenes faecalis.

39. A process according to claim 32 substantially as hereinbefore described.

40. A hydrophilic polyurethane foam whenever prepared by a process as claimed in any one of claims 32 to 39.

41. A process according to any one of claims 1 to 1 8 wherein the foam is one claimed in claim 40.