Patent Publication Number: US-5834259-A

Title: Process and composition for preparing D-aspartic acid

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to a method for preparing D-aspartic acid from racemic aspartic acid. More particularly, the invention relates to a method of preparing D-aspartic acid and β-alanine by enzymatic decarboxylation of racemic aspartic acid. 
     2. Description of the Related Art 
     The L-aspartate-α-decarboxylase enzyme catalyzes the production of β-alanine from L-aspartic acid. Nakano et al. demonstrated the activity of the enzyme in the form of a crude extract from E. coli, B. Nakano, Y. et al., J. Biochem. Vol. 70, pages 327-334 (1971). Williamson et al. further investigated the activity of the enzyme in E. coli purified to apparent homogeneity. Williamson, J. et al., J. Biol. Chem., Vol. 254(16), pages 8074-8082 (August 1979). These disclosures do not demonstrate the production of the enzyme in any useful amount beyond experimental curiosity. As discussed more fully below, this invention utilizes a composition having between 100 and 2000 times the L-aspartate-α-decarboxylase activity of that disclosed in the Nakano et al. and Williamson et al. references. 
     U.S. Pat. No. 5,019,509 to Rozzell discloses methods and compositions for the production of L-alanine and derivatives thereof. Rozzell, however, discloses the gene encoding the aspartate-β-decarboxylase and its use to produce L-alanine. U.S. Pat. Nos. 5,552,317 and 5,552,318 to Houng, et. al. discloses methods for preparing optically active amino acids and esters thereof using lipase from wheat germ or Candida lipolytica. In contrast, the process of this invention is not a common resolution such as the Houng et al. methods, where it is common that resolution of amino acids is actually performed on derivatives of the amino acids, thus requiring additional chemical steps. Most often, racemic esters are treated with esterases/lipases or N-acetyl-amino acids are treated with acylases. 
     Other resolution procedures are known that use an amidase acting on racemic amino acid amides where the enzyme is just specific for one isomer. See, U.S. Pat. No. 4,880,737. However, the present invention provides a much more simple process because there are no additional chemical modifications to prepare the substrate. 
     This invention provides a facile route to produce two highly useful compounds from an abundant source, D,L-aspartate. D-aspartic acid and its derivatives have been widely documented in pharmaceuticals. These uses include, but are not limited to, the inhibition of arginiosuccinate synthetase activity which is useful to prevent or treat sepsis or cytokine-induced systemic hypotension and as immunosuppressive agents. Other uses of D-aspartic acid include taste modifying compositions for foods and a new sweetener, Alitame™, under development by Pfizer. In addition, β-alanine is used in the synthesis of pantothenic acid, which is necessary for coenzyme A synthesis. β-alanine is also known to be used as a chiral synthon for the synthesis of α-substituted β-amino acids. Derivatives of β-alanine have been used as a buffer in the electroplating industry. 
     SUMMARY OF THE INVENTION 
     A racemic mixture of aspartic acid isomers (D and L) are incubated with a microorganism containing the panD gene encoding the enzyme L-aspartate-α-decarboxylase (E.C. 4.1.1.11) or a suitable source of enzyme. The enzyme catalyzes the following reaction: ##STR1## 
     The enzyme stereospecifically decarboxylates L-aspartate to β-alanine and CO 2  whereas D-aspartate remains unreacted. Because this reaction is essentially irreversible with the release of CO 2 , a high conversion of product is realized. This reaction can be carried out under mild conditions, i.e., room temperature and neutral pH, thus making this process very environmentally friendly and highly desirable. 
    
    
     BRIEF DESCRIPTION OF THE FIGURES 
     FIG. 1 shows the production of Plasmid pIF309. 
     FIG. 2 shows the bioconversion of D,L-aspartate substrate solution to D-aspartate and β-alanine, as demonstrated by the reduction in the amount of L-aspartate remaining in solution, catalyzed by the L-aspartate-α-decarboxylase enzyme preparation of the invention. 
     FIG. 3 shows the activity of fresh cells of the L-aspartate-α-decarboxylase enzyme preparation on the supernatant fluid left after completion of the reaction demonstrated in FIG. 2. 
     FIG. 4 shows the activity of previously used L-aspartate-α-decarboxylase enzyme on a fresh D,L-aspartate substrate solution. 
     FIG. 5 shows the activity of L-aspartate-α-decarboxylase enzyme different loads, 50 g/l and 100 g/l, on a 1.0M solution of D,L-aspartate. 
     FIG. 6 shows the activity of L-aspartate-α-decarboxylase enzyme on NH 4  OH-neutralized D,L-aspartate substrate. 
     FIG. 7 compares the activity of L-aspartate-α-decarboxylase enzyme on various amino acid substrates, D,L-aspartate, D,L-glutamate and D,L-alanine. 
     FIG. 8 compares the results of bioconversion of D,L-aspartate at temperatures of 37° C., 45° C. and 50° C. 
    
    
     DETAILED DESCRIPTION 
     The present invention provides a process for preparing D-aspartic acid from D,L-aspartic acid which comprises incubation of a solution of D,L-aspartic acid or a salt thereof with a composition comprising cells of a microorganism or extracts thereof, wherein the composition has an L-aspartate-α-decarboxylase activity of greater than 100 μmol L-aspartate used per hour per gram of cells, under appropriate conditions to produce D-aspartic acid and β-alanine. 
     The enzyme L-aspartate-α-decarboxylase, also referred to herein as L-aspartate-1-decarboxylase, aspartate-1-decarboxylase or E.C. 4.1.1.11, is used in the present invention to prepare D-aspartic acid from racemic aspartic aid according to the following reaction scheme: ##STR2## 
     The substrate and reaction products are alternatively referred to herein by their acid and salt forms. For example, the substrate D,L-aspartic acid is also referred to herein as D,L-aspartate. Those of ordinary skill in the art recognize that the form, and therefore the nomenclature, of an acid differs depending on the pH of the solution in which it is found. As will be discussed more fully below, the process of this invention is useful over a wide pH range which provides for the use of both acid and salt forms of substrate. For the purposes of the present disclosure, the acid and salt-based nomenclature used herein is intended to be synonymous. 
     The panD gene of E. coli K12, encoding the enzyme was cloned into E. coli on a multi copy plasmid vector which is used in a whole cell biotransformation system to produce β-alanine and D-aspartic acid. &#34;Whole cell biotransformation system&#34; refers to the use of cells containing the enzyme which have not been extracted to purify the enzyme but which may be ultrafiltered or otherwise separated from the fermentation media and added directly to the solution to catalyze the reaction to produce β-alanine and D-aspartic acid. Other &#34;Systems&#34; can be used to practice the claimed invention without departing from the scope of the invention. Such methods include, but are not limited to, the use of immobilized cell fractions, immobilized whole enzyme or enzyme fraction. Methods of forming cell fractions and purified enzyme and enzyme fractions are well known to those of ordinary skill in the art. Preferred methods of immobilization are discussed below. 
     The enzyme L-aspartate-α-decarboxylase, also referred to herein as L-aspartate-1-decarboxylase or EC 4.1.1.11, catalyzes the removal of the alpha carboxyl group from L-aspartate to produce β-alanine. 
     The present invention also provides a composition comprising cells of a microorganism or extracts thereof, wherein the composition has an L-aspartate-α-decarboxylase activity of greater than 100 μmol L-aspartate used per hour per gram of cells. 
     In one embodiment the composition comprises cells or extracts of cells of a microorganism transformed with a vector comprising the panD gene which encodes for the L-aspartate-α-decarboxylase enzyme. For example the vector can be a plasmid, such as pIF309 described herein. The vector is then used to transform a microorganism such as a bacterium, for example E. coli. In a preferred embodiment the composition comprises cells or extracts of cells of E. coli NS3291 ATCC Accession NO. 98675. 
     The composition of this invention has an L-aspartate-α-decarboxylase activity of greater than 100 μmol L-aspartate used per hour per gram of cells, and preferably, between about 100 and 2000 μmol L-aspartate used per hour per gram of cells. The enzyme activity of the claimed compositions is calculated using the activity demonstrated in Williamson et al. noted above as a reference point. In that reference, the crude extract showed an activity of 6700 U from 400 grams of cells where 1 U was equal to 1 nmol of CO 2  liberated per minute at 42° C. Thus, 16.75 U/g cells is equal to 16.75 nmol CO 2  liberated per minute per gram of cells. Assuming complete conversion of one mol of L-aspartate to one mol of β-alanine and one mol of CO 2 , 16.75 nmol L-aspartate used per minute per gram of cells would result in approximately 1 μmol L-aspartate utilized per hour per gram cells. As demonstrated in the Examples herein, the composition of the present invention provides an activity of greater than 100 times the activity of the enzyme preparations known in the prior art. From Table III of the Williamson paper the specific activity of the purified enzyme was 650 U/mg protein. Assuming that 50% of the total cell protein has this activity, the present process and composition provide L-aspartate-α-decarboxylase activity up to 2000 μmol L-aspartate used per hour per gram of cells. In a preferred embodiment the composition has an L-aspartate-α-decarboxylase activity of between about 100 and 2000 μmol L-aspartate used per hour per gram of cells. In another embodiment, the composition has an L-aspartate-α-decarboxylase activity of between about 100 and 1000 μmol L-aspartate used per hour per gram of cells. 
     Cloning of the L-aspartate-α-decarboxylase gene can be accomplished by the isolation of a DNA fragment encoding the polypeptide carrying the L-aspartate-α-decarboxylase activity from a suitable donor microorganism, and incorporating the DNA fragment into a suitable vector, known to those of ordinary skill in the art. Suitable donor microorganisms include all microorganisms carrying a gene encoding a polypeptide which catalyzes the α-decarboxylation of L-aspartate to β-alanine. Examples include, but are not limited to, bacteria from the genus Escherichia coli (&#34;E. coli&#34;), including E. coli B, E. coli K12, E. coli NIHJ and E. coli Tennessee; Proteus vulgaris; Bacterium cadaveris; Azotobacter vinelandii; Rhizobium leguminosarum; R. trifolii; or bacterium from the genus Bacillus. 
     Suitable cloning vectors useful in the practice of this invention normally contain an origin of replication and a selectable marker to maintain the stability of the vector in the host cell and to facilitate the identification of transformants. A description of some methods and materials useful in the cloning of the L-aspartate-α-decarboxylase gene can be found in Molecular Cloning: A Laboratory Manual  T. Maniatis, E. Fritsch, and J. Sambrook, Cold Spring Harbor Laboratory (1982)! and references therein, which are hereby incorporated by reference. 
     The DNA fragment encoding the polypeptide displaying L-aspartate-α-decarboxylase activity is then expressed. The general strategy for expression of the L-aspartate-α-decarboxylase gene involves the ligation of the DNA fragment encoding the polypeptide displaying L-aspartate-α-decarboxylase activity into an expression vector suitable for the desired host cell, many examples of which are well known in the art. Suitable expression vectors are normally characterized by the presence of an origin of replication, a promoter or other transcription enhancing sequences, a ribosome binding site, and a transcriptional termination sequence. The expression vector may also include a gene conferring resistance to an antibiotic as a selectable marker; however, plasmids containing any other gene encoding a protein required by the host microorganism for growth and viability may be used as a selectable marker, if desired. Examples of promoters useful in recombinant DNA constructions include, but are not limited to, tryptophan, alkaline phosphatase, beta-galactosidase, beta-lactamase, and P L  promoter systems; and hybrid promoter systems composed of components or structures from two or more known promoter systems such as tac. While these are the most common promoters used in bacterial host strains, other strains and microbial species amenable to genetic manipulation and other promoters useful in those host strains may also be used. 
     After the ligation, the resultant vector of this invention contains the L-aspartate-α-decarboxylase gene in operative association with a promoter such that host cells transformed with the vector are capable of directing the production of L-aspartate-α-decarboxylase. Suitable hosts for transforming with the vector include any microorganism, for example a bacterium, which allows for the expression of the gene encoding L-aspartate-α-decarboxylase. Examples include, but are not limited to, bacteria of the genera Escherichia, Bacillus, Klebsiella, Pseudomonas, Salmonella, Proteus, Azotobacter, and Rhizobium. 
     In a preferred embodiment of this invention, the host strain for expression of the L-aspartate-α-decarboxylase gene is any strain of E. coli, in particular E. coli W3110, and the promoter system is the modified pheA promoter described in U.S. Pat. No. 5,120,837 (Fotheringham et al.) the contents of which are incorporated herein by reference. Expression of the gene is thus accomplished by ligation of the gene into a vector for gene expression in the chosen host strain. In an especially preferred embodiment of this invention, the vector used for the expression of the L-aspartate-α-decarboxylase gene in E. coli is pBR322 or a plasmid which is derived from pBR322, such as pIF309. The plasmid pBR322 contains genes encoding ampicillin and tetracycline resistance, allowing the facile identification of transformed cells. 
     If the preferred embodiment pIF309 is used, expression of the enzyme is constitutive. If a heat sensitive promoter is used, for example the λcI857 repressor/promoter system, induction of the synthesis of L-aspartate-α-decarboxylase can be accomplished by a temperature shift, for example, raising the fermentation temperature from 30° C. to 40° C. L-aspartate-α-decarboxylase enzyme would be expected to be produced at levels of from about 5% to about 40% of the total protein in the cell, thus yielding whole cells containing an enzyme composition with very high L-aspartate-α-decarboxylase activity. 
     It is not critical to this invention whether the expression of the gene results in enzyme produced intracellularly or extracellularly, since the desired product may be conveniently recovered and, if desired, further purified in either case by methods known to those of ordinary skill in the art. In a separately preferred embodiment, the gene can be incorporated into a thermostable microorganism to identify mutations in the gene which enhance the thermal stability of the enzyme thus allowing for the operation of this process at elevated temperatures. 
     In the practice of this invention, the cells producing elevated levels of L-aspartate-α-decarboxylase enzyme may be contacted with a solution containing D,L-aspartic acid or a salt thereof, with the resulting conversion of at least a portion of the L-aspartic acid in the reaction mixture to β-alanine, liberating CO 2  and leaving D-aspartic acid. The cells may be permeabilized to facilitate diffusion of the substrates and products into and out of the cells. This permeabilization can be accomplished by treating cells with a low concentration of a surfactant, including but not limited to Tween 80®, Triton X-100®, Nonidet P40®, cetylpyridinium chloride, deoxycholic acid, hexadecyltrimethylammonium bromide or benzalkonium chloride. Further, organic solvents, including but not limited to N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), ethanol or acetone at low concentrations have also been used to increase permeabilization. L-aspartate-α-decarboxylase may also be added to the D,L-aspartate-containing reaction mixture in the form of cell extracts containing crude, partially purified, or purified L-aspartate-α-decarboxylase enzyme. Cell extracts are prepared by methods known to those skilled in the art which provide for cell disruption and recovery of the enzyme. Cell disruption, can be accomplished by mechanical or non-mechanical means. Most often, for bacterial suspensions mechanical devices such as a French pressure cell, ultrasonication, bead mill or Manton-Gaulin homogenizer is used with the specifics of the method known to those of ordinary skill in the art. See, Scopes, R. K. &#34;Protein Purification&#34;, (1982) (Springer-Verlag, N.Y.). The reaction using the cell extract is then carried out in similar fashion to the whole cell method discussed above. 
     The L-aspartate-α-decarboxylase-containing cells, or extracts thereof or purified enzyme or enzyme fractions, may also be immobilized, if desired. Immobilization methods which may be used in the practice of this invention include well-known methods such as entrapment in polymeric gels, covalent attachment, crosslinking, adsorption, and encapsulation. Some examples of these methods are described by A. M. Klibanov in Science, 219:722-727 (1983) and the references therein and in Methods in Enzymology (1976), Volume 44, (K. Mosbach editor) which are hereby incorporated by reference. In one method of immobilization disclosed in U.S. Pat. No. 5,019,509, a support material containing at least 20% by weight of silica or alumina is contacted with aminoalkyl compound such as an aminoalkyl silane, polyethyleneimine, or a polyalkylamine, followed by activation with glutaraldehyde. The enzyme-containing solution is then contacted with the activated support to produce an immobilized enzyme composition having L-aspartate-α-decarboxylase activity. Other immobilization supports useful in the practice of this invention include, but are not limited to, porous glass and porous ceramics, bentonite, diatomaceous earth, charcoal Sepharose® and Sepharose® derivatives, cellulose and cellulose derivatives, polyacrylamide and polyacrylamide derivatives, polyazetidine, alginate, carrageenan, and Chromosorb®. Sepharose® (Pharmacia Fine Chemicals, Uppsala Sweden) is a bead-formed gel prepared from agarose. The manufacturer&#39;s product literature reports that in its natural state, agarose occurs as part of the complex mixture of charged and neutral polysaccharides referred to as agar. The agarose used to make Sepharose® is obtained by a purification process which removes the charged polysaccharides to give a gel with only a very small number of residual charged groups. Those of ordinary skill in the art will appreciate that a number of other materials suitable for the immobilization of cells or extracts derived therefrom may also be useful for the immobilization of the L-aspartate-α-decarboxylase of the present invention. These supports can be activated, if desired, by techniques well-known in the art. 
     The reaction to produce D-aspartic acid utilizing cells containing L-aspartate-α-decarboxylase, or compositions comprising extracts derived from said cells, is carried out by contacting a solution containing D,L-aspartate with the L-aspartate-α-decarboxylase under conditions permitting the conversion of at least a portion of the L-aspartate to β-alanine. The concentration of D,L-aspartate is not critical in this reaction and concentrations as high as 2M would be anticipated to be useful. In a preferred embodiment of the process the cells or extracts of cells contact an aqueous solution D,L-aspartate having a concentration of 0.5M. In a separately preferred embodiment, the solution of D,L-aspartate has a concentration of 1.0M. 
     The enzymatic reactions of this invention are carried out at temperatures in the range of from about 4° C. to about 70° C., and preferably at temperatures ranging from about 20° C. to about 60° C. In a preferred embodiment, the temperature of the reaction is ambient temperature. For the purposes of this invention, &#34;ambient temperature&#34; means that no methods or devices are employed to either raise or lower the temperature of the reaction vessel from the temperature of the reaction vessel at rest in its natural environment. The optimal pH for the reaction ranges from about 2.0 to about 12.0, and more preferably from about 4.0 to about 9.0, with a pH of 7.0 being most preferred. 
     The D-aspartate and β-alanine may be separated and recovered by methods known in the art, or the D-aspartate/β-alanine mixture may be recovered or used directly as a mixture. The L-aspartate-α-decarboxylase compositions of this invention are further applicable to the production of β-alanine or β-alanine/D-aspartate mixtures containing radioactive or non-radioactive isotopic labels. Such products are readily produced by using an appropriately labelled aspartate precursor, and/or by carrying out the reaction in the presence of isotopically labelled solvent. Examples of isotopic labels which can be incorporated into the β-alanine and/or D-aspartate products include, but are not limited to,  14  C,  13  C,  13  N,  15  N,  2  H,  3  H,  17  O, and  18  O. 
     The invention will now be further illustrated by the following examples, which are not intended, and should not be interpreted, to limit the scope of the invention which is defined in the claims which follow thereafter. 
     EXAMPLE 1 
     Preparation of Plasmid pIF309 
     The pad gene encoding L-aspartate-1-decarboxylase was isolated from the chromosome of E. coli K12 by PCR using the following methods. 
     A. Chromosomal DNA Preparation 
     E. coli strain W3110 was obtained from the Coli Genetic Stock Center (Yale University, Newhaven, Conn.) The strain was recovered as directed in the supplier&#39;s instructions and cultured overnight with shaking in a 50 ml volume of Luria Broth at 37° C. in a 500 ml flask. A 10 ml aliquot of culture was centrifuged at 10,000×G for 4 minutes. The supernatant fluid was discarded and the pellet resuspended in 1 ml of a solution containing 50 mM Tris/HCl at pH 8.0, 10 mM EDTA and 100 μg/ml RNase A (Sigma). This was incubated for 10 minutes at room temperature. To this was added 2 ml of solution containing 0.4% (w/v) sodium dodecyl sulfate (&#34;SDS&#34;) and 100 μg/ml protease K (Sigma). This was incubated for 20 minutes at 37° C. To this was added sodium acetate at pH 5.2 to a final concentration of 300 mM. This solution was then extracted 3 times with an equal volume of phenol at 37° C. and precipitated with 2.5 volumes of ethanol. The DNA was then removed with a sterile loop and redissolved in 400 μl of 300 mM sodium acetate at pH 5.2. The DNA was then re-precipitated in 2.5 volumes of ethanol, removed as before, dried and taken up in 500 μl of 10 mM Tris pH 8.0 and 1 mM EDTA. The final concentration was approximately 200 μg/ml. 
     B. Amplification of panD by PCR 
     Amplification of the E. coli panD gene by PCR was accomplished using a 0.2 ml MicroAmp™ reaction tube (Perkin Elmer, Norwalk, Conn.) to which was added 100 ng E. coli chromosomal DNA prepared as above, 2 μl each of dATP, dGTP, dCTP and dTTP (10 mM each), 10 μl of buffer comprising 15 mM MgCl 2 , 500 mM KCl, 100 mM Tris pH 8.3, and 0.01% (w/v) gelatin, 5 U of Taq DNA polymerase (AmpliTaq™) and 5 μl of a 10 nmol/ml solution of each of the following oligonucleotide primers which were synthesized according to known methods using an Applied Biosystems 380B DNA synthesizer. 
     (MB1682) 
     5&#39; CGC GGA TCC ACT ATG ATT CGC ACG ATG CTG CAG GGC 3&#39; 
     (MG1683) 
     5&#39; CAG CGT GCA TGC TCA AGC AAC CTG TAC CGG AAT CGC 3&#39; 
     The reaction was carried out in a Perkin Elmer 9600™ PCR Thermal Cycler (Perkin Elmer). Amplification was carried out by pre-heating at 94° C. for 3 minutes followed by 30 cycles of denaturation at 94° C. for 30 seconds, annealing at 50° C. for 30 seconds and extension at 72° C. for 2 minutes. The reaction mixture was then stored at 4° C. 
     C. Cloning of panD 
     The amplified panD gene was cloned on a plasmid vector derived from plasmid pBR322. This plasmid was named pIF309. The fragment containing the panD gene was digested to completion with the restriction enzymes BamHI and SphI and ligated between the unique BamHI and SphI sites of the vector which had been similarly cleaved. Between the HindIII and BamHI sites was inserted a synthetic DNA fragment which carried a modified version of the E. coli K12 pheA promoter region. This fragment was completely synthesized on an Applied Biosystems 380B DNA synthesizer and derived from that characterized in co-owned U.S. Pat. No. 5,120,837 to Fotheringham et al. such that the sequence was as follows: 
     HindIII 
     AAGCTTTTTTGTTGACAGCGTGAAAACAGTACGGGTATAATACT AAAGTCACAAGGAGGATCC 
     BamHI 
     Plasmid pIF309 is shown in FIG. 1. Restriction digests were carried out using enzymes supplied by New England Biolabs and used according to the manufacturer&#39;s specifications (NEB, Beverly, Mass.). Ligation of DNA fragments was carried out using a ligation kit supplied by Takara Biochemicals (Panvera Corp., Madison Wis.) according to the manufacturer&#39;s specifications. 
     Expression of panD was determined by biological assay of L-aspartate-1-decarboxylase activity in extracts prepared from cultures of strain NS3291 grown as described below. 
     EXAMPLE 2 
     Production and Fermentation of E. coli Strain NS3291 
     Plasmid pIF309 was used to transform E. coli strain W3110, using conditions described in U.S. Pat. No. 5,354,672 the contents of which are hereby incorporated by reference, resulting in the strain NS3291. Strain NS3291 was deposited with the American Type Culture Collection (ATCC) 12301 Parklawn Drive, Rockville, Md. 20852, USA. The strain is designated ATCC Accession No. 98675. 
     The strain NS3291 was inoculated into a 2800 ml Fernbach flask containing 1 L of the following growth medium: 
     
         ______________________________________                                    
Potassium phosphate (dibasic)                                             
                          13     g                                        
Potassium phosphate (monobasic)                                           
                          2      g                                        
Ammonium phosphate        4      g                                        
Ferric ammonium citrate   0.24   g                                        
Yeast extract             2      g                                        
Magnesium sulfate (7 · H.sub.2 O)                                
                          1      g                                        
Water                     930    ml                                       
After Sterilization the following were added:                             
Glucose (50% W/V stock)   70     ml                                       
Ampicillin                0.2    g                                        
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     The flask was incubated on a shaker incubator at 37° C. with agitation. The strain was grown to 800-1100 Klett units and used to inoculate the fermentor. The fermentor was a Biolafite 78-100 (St. Germain-en Laye, France) 20 L. The fermentor was operated under the following conditions: 
     
         ______________________________________                                    
Agitation             500    rpm                                          
Temperature           37°                                          
                             C.                                           
Back Pressure         0.7    Bar                                          
pH (adjusted with NH.sub.3)                                               
                      7.2                                                 
Aeration              1      vvm                                          
Set Volume            10     L                                            
Inoculation           100    ml                                           
Run Time              16     hr                                           
______________________________________                                    
 
    
     The fermentation medium was comprised of the following, per liter of medium unless otherwise noted: 
     
         ______________________________________                                    
Magnesium Sulfate (7 · H.sub.2 O)                                
                      5.35    g                                           
Ferric ammonium citrate                                                   
                      0.13    g                                           
Potassium phosphate (dibasic)                                             
                      4.6     g                                           
Manganese sulfate     0.023   g                                           
Potassium iodide      0.74    mg                                          
Nickel sulfate        0.74    mg                                          
Antifoam (Mazur Mazu DF204)                                               
                      0.4     ml                                          
Yeast extract         5       g                                           
L-aspartic acid       10      g                                           
Tap water             10      L                                           
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     Prior to inoculation, glucose was added to a concentration of 25 g/l. After the initial glucose was completely depleted, glucose was fed at a variable rate to achieve less than 1 g/l for the remaining time for a total of 290 grams of glucose in 16 hours. The final volume of the tank was 12.2 L. The fermentation reached 10,352 Klett units and a dry cell weight of 24.2 g/l. The fermentor broth was cooled to below 10° C. and ultrafiltered using a hollow fiber cartridge (500,000 Mol Wt. cutoff) to a dry cell weight of 158 g/l. The concentrated cell cream was stored at 4° C. 
     This strain was used in the following Examples. 
     EXAMPLE 3 
     Bioconversion of D/L-aspartate with L-aspartate-α-decarboxylase 
     66.5 g (0.5M) D/L-aspartate (Sigma, A-9006) was weighed into a 2 L beaker and the pH adjusted to 7.0 with 10N NaOH. The volume was brought to 963 ml with deionized H 2  O. 137 ml of cells (NS 3291, described above) were added at a packed cell volume (&#34;PCV&#34;) of 7.31 g/10 ml cells resulting in a final volume of 1.1 L. Samples (approximately 1 ml each) were removed over time, centrifuged and the supernatant fluid was analyzed by HPLC for D- and L-aspartic acid content. 
     In all of the Examples herein, D- and L-aspartic acid isomers were quantified by HPLC using the following precolumn derivatization method. Aspartic acid was converted into diastereomers using ortho-phthalaldehyde and N-acetyl-L-cysteine. Separation of the diastereomers was achieved on a Supelcosil™ LC-18DB, 3μ, 150×4.6 mm column using a mobile phase of A: 20% methanol/80% triethylamine phosphate and B: methanol, and a step gradient as follows: 
     
         ______________________________________                                    
Time (min)       A (%)   B (%)                                            
______________________________________                                    
 6.5             100      0                                               
 7.0              40     60                                               
11.5              40     60                                               
12.0             100      0                                               
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     The derivatization was carried out automatically using a Hewlett-Packard 1090M chromatograph and detection was at 338 nm (bandwidth 4) and a flow rate of 1.0 ml/minute. Retention times were 4.19 minutes and 4.54 minutes for D-aspartic acid and L-aspartic acid, respectively. 
     Samples from bioconversions were centrifuged to remove cells in a microcentrifuge and the supernatant fluid diluted appropriately such that the value determined from the HPLC fell within the linear region of the standard curve. These dilutions were usually between 1:100 and 1:200, made with deionized water. 
     Throughout the Examples the terms &#34;D-Asp&#34; and &#34;L-Asp&#34; are used as short hand notation for the respective isomers of aspartic acid. 
     
                       TABLE 1                                                     
______________________________________                                    
Time      D-Asp   D-Asp    L-Asp  L-Asp                                   
(hr)      (mg/ml) (%)      (mg/ml)                                        
                                  (%)                                     
______________________________________                                    
0    0.0000   29.300  100.00 28.870 100.00                                
1    5.5000   29.320  100.07 12.050 41.739                                
2   24.500    29.250  99.829 0.17000                                      
                                    0.58885                               
3   26.500    27.740  94.676 0.12000                                      
                                    0.41566                               
4   32.500    28.090  95.870 0.060000                                     
                                    0.20783                               
5   47.500    28.060  95.768 0.030000                                     
                                    0.10391                               
6   52.000    28.390  96.894 0.020000                                     
                                     0.069276                             
7   68.000    25.520  87.099 0.43000                                      
                                    1.4894                                
______________________________________                                    
 
    
     The data in Table 1 are presented graphically in FIG. 2. At approximately 24 hours, the percent L-aspartate remaining is essentially negligible, about 0.17 mg/ml. The enzyme activity of the composition used in the bioconversion is calculated as follows: ##EQU1## which equals 114.6 μmol L-aspartic acid used per hour per gram cells, or more than 100 times the enzyme activity known in the prior art. 
     The enantiomeric enrichment at this time is calculated as follows: ##EQU2## 
     Following the whole-cell bioconversion reaction the cells were removed by centrifugation or ultrafiltration. The pH of the cell-free bioconversion mixture was adjusted with mineral acid, usually sulfuric acid, to a pH where D-aspartic acid precipitates as a solid and the β-alanine remains in solution, typically between pH 2.0 and 2.5. The solid was collected by filtration or other means and washed with cold water to afford D-aspartic acid. Recovery was between 50% and 90%. 
     EXAMPLE 4 
     Re-Treatment of Supernatant Fluid with Fresh Cells 
     In this Example the supernatant fluid from Example 3 was re-treated with fresh cells. The reaction from Example 3, stopped at 68 hours, was centrifuged for 45 minutes in a GS-3 rotor at 7000 rpm. The supernatant fluid, 990 ml, was separated from the cells. 963 ml of this supernatant fluid was mixed with 137 ml fresh cells and placed in a 2 L fermentor. Bioconversion took place with mixing at 300 rpm, without automatic pH control and at a temperature of 37° C. Samples, approximately 1 ml, were removed over time and analyzed as in Example 3. 
     
                       TABLE 2                                                     
______________________________________                                    
Time        D-Asp   D-Asp     L-Asp  L-Asp                                
(hr)        (mg/ml) (%)       (mg/ml)                                     
                                     (%)                                  
______________________________________                                    
0     0.0000    21.450  100.00  0.43000                                   
                                       100.00                             
1     2.7500    21.350  99.534  0.11000                                   
                                       25.581                             
2     13.500    21.190  98.788  0.090000                                  
                                       20.930                             
3     15.000    20.770  96.830  0.080000                                  
                                       18.605                             
4     19.500    21.480  100.14  0.090000                                  
                                       20.930                             
5     23.500    20.880  97.343  0.070000                                  
                                       16.279                             
6     39.000    19.080  88.951  0.10000                                   
                                       23.256                             
7     47.000    17.280  80.559  0.16000                                   
                                       37.209                             
8     51.750    17.010  79.301  0.23000                                   
                                       53.488                             
9     68.500    14.900  69.464  0.55000                                   
                                       127.91                             
______________________________________                                    
 
    
     The data in Table 2 are represented graphically in FIG. 3. Although the concentration of L-aspartic acid at t=0 was not high (0.43 mg/ml), it was essentially reduced to nothing (0.07 mg/ml) by 23.5 hours. It is believed that the increases shown for concentration of L-aspartic acid after 23.5 hours is due to evaporation, although concomitant synthesis from extraneous enzymes could not be ruled out. At the end of the reaction (68.5 hours) there was only 0.55 mg/ml L-aspartic acid. 
     EXAMPLE 5 
     Re-use of Cells 
     Cells that had already been used in Example 3 were put in fresh substrate to determine if they were still active. 
     Fresh D/L-aspartate substrate was prepared as previously described in Example 3. The cells from Example 3 were resuspended in deionized H 2  O and made up to 100 ml. The pH of the substrate (900 ml, 0.5M D/L-Asp) was adjusted to 7.0 with NaOH. After placing in the 2 L fermentor, the cells (100 ml) were added. There was no pH control and deionized H 2  O was used for substrate. Samples were removed over time, centrifuged and the supernatant fluid diluted appropriately for both D- and L-aspartic acid determination. Temperature was set at 37° C. 
     
                       TABLE 3                                                     
______________________________________                                    
Time        D-Asp   D-Asp     L-Asp  L-Asp                                
(hr)        (mg/ml) (%)       (mg/ml)                                     
                                     (%)                                  
______________________________________                                    
0     0.0000    30.850  100.00  24.700 100.00                             
1     6.2500    30.820  99.903  17.130 69.352                             
2     15.250    29.870  96.823  15.620 63.239                             
3     25.750    28.250  91.572  10.870 44.008                             
4     28.000    27.980  90.697  9.5300 38.583                             
5     44.250    25.000  81.037  4.4200 17.895                             
6     63.500    21.760  70.535  0.98000                                   
                                       3.9676                             
7     65.000    20.340  65.932  0.95000                                   
                                       3.8462                             
______________________________________                                    
 
    
     The data in Table 3 are also graphically represented in FIG. 4. These results show that even after one cycle, the L-aspartate-α-decarboxylase still remains active, thus indicating that this biocatalyst can be recycled. 
     EXAMPLE 6 
     D/L-aspartate as Substrate for Various Enzyme Cell Loads 
     1.0M D/L-aspartate was tested as a substrate for two enzyme cell loads, 100 g/l cells, referred to here as reaction &#34;A&#34;, and 50 g/l cells, referred to here as reaction &#34;B&#34;. 
     133.1 g of D/L-aspartic acid was dissolved in approximately 600 ml deionized H 2  O and pH was adjusted to 7.0 with NaOH, using 100 ml of 10N NaOH and a small amount of 1.0N NaOH. The substrate for reaction A was brought to 963 ml with deionized H 2  O. The reaction was started with 137 ml cells that were stored in the cold room. For reaction B, the substrate was brought to a volume of 981.5 ml. The reaction was started with 65.0 ml cells. 
     Both reactions were carried out in 2 L fermentors at pH 7.0 without constant adjustments and at 37° C. Samples were removed over time, centrifuged and the supernatant fluid was diluted and submitted for D- and L-aspartic acid HPLC analysis. Table 4 shows the results of reaction A and Table 5 shows the results of reaction B. 
     
                       TABLE 4                                                     
______________________________________                                    
Time        D-Asp   D-Asp     L-Asp  L-Asp                                
(hr)        (mg/ml) (%)       (mg/ml)                                     
                                     (%)                                  
______________________________________                                    
0     0.0000    53.600  100.00  54.850 100.00                             
1     2.0000    50.680  94.552  49.340 89.954                             
2     4.5000    44.920  83.806  50.710 92.452                             
3     7.0000    42.260  78.843  50.230 91.577                             
4     22.500    35.680  66.567  54.490 99.344                             
5     24.000    34.420  64.216  50.780 92.580                             
6     31.000    32.660  60.933  51.450 93.801                             
7     37.500    27.950  52.146  53.850 98.177                             
8     47.000    20.710  38.638  52.370 95.479                             
9     50.500    18.490  34.496  51.080 93.127                             
10    53.800    13.730  25.616  46.500 84.777                             
11    58.000    13.410  25.019  50.040 91.231                             
12    70.000    6.1300  11.437  52.400 95.533                             
13    72.000    5.3900  10.056  50.430 91.942                             
______________________________________                                    
 
    
     
                       TABLE 5                                                     
______________________________________                                    
Time        D-Asp   D-Asp     L-Asp  L-Asp                                
(hr)        (mg/ml) (%)       (mg/ml)                                     
                                     (%)                                  
______________________________________                                    
0     0.0000    56.730  100.00  57.440 100.00                             
1     2.0000    52.420  .92.403 53.770 93.611                             
2     4.5000    51.340  90.499  52.310 91.069                             
3     7.0000    50.470  88.965  53.860 93.767                             
4     22.500    44.260  78.019  53.650 93.402                             
5     24.000    44.280  78.054  53.460 93.071                             
6     31.000    40.590  71.549  51.740 90.077                             
7     37.500    38.650  68.130  55.000 95.752                             
8     47.000    35.420  62.436  53.410 92.984                             
9     50.500    34.920  61.555  54.310 94.551                             
10    53.800    31.780  56.020  55.060 95.857                             
11    58.000    30.860  54.398  53.420 93.001                             
12    70.000    24.300  42.834  54.060 94.116                             
13    72.000    26.830  47.294  54.230 94.412                             
______________________________________                                    
 
    
     The data in Tables 4 and 5 are graphically presented in FIG. 5. Results show that the conversion rates for 100 g/l are faster than with 50 g/l. However, the rate at a substrate concentration of 1.0M D/L-aspartate is slower that at 0.5M. (See Example 3 and FIG. 2). Also, at 1.0M substrate, after 72.0 hours, the L-aspartate was reduced to 5.39 mg/ml from a starting concentration of 53.6 mg/ml, a 90% reduction. 
     EXAMPLE 7 
     L-aspartate-α-decarboxylase NH 4  OH Neutralized Substrate 
     This Example shows the effect of neutralizing the substrate, D/L-aspartate, with NH 4  OH instead of NaOH as done in the prior examples. Cells were stored frozen at -80° C. 133.1 g (1 mol) D/L-aspartic acid was dissolved in approximately 600 ml deionized H 2  O and adjusted to a pH of about 7 with concentrated NH 4  OH. The final volume of substrate was brought to 963 ml to which was added 137 ml cells, freshly thawed. The mixture was placed in the 2 L fermentor and the temperature adjusted to 37° C. No automatic pH control was used, however, the pH was monitored as samples were removed and the pH adjusted with 50% NH 4  OH. Samples, approximately 1 ml, were removed periodically, centrifuged, and the supernatant fluid was stored frozen prior to diluting and HPLC analysis. 
     
                       TABLE 6                                                     
______________________________________                                    
Time        D-Asp   D-Asp     L-Asp  L-Asp                                
(hr)        (mg/ml) (%)       (mg/ml)                                     
                                     (%)                                  
______________________________________                                    
0     0.0000    55.260  100.00  55.740 100.00                             
1     3.0000    54.670  98.932  53.380 95.766                             
2     6.0000    49.100  88.853  56.370 101.13                             
3     9.0000    47.810  86.518  56.680 101.69                             
4     19.000    43.490  78.701  59.270 106.33                             
5     23.500    39.120  70.793  54.930 98.547                             
6     28.000    36.290  65.671  52.620 94.403                             
7     32.500    33.540  60.695  50.900 91.317                             
8     43.000    30.370  54.958  53.520 96.017                             
9     49.000    21.580  39.052  51.i00 91.676                             
______________________________________                                    
 
    
     The data in Table 6 is presented graphically in FIG. 6. The rate of L-Asp utilization is shown to be slower with the NH 4   +  salt as compared to the Na +  salt. 
     EXAMPLE 8 
     Activity of L-aspartate-α-decarboxylase towards D/L-glutamate and D/L-alanine 
     This Example shows the activity of L-aspartate-α-decarboxylase towards two potential amino acid substrates, D/L-glutamate and D/L-alanine. D/L-aspartate was run as a control. 
     Substrates were prepared by dissolving solid amino acid racemate in approximately 75 ml deionized H 2  O and adjusting the pH to 7.0 with 10N NaOH. The volume was then brought to 100 ml with deionized H 2  O. The cells used were stored frozen for about 1 month and then thawed and stored in a refrigerator for about 1 month prior to running the experiments. 
     The reaction was run the same as described in the prior Examples. Samples of supernatant fluid, approximately 0.20 ml, were removed over time, centrifuged and stored frozen before analysis by HPLC as described in the prior Examples. Table 7 shows the results of the reactions. 
     
                                           TABLE 7                                 
__________________________________________________________________________
Time L-Glu                                                                
         D-Glu                                                            
             L-Ala                                                        
                 D-Ala                                                    
                     L-Glu                                                
                         D-Glu                                            
                             L-Ala                                        
                                 D-Ala                                    
                                     L-Asp                                
                                         D-Asp                            
                                             L-Asp                        
                                                  D-Asp                   
(hr) (mg/ml)                                                              
         (mg/ml)                                                          
             (mg/ml)                                                      
                 (mg/ml)                                                  
                     (%) (%) (%) (%) (mg/ml)                              
                                         (mg/ml)                          
                                             (%)  (%)                     
__________________________________________________________________________
0 0  29.350                                                               
         29.440                                                           
             19.320                                                       
                 19.190                                                   
                     100.00                                               
                         100.00                                           
                             100.00                                       
                                 100.00                                   
                                     26.810                               
                                         29.890                           
                                             100.0                        
                                                  100.0                   
1 3.5                                                                     
     28.400                                                               
         29.010                                                           
             18.960                                                       
                 18.590                                                   
                     96.763                                               
                         98.539                                           
                             98.137                                       
                                 96.873                                   
                                     14.050                               
                                         29.850                           
                                             52.406                       
                                                  99.866                  
2 8.5                                                                     
     28.740                                                               
         29.520                                                           
             18.730                                                       
                 18.370                                                   
                     97.922                                               
                         100.27                                           
                             96.946                                       
                                 95.727                                   
                                     12.660                               
                                         29.900                           
                                             47.221                       
                                                  100.03                  
3 24 28.200                                                               
         29.350                                                           
             18.320                                                       
                 17.930                                                   
                     96.082                                               
                         99.694                                           
                             94.824                                       
                                 93.434                                   
                                     9.4700                               
                                         29.740                           
                                             35.323                       
                                                  99.498                  
4 55 28.000                                                               
         29.840                                                           
             17.830                                                       
                 17.580                                                   
                     95.400                                               
                         101.36                                           
                             92.288                                       
                                 91.610                                   
                                     0.3300                               
                                         30.120                           
                                             1.2309                       
                                                  100.77                  
__________________________________________________________________________
 
    
     The data in Table 7 are presented graphically in FIG. 7. The results show the specificity of the claimed method and enzyme preparation in that all isomers of substrates used except L-aspartic acid are stable up to 55 hours in the presence of the enzyme, indicating that L-aspartic acid is the only substrate for the enzyme. L-aspartic acid, as demonstrated throughout the specification, decreased to 1.23% of starting material. 
     EXAMPLE 9 
     Temperature Study 
     This Example shows the activity of the L-aspartate-α-decarboxylase reaction at 37° C., 45° C. and 50° C. 
     133.1 g (1 mol) of D/L-aspartic acid was dissolved in deionized H 2  O and the pH was adjusted to 7.0 with 10N NaOH, approximately 100 ml. Cells, freshly thawed from -80° C., were added to achieve a final concentration of 100 g/l. Three 2 L fermentors were each charged with 963 ml of substrate solution and 137 ml of cells and then heated to 37° C. and 45° C. while mixing at 300 rpm or heated to 50° C. while mixing at 450 rpm. Samples, approximately 1 ml, were removed over time and pH adjusted to 7.0 with either NaOH or HCl. After centrifugation to remove cells, the supernatant fluid was analyzed for both D- and L-aspartic acid. 
     Table 8 shows the results of the reaction at 37° C., Table 9 shows the results at 45° C. and Table 10 shows the results at 50° C. 
     
                       TABLE 8                                                     
______________________________________                                    
Time        D-Asp   D-Asp     L-Asp  L-Asp                                
(hr)        (mg/ml) (%)       (mg/ml)                                     
                                     (%)                                  
______________________________________                                    
0     0.0000    55.000  100.00  53.700 100.00                             
1     2.5000    54.310  98.745  48.940 91.136                             
2     4.0000    54.440  98.982  47.600 88.641                             
3     6.0000    54.470  99.036  46.170 85.978                             
4     8.0000    54.760  99.564  44.810 83.445                             
5     10.000    55.840  101.53  42.120 78.436                             
6     12.000    56.760  103.20  42.120 78.462                             
7     20.000    55.460  100.84  35.960 66.965                             
8     24.000    55.390  100.71  34.840 64.879                             
9     29.000    53.990  98.164  28.040 54.078                             
10    34.000    53.440  97.164  28.040 54.078                             
11    44.000    52.960  96.291  21.670 40.354                             
12    48.000    52.940  96.255  18.440 34.339                             
13    68.000    49.940  90.800  5.9700 11.117                             
14    92.000    50.670  92.127  0.91500                                   
                                       1.7039                             
______________________________________                                    
 
    
     
                       TABLE 9                                                     
______________________________________                                    
Time        D-Asp   D-Asp     L-Asp  L-Asp                                
(hr)        (mg/ml) (%)       (mg/ml)                                     
                                     (%)                                  
______________________________________                                    
0     0.000     55.280  100.00  54.410 100.00                             
1     2.5000    54.820  99.168  50.i00 92.079                             
2     4.0000    53.960  97.612  47.110 86.583                             
3     6.0000    53.460  96.708  43.130 79.269                             
4     8.0000    54.040  97.757  39.310 72.248                             
5     10.000    54.870  99.258  36.800 67.635                             
6     12.000    54.060  97.793  34.540 63.481                             
7     20.000    52.360  94.718  24.690 45.378                             
8     24.000    51.310  92.818  23.210 42.658                             
9     29.000    53.830  97.377  21.700 39.882                             
10    34.000    52.880  95.658  19.310 35.490                             
11    44.000    53.640  97.033  15.800 29.039                             
12    48.000    52.730  95.387  16.700 30.822                             
13    68.000    53.560  96.889  16.050 29.498                             
14    92.000    53.560  96.889  13.370 24.573                             
______________________________________                                    
 
    
     
                       TABLE 10                                                    
______________________________________                                    
Time        D-Asp   D-Asp     L-Asp  L-Asp                                
(hr)        (mg/ml) (%)       (mg/ml)                                     
                                     (%)                                  
______________________________________                                    
0     0.000     54.89   48.47   100.0  100.0                              
1     1.000     54.30   52.78   98.93  108.9                              
2     3.000     58.01   40.61   105.7  83.78                              
3     5.0000    58.16   36.97   106.0  73.63                              
4     7.000     58.51   35.69   106.6  73.63                              
5     9.000     58.88   33.99   107.3  70.13                              
6     13.50     59.62   34.19   108.4  70.54                              
7     23.00     56.71   31.98   103.3  65.98                              
8     25.00     59.85   31.86   109.0  65.73                              
9     27.00     56.87   26.89   103.6  55.48                              
10    32.00     57.30   25.85   104.4  53.33                              
11    37.50     58.48   27.54   106.5  58.82                              
12    47.00     57.93   25.58   10s.s  52.77                              
13    52.00     58.74   27.28   107.0  56.28                              
14    57.00     65.56   32.42   119.4  66.89                              
15    71.00     57.17   25.52   104.2  52.65                              
16    77.00     57.85   25.86   105.4  53.35                              
17    81.00     57.41   25.54   104.6  52.69                              
18    99.75     59.36   26.14   108.1  53.93                              
______________________________________                                    
 
    
     The data from Tables 8, 9 and 10 are presented graphically together in FIG. 8. These results show that at all three temperatures, the D-isomer is stable. At 37° C., the L-isomer is almost completely consumed by 99.75 hours. At 45° C. and 50° C. the initial rate is shown to be increased with the higher temperature, but it appears that the enzyme becomes unstable and inactivates after an extended period of time. Although moderate temperatures are preferred for the claimed process and composition, the reaction rate can be increased by raising the reaction temperature.