US00782.9324B2

(12) United States Patent (10) Patent No.: US 7,829,324 B2 Glick et al. (45) Date of Patent: Nov. 9, 2010

(54) METHOD FOR THE MICROBIOLOGICAL Schnell, B., et al., “Enzymatic Racemisation and its Application to SOMERISATION OF ALPHA-HYDROXY Synthetic Biotransformations'. Advanced Synthesis & Catalysis, CARBOXYLIC ACDS 2003, vol. 345, pp. 653-666. Liu, S-Q., “Practical Implications of Lactate and Pyruvate Metabo (75) Inventors: Silvia Glick, Graz (AT); Barbara lism by Lactic Acid Bacteria in Food and Beverage Fermentations'. Schnell, Graz (AT); Monika Pirker, International Journal of Food Microbiology, 2003, vol. 83, pp. 115 Graz (AT); Kurt Faber, Graz (AT) 131. Strauss. U.T., et al., “Deracemization of (+)-Mandelic Acid Using a (73) Assignee: BASFSE, Ludwigshafen (DE) Lipase-Mandelate Racemase Two-enzyme System”. Tetrahedron: Asymmetry, 1999, vol. 10, pp. 4079-4081. (*) Notice: Subject to any disclaimer, the term of this Felfer, U., et al., “Substrate Spectrum of Mandelate Racemase Part 2. patent is extended or adjusted under 35 (Hetero)-Aryl-Substituted Mandelate Derivatives and Modulation of U.S.C. 154(b) by 457 days. Activity”. Journal of Molecular Catalysis B: Enzymatic, 2001, vol. 15, pp. 213-222. (21) Appl. No.: 10/560,455 Schafer, S.L., et al., “Mechanism of the Reaction Catalyzed by Mandelate Racemase: Structure and Mechanistic Properties of the (22) PCT Filed: Jun. 17, 2004 D27ON Mutant”, Biochemistry, 1996, vol. 35, pp. 5662-5669. Garcia-Viloca, M., et al., “A QM/MM Study of the Racemization of (86). PCT No.: PCT/EP2004/OO6564 Vinylglycolate Catalyzed by Mandelate Racemase Enzyme'. Journal of the American Chemical Society, 2001, vol. 123, pp. 709-721. S371 (c)(1), Li, R., et al., “Racemization of Vinylglycolate Catalyzed by (2), (4) Date: Dec. 14, 2005 Mandelate Racemase'. Journal of Organic Chemistry, 1995, vol. 60, pp. 3347-3351. (87) PCT Pub. No.: WO2004/111257 Kenyon, G.L., et al., “Mandelate Racemase: Structure—Function Studies of a Pseudosymmetric Enzyme'. Acc. Chem.Res., 1995, vol. PCT Pub. Date: Dec. 23, 2004 28, pp. 178-186. Pearson, W.R., et al., “ImprovedTools for Biological Sequence Com (65) Prior Publication Data parison”. Proc. Natl. Acad. Sci. USA, 1988, vol. 85, pp. 2444-2448. US 2006/0148051A1 Jul. 6, 2006 Narang, S.A., “DNA Synthesis”, Tetrahedron, 1983, vol.39, No. 1, pp. 3-22. (30) Foreign Application Priority Data Itakura, K., et al., “Synthesis and Use of Synthetic Oligonucleotides'. Ann. Rev. Biochem., 1984, vol. 53, pp. 323-356. Jun. 18, 2003 (DE) ...... 103 27 582 Itakura, K., et al., Expression in Escherichia coli of a Chemically Synthesized Gene for the Hormone Somatostatin, 1977, vol. 198, pp. (51) Int. Cl. 1056-1063. CI2P 7/40 (2006.01) Ike. Y., et al., “Solid Phase Synthesis of Polynucleotides. VIII. Syn (52) U.S. Cl...... 435/280; 435/252.9; 435/136 thesis of Mixed Oligodeoxyribonucleotides by the Phosphotriester

(58) Field of Classification Search ...... None Solid Phase Method’, Nucleic Acids Research, 1983, vol. 11, No. 2, See application file for complete search history. pp. 477-488. Arkin, A.P. et al., “An Algorithm for Protein Engineering: Simula (56) References Cited tions of Recursive Ensemble Mutagenesis'. Proc. Natl. Acad. Sci. U.S. PATENT DOCUMENTS USA, 1992, vol. 89, pp. 7811-7815. 5,534,436 A * 7/1996 Seufer-Wasserthal et al. .... 435/ (Continued) Primary Examiner Irene Marx 6,605,430 B1* 8/2003 Affholter et al...... so (74) Attorney, Agent, or Firm—Connolly Bove Lodge & Hutz, LLP FOREIGN PATENT DOCUMENTS (57) ABSTRACT EP 0596466 5, 1994

OTHER PUBLICATIONS The present invention relates to a method for the microbio Dennis et al., Annals of the New York Academy of Sciences, Jul. logical isomerization of alpha-hydroxycarboxylic acids 1965, vol. 119, No. 3 : pp. 868-876.* using an alpha-hydroxycarboxylic acid racemase, the Hiyama et al., J Biochem. 1968; 64:99-107.* enzymes used for this method and microorganisms which Stetter et al., Arch Mikrobiol. Dec. 31, 1973:94(3):221-47.* express a suitable racemase activity, a screening method for DSMZ. Catalogue on line, accessed Jun. 26, 2008 (1 page) http:// microorganisms with alpha-hydroxycarboxylic acid race www.dsmz.de/microorganisms/html/strains/strain.dsm020017. mase activity, the nucleic acid sequences encoding this html. enzyme, expression vectors, recombinant microorganisms DSMZ. Catalogue on line, accessed Jun. 26, 2008 (1 page) http:// which express this racemase, and methods for the production www.dsmz.de/microorganisms/html/strains/strain.dsm020207. or isolation of a protein with alpha-hydroxycarboxylic acid html. racemase activity. Glueck, S. M., et al., “Lactate Racemase. As a Versatile Tool for the Racemization of C-Hydroxycarboxylic Acids”. Chem. Listy, 2003, vol.97, p. 431. 10 Claims, No Drawings US 7,829,324 B2 Page 2

OTHER PUBLICATIONS Stockland, A.E., et al., “Multiple Forms of Lactate Dehydrogenase in Delagrave, S., et al., “Recursive Ensemble Mutagenesis'. Protein Staphylococcus aureus”, Journal of Bacteriology, 1969, vol. 100, No. Engineering, 1993, vol. 6, No. 3, pp. 327-331. 1, pp. 347-353. Thomas, K.R., et al., “Site-Directed Mutagenesis by Gene Targeting Hino, T., et al., “Presence of Lactate Dehydrogenase and Lactate in Mouse Embryo-Derived Stem Cells”. Cell, 1987, vol. 51, pp. Racemase in Megasphaera elsdenii Grown on Glucose or Lactate'. SO3-5 12. Applied and Environmental Microbiology, 1993, vol. 58, No. 1, pp. Ebbers, E.J., et al., “Controlled Racemization of Optically Active 255-259. Organic Compounds: Prospects for Asymmetric Transformation'. Pepple, J. S., et al., “Lactate Racemase: Hydroxylamine-Dependent Tetrahedron, 1997, vol. 53, No. 28, pp. 9417-9476. 'O Exchange of the O-Hydroxyl of Lactic Acid”, Biochimica et van Rantwijk, F., et al., “Lipase-Catalyzed Synthesis of Carboxylic Biophysica Acta, 1976, vol. 429, pp. 1036-1040. Amides: Nitrogen Nucleophiles as Acyl Acceptor'. Monatshefte für Chemie, 2000, vol. 131, pp. 549-569. * cited by examiner US 7,829,324 B2 1. METHOD FOR THE MICROBOLOGICAL SOMERISATION OF ALPHA-HYDROXY CARBOXYLCACDS HOyer (I) RELATED APPLICATIONS R CO2H This application is a national stage application (under 35 U.S.C. 371) of PCT/EP2004/006564 filed Jun. 17, 2004 where which claims benefit to German application 103 27 582.7 R is straight-chain or branched lower alkyl or lower alk filed Jun. 18, 2003. 10 enyl, preferably C-C alkyl, or —(CH2)-Cyc, where n is an The present invention relates to a method for the microbio integer of 0 to 4, preferably 1, 2 or 3, and Cyc is an unsubsti logical isomerization of alpha-hydroxycarboxylic acids tuted or mono- or poly Substituted, mono- or binuclear carbo using an alpha-hydroxycarboxylic acid racemase, the or heterocyclic ring such as, for example, an unsubstituted or enzymes used for this method and microorganisms which Substituted aromatic or heteroaromatic ring, preferably an express a suitable racemase activity, a screening method for 15 unsubstituted or Substituted mononuclear aromatic ring, microorganisms with alpha-hydroxycarboxylic acid race where a Substrate comprising essentially a first stereoiso mase activity, the nucleic acid sequences encoding this meric form ((R) or (S) form) of an alpha-hydroxycarboxylic enzyme, expression vectors, recombinant microorganisms acid of the formula (I) is isomerized with the aid of an enzyme which express this racemase, and methods for the production with alpha-hydroxycarboxylic acid racemase activity and, if or isolation of a protein with alpha-hydroxycarboxylic acid appropriate, the resulting isomer mixture ((R)/(S)) or a result racemase activity. ing second stereoisomer (S) or (R) form) is isolated, or a resulting second stereoisomer is removed from the reaction BACKGROUND OF THE INVENTION equilibrium. The enzymatic isomerization is preferably carried out by The prior art knows what are known as microbial mande 25 reacting the substrate with purified enzyme, for example with late racemases, which are capable of racemizing mandelic a degree of purity of >50%, such as, for example, >80% acid in vitro or in vivo (G. L. Kenyon, J. A. Gerlt, G. A. Petsko or >90%, or 90 to 99% on protein basis, an enzyme-contain and J. W. Kozarich, “Mandelate Racemase: Structure-Func ing cell extract (for example crude extract following cell tion Studies of a Pseudosymmetric Enzyme'. Accts. Chem. disruption and removal of cell debris) or in the presence of Res., 28, 178-186 (1995); R. Li, V. M. Powers, J. W. Kozarich intact cells which express at least one enzyme with alpha and G. L. Kenyon, “Racemization of Vinylglycolate Cata 30 hydroxycarboxylic racemase activity. lyzed by Mandelate Racemase'. J. Org. Chem. 60,3347 The enzyme with alpha-hydroxycarboxylic acid racemase 3351 (1995); S. S. Schafer, A. T. Kallarakal, J. W. Kozarich, activity can preferably be isolated from microorganisms J. A. Gerlt, J. R. Clifton and G. L. Kenyon, “Mechanism of the which form or metabolize lactate. Such as, for example, lactic Reaction Catalyzed by Mandelate Racemase: The Structure 35 acid bacteria or propionic acid bacteria. Particularly suitable and Mechanistic Properties of the D270N Mutant”, Biochem Sources are bacteria of the genus Lactobacillus and Lactococ istry, 35,5662-5669 (1996)). C.S. At the same time, the simple production of chiral, nonra In accordance with a preferred variant, the reaction is car cemic Substances, for example for the production of active ried out in the presence of intact cells of microorganisms of ingredients in the pharmaceuticals industry, is greatly needed. 40 the genus Lactobacillus, or intact cells of a recombinant The enzyme-catalyzed racemization of a stereoisomeric form microorganism which express an alpha-hydroxycarboxylic of a compound, which leads to the desired enantiomer, would acid racemase activity according to the invention. constitute a suitable route for producing the desired, chiral, Preferred microorganisms are selected from among L. nonracemic materials. However, the racemases known to date are distinguished by their high Substrate specificity. Thus, paracasei, L. delbrueckii, L. Sakel and L. Oris, in particular 45 among the strains L. paracasei DSM 20207 and DSM 2649, mandelate racemase is only Suitable for the biocatalytic race L. delbrueckii DSM20074, L. Sakei DSM 20017 and L. Oris mization of betagamma-unsaturated alpha-hydroxyacids. DSM 4864. Saturated alpha-hydroxycarboxylic acids are not accepted In a further preferred process variant, the enzyme is a (cf. Felfer, U. et al., J. Mol. Catal. B: Enzymatic 15, 213 lactate racemase (E.C.5.1.2.1) with a widened substrate spec (2001)). 50 trum, i.e. an enzyme which, in addition to (R)- or (S)-lactate SUMMARY OF THE INVENTION racemizes at least one further (R)- or (S)-alpha-hydroxycar boxylic acid of the above formula I. Enzymes with racemase activity which have a different, or An enzyme activity which is useful in accordance with the widened, Substrate spectrum in comparison with known race invention encompasses, for example, the racemization of at mases are therefore required. 55 least one selected from among the (R) and/or (S) form of It is an object of the present invention to provide microor phenyl lactate, 4-fluorophenyl lactate, 2-hydroxy-4-phenyl ganisms and enzyme preparations with a novel racemase butyric acid, 2-hydroxy-4-methylpentanecarboxylic acid and activity, and to provide methods for the racemization of ste 2-hydroxy-3-methylbutyric acid. reoisomeric chemical compounds using these enzymes or In a preferred embodiment of the above process, the microorganisms. 60 desired stereoisomer is essentially removed, for example by Surprisingly, we have found that this object is achieved by chromatography, chemical or enzymatic stereoselective Sub a method for the microbiological isomerization of alpha sequent reaction and, if appropriate, further separation of the hydroxycarboxylic acids using an enzyme with alpha-hy downstream product, from the isomer mixture formed, pref droxycarboxylic racemase activity. erably after isolation of the mixture from the reaction A first aspect of the invention relates to a method for the 65 medium, for example by chromatography, and the residue microbiological isomerization of alpha-hydroxycarboxylic which essentially contains the undesired Stereoisomer, is acids of the formula I again isomerized. This can be repeated any number of times US 7,829,324 B2 3 4 until a complete conversion into the desired stereoisomer, or The invention furthermore relates to expression vectors the desired downstream product thereof, is obtained. comprising at least one coding nucleic acid sequence for an In a further preferred variant of the above process, the alpha-hydroxycarboxylic acid racemase in operable linkage isomer mixture formed is Subjected to a chemical or enzy with at least one regulatory nucleic acid sequence. matic stereoselective Subsequent reaction, preferably after 5 Aspect of the invention are also recombinant prokaryotic the mixture has been isolated from the reaction medium, for or eukaryotic microorganisms comprising at least one nucleic example by chromatography, and the reaction mixture acid sequence as defined above or at least one expression obtained, which also contains the unreacted isomer of the vector as defined above. hydroxycarboxylic acid, is subjected to another isomeriza A further aspect of the invention relates to a method for tion according to the invention. This can be repeated any 10 producing a protein with alpha-hydroxycarboxylic acid race number of times until a complete conversion into the desired mase activity, in which a recombinant microorganism as stereoisomer, or the desired downstream product thereof, is defined above, which expresses an enzyme with the desired obtained. racemase activity, is cultured and the protein is isolated from In a further preferred variant of the above process, the the culture. isomerization reaction is coupled with a chemical or enzy 15 Finally, the invention relates to a method for isolating a matic, enantioselective Subsequent reaction, preferably in protein with alpha-hydroxycarboxylic acid racemase activity, what is referred as a “one-pot reaction', where the resulting in which a nonrecombinant microorganism as defined above, desired stereoisomer of the alpha-hydroxycarboxylic acid is in particular of the genus Lactobacillus, which has tested removed stepwise or continuously from the reaction equilib positively for racemase activity is disrupted, cell wall debris 1. is separated off and the protein with the desired enzyme Preferred chemical or enzymatic, enantioselective subse activity is isolated. quent reactions after the isomerization step according to the invention are selected from among the esterification and the DETAILED DESCRIPTION OF THE INVENTION amidation of a stereoisomeric form of the alpha-hydroxycar 25 boxylic acid. The Subsequentenantioselective reaction can be A. General Terms and Definitions performed, in particular, on a carboxyl or hydroxyl group. Unless specified otherwise, the following general condi A further aspect of the invention relates to a screening tions apply: method for microorganisms which express an enzyme with “Racemates' represent equimolar mixtures of the two alpha-hydroxycarboxylic acid racemase activity, in which a 30 enantiomers of an optically active compound. microorganism in which the racemase activity is suspected is In accordance with the invention, "racemization' or cultured in the presence of a Substrate comprising essentially "isomerization” takes place at the alpha-carbon atom of the a stereoisomeric form of an alpha-hydroxycarboxylic acid of hydroxycarboxylic acid. the above formula I and the reaction medium is examined for racemization (for example diminishing quantity of one stere "Halogen' is fluorine, chlorine, bromine or iodine, in par 35 ticular fluorine or chlorine. oisomeric form and/or increasing quantity of the other stere “Lower alkyl is preferably straight-chain or branched oisomeric form) of the substrate. alkyl radicals having 2 to 8, in particular 2 to 6, carbon atoms The screening method according to the invention is not Such as ethyl, i- or n-propyl. n-, i-, sec- or tert-butyl, n-pentyl limited to specific microorganisms. In principle, it can be or 2-methylbutyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, carried out with all known eukaryotic and prokaryotic micro 40 2-ethylbutyl. organisms, animal cells or plant cells. However, those micro organisms which form or metabolize lactate Such as, for “Lower alkenyl is the mono- or polyunsaturated, prefer example, lactic acid bacteria or propionic acid bacteria, are ably monounsaturated, analog of the abovementioned alkyl preferred. Particularly suitable sources are bacteria of the radicals having 2 to 8, in particular 2 to 6, carbon atoms, it genus Lactobacillus. being possible for the double bond to be in any position of the Microorganisms which are preferably screened are those 45 carbon chain. which racemize the essentially stereoisomeric substrate to 1 Aryl is a mono- or polynuclear, preferably mono- or to 100%, preferably to 20 to 100%, in particular 50 to 100% binuclear, unsubstituted or Substituted aromatic radical, in or 80 to 100% (racemization (%)=2R/(R+S)x100: particular phenyl or else naphthyl which is bonded via any R=concentration of the R form; S=concentration of the S ring position, Such as 1- or 2-naphthyl. If appropriate, these form). Suitable conversion rates are in the range of from 1 to 50 aryl radicals can have attached to them 1 or 2 identical or 50%, preferably 5 to 50%, in particular 15 to 50% or 30 to different Substituents selected from among halogen, lower 50% (conversion rate (%)=R/(R+S)x100. alkyl, lower alkoxy as defined above or trifluoromethyl. A further aspect of the invention relates to alpha-hydroxy B. Alpha-Hydroxycarboxylic Acids Capable of Undergoing carboxylic acid racemases which are capable of racemizing/ 55 Racemization isomerizing at least one compound of the above formula I and alpha-Hydroxycarboxylic acids which, in accordance with which are obtainable by culturing a microorganism which has the invention, can be converted by racemization are those tested positively for racemase activity in a screening method alpha-hydroxycarboxylic acids of the above formula (I) as defined above and isolating the alpha-hydroxycarboxylic where R is straight-chain or branched lower alkyl or lower acid racemate from the culture. 60 alkenyl or —(CH2)-Cyc where n is an integer of 0 to 4 and Especially preferred alpha-hydroxycarboxylic acid race Cyc is an unsubstituted or mono- or polysubstituted, mono- or mases are those which racemize at least one alpha-hydroxy binuclear carbo- or heterocyclic ring. These compounds can carboxylic acid of the above formula I to 1 to 100%, prefer be racemized in accordance with the invention in an enantio ably 20 to 100%, in particular 50 to 100%. merically pure form, i.e. as the R or S enantiomer, or as A further aspect of the invention relates to nucleic acid 65 nonracemic mixture of the two enantiomers. sequences encoding at least one alpha-hydroxycarboxylic The alpha-hydroxycarboxylic acids of the formula I which acid racemase as defined above. are used for the enzymatic synthesis are compounds which US 7,829,324 B2 5 6 are known perse and which can be obtained using generally Also encompassed by the invention are “functional equiva known methods of organic synthesis. lents’ of the natural enzymes with alpha-hydroxycarboxylic Examples of carbo- and heterocyclic groups Cyc which acid racemase activity which can be isolated from the above must be mentioned are, in particular, mono- or binuclear, organisms. preferably mononuclear, groups having up to 4. Such as, for 5 "Functional equivalents’ or analogs of the natural race example, 0, 1 or 2, identical or different ring heteroatoms mases are, for the purposes of the present invention, polypep selected among O, N and S. tides which differ from them but retain the desired biological These carbo- or heterocyclic rings encompass in particular activity Such as, for example, Substrate specificity. Thus, for 3 to 12, preferably 4, 5 or 6, ring carbon atoms. Examples example, “functional equivalents' are understood as meaning which may be mentioned are cyclopropyl, cyclobutyl, cyclo 10 enzymes which racemize at least one compound selected pentyl, cyclohexyl, cycloheptyl, their mono- or polyunsatu from among phenyl lactate, 4-fluorophenyl lactate, 2-hy rated analogs such as cyclobutenyl, cyclopentenyl, cyclopen droxy-4-phenylbutyric acid, 2-hydroxy-4-methylpentanecar tadienyl, cyclohexenyl, cycloheptenyl, cyclohexadienyl, boxylic acid and 2-hydroxy-3-methylbutyric acid and which cycloheptadienyl, and phenyl; and 5- to 7-membered Satu have at least 20%, preferably at least 50%, especially prefer rated or mono- or polyunsaturated heterocyclic radicals hav 15 ably at least 75%, very especially preferably at least 90% of ing 1 to 4 heteroatoms which are selected from among O. N the activity of a natural racemase enzyme from one of the and S. Heterocyclic radicals derived from pyrrolidine, tet abovementioned Lactobacillus Strains or a higher activity rahydrofuran, piperidine, morpholine, pyrrole, furan, than the latter. Moreover, functional equivalents are prefer thiophene, pyrazole, imidazole, oxazole, thiazole, pyridine, ably stable from about pH 4 to 10 and advantageously have a pyran, pyrimidine, pyridazine and pyrazine must be men pH optimum between pH 5 and 8 and a temperature optimum tioned in particular. in the range of from 20° C. to 80° C. Others to be mentioned are binuclear radicals in which one “Functional equivalents' are, in accordance with the inven of the abovementioned carbocycles or heterocycles is fused to tion, in particular also understood as meaning mutants which a further heterocycle or carbocycle. Such as, for example, have an amino acid which is not the natural amino acid in at radicals which are derived from cumarone, indole, quinoline 25 least one sequence position of the natural amino acid and naphthalene. sequences while retaining one of the abovementioned bio In this context, the radicals Cyc can be bonded via any ring logical activities. Thus, “functional equivalents' encompass position, preferably via a ring carbon atom. the mutants obtainable by one or more amino acid additions, Examples of Suitable Cyc radicals are phenyl, naphthyl, Substitutions, deletions and/or inversions, it being possible 2-thienyl, 3-thienyl: 2-furanyl, 3-furanyl: 2-pyridyl, 3-py 30 for the abovementioned modifications to occur in any ridyl or 4-pyridyl: 2-thiazolyl, 4-thiazolyl or 5-thiazolyl: sequence position as long as they lead to a mutant with the 4-methyl-2-thienyl, 3-ethyl-2-thienyl, 2-methyl-3-thienyl, property profile according to the invention. In particular, 4-propyl-3-thienyl, 5-n-butyl-2-thienyl, 4-methyl-3-thienyl, functional equivalents also exist when the reactivity pattern 3-methyl-2-thienyl: 3-chloro-2-thienyl, 4-bromo-3-thienyl, between mutant and unmodified polypeptide agree in terms 2-iodo-3-thienyl, 5-iodo-3-thienyl, 4-fluoro-2-thienyl, 35 of quality, i.e. for example when identical Substrates are con 2-bromo-3-thienyl, and 4-chloro-2-thienyl. verted at different rates. The radicals Cyc can furthermore be monosubstituted or “Functional equivalents’ in the above sense are also “pre poly Substituted. Such as, for example, monosubstituted or cursors” of the polypeptides described, and “functional disubstituted. Preferably, the substituents are attached to a derivatives” and “salts' of the polypeptides. ring carbon atom. Examples of suitable Substituents are halo 40 In this context, “precursors' are natural or synthetic pre gen, lower alkyl, lower alkenyl, lower alkoxy, —OH, -SH, cursors of the polypeptides with or without the desired bio -NO or NRR, where R and R independently of one logical activity. another are H. methyl or ethyl. Preferred substituents are The term "salts' is understood as meaning not only salts of halogen radicals. carboxylic acid groups, but also addition salts of amino A further preferred group of Cyc radicals are aryl radicals 45 groups of the protein molecules according to the invention. as defined above. Salts of carboxyl groups can be prepared in a manner known per se and encompass inorganic salts such as, for example, C. Enzymes with Alpha-Hydroxycarboxylic Acid Racemase salts of sodium, calcium, ammonium, iron and Zinc, and salts Activity with organic bases such as, for example, amines, such as The enzymes with alpha-hydroxycarboxylic acid race 50 triethanolamine, arginine, lysine, piperidine and the like. mase activity in accordance with the invention can be Acid addition salts such as, for example, salts with mineral obtained in particular from microorganisms of the genus Lac acids such as hydrochloric acid or Sulfuric acid, and salts with tobacillus. organic acids such as acetic acid and Oxalic acid, are likewise Preferred enzymes can be isolated from the Lactobacillus subject matter of the invention. strains L. paracasei DSM 20207 (DSM 15755) and DSM 55 “Functional derivatives of enzymes according to the 2649 (DSM 15751), L. delbrueckii DSM 20074 (DSM invention can likewise be prepared on functional amino acid 15754), L. sakei DSM 20017 (DSM 15753) and L. oris DSM side groups or their N- or C-terminal end with the aid of 4864 (DSM 15752). These strains are available from the known techniques. Such derivatives encompass, for example, DSMZ (Deutsche Sammlung von Mikroorganismen and aliphatic esters of carboxyl groups, amides of carboxyl Zellkulturen GmbH; German Collection of Microorganisms 60 groups, obtainable by reaction with ammonia or with a pri and Cell Cultures, Inhoffenstrafe 7B, 38.124 Braunschweig, mary or secondary amine; N-acyl derivatives of free amino Germany). groups, prepared by reaction with acyl groups; or O-acyl The enzymes can be isolated from cell cultures using cus derivatives of free hydroxyl groups, prepared by reaction with tomary preparative biochemical methods. Then, first amino acyl groups. acid sequence information can be deduced from the isolated 65 Naturally, “functional equivalents' also encompass enzymes in the customary manner, for example by peptide polypeptides which can be obtained from other organisms, fragmentation and N-terminal sequencing. and naturally occurring variants. For example, regions of US 7,829,324 B2 7 8 homologous sequences can be determined by sequence align Yourvan (1992) PNAS 89:7811-7815; Delgrave et al. (1993) ment, and equivalent enzymes can be established on the basis Protein Engineering 6(3):327-331). of the specific requirements of the invention. “Functional equivalents’ are additionally fusion proteins D. Coding Nucleic Acid Sequences which have one of the natural racemase sequences or func The invention also relates to nucleic acid sequences which tional equivalents derived therefrom and at least one other encode an enzyme with alpha-hydroxycarboxylic acid race heterologous sequence which is functionally different there mase activity. Preferred nucleic acid sequences are those from in functional N- or C-terminal linkage (i.e. without which comprise the nucleic acid sequences derived from the substantial mutual impairment of the function of the fusion natural amino acid sequences of the racemases which can be protein moieties). Nonlimiting examples of Such heterolo 10 isolated from the above microorganisms. gous sequences are, for example, signal peptides or enzymes. All of the nucleic acid sequences according to the invention "Functional equivalents' also encompassed by the inven (single- and double-stranded DNA and RNA sequences such tion are homologs of the natural proteins. These have at least as, for example, cDNA and mRNA) can be generated from the 60%, preferably at least 75%, in particular at least 85%, such nucleotide units in a manner known perse by chemical Syn as, for example, 90%. 95% or 99%, homology to one of the 15 thesis, Such as, for example, by fragment condensation of natural amino acid sequences, calculated by the algorithm of individual overlapping complementary nucleic acid units of Pearson and Lipman, Proc. Natl. Acad, Sci. (USA) 85(8), the double helix. The chemical synthesis of oligonucleotides 1988, 2444-2448. A percentage homology of a homologous can be carried out for example in the known manner using the polypeptide according to the invention means, in particular, phosphoamidite method (Voet, Voet, 2" Edition, Wiley Press percentage identity of the amino acid residues based on the New York, pages 896-897). Annealing synthetic oligonucle total length of one of the amino acid sequences of an enzyme otides and filling in gaps with the aid of the DNA polymerase according to the invention or an enzyme subunit. Klenow fragment, and ligation reactions, as well as general In the event of protein glycosylation, “functional equiva cloning methods, are described in Sambrook et al. (1989), lents' according to the invention encompass proteins of the Molecular Cloning: A laboratory manual, Cold Spring Har above-specified type in deglycosylated or glycosylated form, 25 bor Laboratory Press. and modified forms obtainable by changing the glycosylation In particular, the invention relates to nucleic acid pattern. sequences (single- and double-stranded DNA and RNA Homologs of the proteins or polypeptides according to the sequences such as, for example, cDNA and mRNA) encoding invention can be generated by mutagenesis, for example by one of the enzymes according to the invention and their func point mutation or truncation of the protein. 30 tional equivalents which are obtainable, for example, by Homologs of the racemases according to the invention can using artificial nucleotide analogs. be identified by screening combinatorial libraries of mutants The invention relates to both isolated nucleic acid mol Such as, for example, truncation mutants. For example, it is ecules which encode polypeptides or proteins according to possible to generate a variegated library of protein variants by the invention or biologically active segments thereof, and to combinatorial mutagenesis at the nucleic acid level. Such as, 35 nucleic acid fragments which can be used, for example, for for example, by enzymatic ligation of a mixture of synthetic use as hybridization probes or primers for identifying or oligonucleotides. There is a large number of methods which amplifying coding nucleic acids according to the invention. can be used to generate libraries of potential homologs for a The nucleic acid molecules according to the invention may degenerate oligonucleotide sequence. Chemical synthesis of additionally comprise untranslated sequences from the 3' and/ a degenerate gene sequence can be carried out in an automatic 40 or 5' end of the coding region of the gene. DNA synthesizer, and the synthetic gene can then be ligated The invention furthermore encompasses the nucleic acid into a suitable expression vector. The use of a degenerate set molecules which are complementary to the nucleotide of genes makes it possible to provide, in one mixture, all sequences described individually, or a segment of Such a sequences which encode the desired set of potential protein nucleic acid molecule. sequences. Methods for synthesizing degenerate oligonucle 45 The nucleic acid sequences according to the invention otides are known to the skilled worker (for example Narang, make possible the generation of probes and primers which S. A. (1983) Tetrahedron 39:3: Itakura et al. (1984) Annu. can be used for identifying and/or cloning homologous Rev. Biochem. 53:323: Itakura et al., (1984) Science 198: sequences in other cell types and organisms. Such probes and 1056; Ike et al. (1983) Nucleic Acids Res. 11:477). primers usually comprise a nucleotide sequence region which The prior art knows a variety of techniques for screening 50 hybridizes under stringent conditions (see hereinbelow) with gene products of combinatorial libraries which have been at least approximately 12, preferably at least approximately generated by point mutation or truncation, and for Screening 25, such as, for example, approximately 40, 50 or 75, con cDNA libraries for gene products with a selected character secutive nucleotides of a sense strand of a nucleic acid istic. These techniques can be adapted to the rapid screening sequence according to the invention or of a corresponding of the gene libraries which have been generated by combina 55 antisense Strand. torial mutagenesis of homologs according to the invention. An "isolated nucleic acid molecule is separated from The most frequently used techniques for Screening large gene other nucleic acid molecules which are present in the natural libraries undergoing high-throughput analysis comprise the Source of the nucleic acid and may, moreover, be essentially cloning of the gene library into replicable expression vectors, free of other cellular material or culture medium if produced transforming suitable cells with the resulting vector library 60 by recombinant techniques, or free of chemical precursors or and expressing the combinatorial genes under conditions other chemicals if chemically synthesized. under which the detection of the required activity facilitates A nucleic acid molecule according to the invention can be isolation of the vector which encodes the gene whose product isolated by using standard techniques of molecular biology has been detected, Recursive ensemble mutagenesis (REM), and the sequence information provided in accordance with a technique which increases the frequency of functional 65 the invention. For example, cDNA can be isolated from a mutants in the libraries, can be used in combination with the suitable cDNA library by using one of the specifically dis screening tests in order to identify homologs (Arkin and closed complete sequences or a segment thereof as hybrid US 7,829,324 B2 10 ization probe and standard hybridization techniques (such as, Press at Oxford University Press, Oxford; Brown (ed), 1991, for example, described in Sambrook, J., Fritsch, E. F. and Essential Molecular Biology: A Practical Approach, IRL Maniatis, T. Molecular Cloning: A Laboratory Manual. 2" Press at Oxford University Press, Oxford. Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor The invention also relates to derivatives of the specifically Laboratory Press, Cold Spring Harbor, N.Y., 1989). More disclosed or derivable nucleic acid sequences. over, it is possible for a nucleic acid molecule comprising one Thus, further nucleic acid sequences according to the of the disclosed sequences, or a segment of such a molecule, invention can be derived from the natural sequences and differ to be isolated by polymerase chain reaction using the oligo therefrom through addition, substitution, insertion ordeletion nucleotide primers which have been generated on the basis of of one or more nucleotides, while still encoding polypeptides this sequence. The nucleic acid amplified in this way can be 10 with the desired profile of characteristics. cloned into a suitable vector and characterized by DNA Also encompassed in accordance with the invention are sequence analysis. The oligonucleotides according to the those nucleic acid sequences which comprise what are known invention can furthermore be generated by standard synthetic as silent mutations or which are modified, in comparison with method, for example using an automatic DNA synthesizer. a specifically mentioned sequence, in accordance with the In principle, the nucleic acid sequences according to the 15 codon usage of a specific source of origin or host organism, as invention can be identified and isolated from all organisms. are naturally occurring variants such as, for example, splice The nucleic acid sequences according to the invention can variants or allelic variants, thereof. advantageously be isolated from bacteria of the abovemen Sequences which can be obtained by conservative nucle tioned type. otide Substitutions (i.e. the amino acid in question is replaced Nucleic acid sequences according to the invention or by an amino acid with the same charge, size, polarity and/or derivatives thereof, homologs or parts of these sequences can solubility) are also subject matter. be isolated from other microorganisms for example using The invention also relates to the molecules derived from customary hybridization methods or PCR technology, for the specifically disclosed nucleic acids by sequence polymor example via genomic libraries or cDNA libraries. These DNA phisms. These genetic polymorphisms may exist within indi sequences hybridize with the sequences according to the 25 viduals within a population owing to the natural variation. invention understandard conditions. It is advantageous to use These natural variations usually bring about a variance of 1 to short oligonucleotides of the conserved regions, for example 5% in the nucleotide sequence of a gene. the active center, which can be identified via comparisons Derivatives of the nucleic acids according to the invention with alpha-hydroxycarboxylic acid racemase in the manner are understood as meaning, for example, allelic variants with known to the skilled worker, for the hybridization. Alterna 30 at least 40% homology at the deduced amino acid level, tively, longer fragments of the nucleic acids according to the preferably 60% homology, very especially preferably at least invention, or the complete sequences, may be used for the 80% homology, over the entire sequence region (with regard hybridization. Depending on the nucleic acid used (oligo to homology at the amino acid level, reference may be made nucleotide, longer fragment or complete sequence), or to the above explanation regarding polypeptides). Advanta depending on the type of nucleic acid (DNA or RNA) being 35 geously, the homology levels can be higher over part-regions used for the hybridization, these standard conditions vary. of the sequences. Thus, for example, the melting points for DNA:DNA hybrids Derivatives are also understood as meaning homologs of are approximately 10° C. lower than those of DNA:RNA the nucleic acid sequences according to the invention, for hybrids of the same length. example fungal or bacterial homologs, truncated sequences, For example, standard conditions are understood as mean 40 single-strand DNA or RNA of the coding and noncoding ing, depending on the nucleic acid, temperatures between 42 sequence. Thus, for example, homologs to SEQ ID NO: 1 and 58°C. in an aqueous buffer solution at a concentration of have at least 40% homology at the DNA level, preferably at from 0.1 to 5xSSC (1xSSC=0.15 M NaCl, 15 mM sodium least 60%, especially preferably at least 70%, very especially citrate, pH 7.2) or additionally in the presence of 50% forma preferably at least 80% over the entire DNA region stated in mide, such as, for example, 42°C. in 5xSSC, 50% forma 45 SEQID NO: 1. mide. For DNA:DNA hybrids, the hybridization conditions Derivatives are, moreover, understood as meaning for are advantageously 0.1xSSC and temperatures of from example fusions with promoters. The promoters which pre approximately 20° C. to 45° C., preferably from approxi cede the abovementioned nucleotide sequences can have mately 30° C. to 45° C. For DNA:RNA hybrids, the hybrid been modified through one or more nucleotide Substitutions, ization conditions are advantageously 0.1XSSC and tempera 50 insertions, inversions and/or deletions without, however, tures from approximately 30° C. to 55° C., preferably from adversely affecting the functionality or efficacy of the pro approximately 45° C. to 55° C. These temperatures which moters. Moreover, the efficacy of the promoters can be have been stated for the hybridization are melting points increased by modifying their sequence, or the promoters can which have been calculated by way of example for a nucleic be substituted completely by more efficient promoters, acid with a length of approximately 100 nucleotides and a 55 including those from heterologous organisms. G+C content of 50% in the absence of formamide. The Derivatives are also understood as meaning variants whose experimental conditions for DNA hybridization are described nucleotide sequence in the region of from -1 to -1000 bases in relevant textbooks of genetics, such as, for example, Sam upstream of the start codon, or 0 to 1000 bases downstream of brook et al., “Molecular Cloning, Cold Spring Harbor Labo the stop codon, have been modified in Such a way that gene ratory, 1989, and can be calculated using formulae known to 60 expression and/or protein expression is modified, preferably the skilled worker, for example as a function of the length of increased. the nucleic acids, the type of hybrid or the G+C content. The invention furthermore relates to nucleic acid Further information on hybridization can be found by the sequences which hybridize with the abovementioned coding skilled worker in the following textbooks: Ausubel et al. sequences under “stringent conditions'. These polynucle (eds), 1985, Current Protocols in Molecular Biology, John 65 otides can be identified when screening genomic libraries or Wiley & Sons, New York; Hames and Higgins (eds), 1985, cDNA libraries and, if appropriate, amplified by means of Nucleic Acids Hybridization: A Practical Approach, IRL PCR using suitable primers and subsequently isolated, for US 7,829,324 B2 11 12 example with suitable probes. Moreover, polynucleotides Instead, the natural regulatory sequence is mutated in Such a according to the invention may also be synthesized chemi way that regulation no longer takes place and gene expression cally. This characteristic is understood as meaning the ability is increased. of a poly- or oligonucleotide to bind under stringent condi A preferred nucleic acid construct advantageously also tions to an almost complementary sequence, while there are comprises one or more of the abovementioned enhancer no nonspecific bindings between noncomplementary part sequences in operable linkage with the promoter, which make ners under these conditions. For this purpose, the sequences possible enhanced expression of the nucleic acid sequence. At should have 70-100%, preferably 90-100%, complementar the 3' end of the DNA sequences, too, additional advanta ity. The characteristic of complementary sequences being geous sequences, such as further regulatory elements or ter able to bind specifically to one another is exploited, for 10 minators, may be inserted. One or more copies of the nucleic example, in Northern or Southern blottechnique or in PCR or acids according to the invention may be present in the con RT-PCR in the case of primer binding. Usually, oligonucle struct. Further markers, such as resistances to antibiotics, or otides with a length of 30 base pairs or more are employed for auxotrophism-complementing genes, may additionally be this purpose. Stringent conditions are understood as meaning, present, if appropriate, for selection for the construct. 15 Advantageous regulatory sequences for the method for example in the Northern blot technique, the use of a according to the invention are present for example in promot washing solution at a temperature of 50-70° C., preferably erS Such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, laclT7. 60-65° C., for example 0.1xSSC buffer with 0.1% SDS (20x T5, T3, gal, trc, ara, rhap (rhap)SP6, lambda-P or SSC:3M NaCl, 0.3M Na citrate, pH 7.0) for eluting nonspe lambda-P, promoter, which are preferably employed in cifically hybridized cDNA probes or oligonucleotides. In this Gram-negative bacteria. Other advantageous regulatory case, as mentioned above, only nucleic acids with a high sequences are present for example in the Gram-positive pro degree of complementarity remain bound to one another. The moters amy and SPO2, in the yeast or fungal promoters setting up of stringent conditions is known to the skilled ADC1, MFalpha, AC, P-60, CYC1, GAPDH, TEF, rp28, worker and described, for example, in Ausubel et al., Current ADH. Artificial promoters may also be used for the regula Protocols in Molecular Biology, John Wiley & Sons, N.Y. 25 tion. (1989), 6.3.1-6.3.6. For expression in a host organism, the nucleic acid con struct is advantageously inserted into a vector. Such as for E. Constructs According to the Invention example a plasmid or a phage, which makes possible optimal The invention furthermore relates to expression constructs expression of the genes in the host. Vectors are understood as comprising, under the genetic control of regulatory nucleic 30 meaning, in addition to plasmids and phages, all other vectors acid sequences, a nucleic acid sequence encoding an enzyme known to the skilled worker, that is to say, for example, of the invention; and to vectors comprising at least one of viruses such as SV40, CMV, baculovirus and adenovirus, these expression constructs. transposons, IS elements, phasmids, cosmids, and linear or Such constructs according to the invention preferably circular DNA. These vectors are capable of undergoing encompass a promoter 5'-upstream from the particular coding 35 autonomous replication in the host organism or chromosomal sequence and a terminator sequence 3'-downstream, and, replication. These vectors constitute a further embodiment of where appropriate, further customary regulatory elements, in the invention. Suitable plasmids are, for example, in E. coli each case linked operably to the coding sequence. pLG338, p.ACYC184, pBR322, p OC18, p OC19, pKC30, "Operable linkage' is understood as meaning the sequen pRep4, pHS1, pKK223-3, pIDHE19.2, pHS2, pPLc236, tial arrangement of promoter, coding sequence, terminator 40 pMBL24, pI G200, pluR290, pIN-III'-B1.gt11 orpEdCl, and, where appropriate, other regulatory elements in Such a in Streptomyces plJ101, pIJ364, pIJ702 or plJ361, in Bacillus way that each of the regulatory elements is able to comply pUB110, pC194 or pBD214, in Corynebacterium pSAT7 or with the intended function in the expression of the coding pAJ667, in fungip ALS1, pIL2 orpFBB 116, in yeast 2alphaM, sequence. Examples of sequences capable of operable link pAG-1, YEp6, YEp13 or pEMBLYe23 or in plants plGV23, age are targeting sequences, and also enhancers, polyadeny 45 pGHlac", pBIN19, p.AK2004 or pH51. The abovemen lation signals and the like. Other regulatory elements com tioned plasmids are a small selection of plasmids which are prise selectable markers, amplification signals, origins of possible. Other plasmids are well known to the skilled worker replication and the like. Suitable regulatory sequences are and can be found, for example, in the book Cloning Vectors described, for example, in Goeddel, Gene Expression Tech (Eds. Pouwels P. H. et al. Elsevier, Amsterdam-New York nology: Methods in Enzymology 185, Academic Press, San 50 Oxford, 1985, ISBN 0444904018). Diego, Calif. (1990). For the expression of the further genes which are present, A nucleic acid construct according to the invention is the nucleic acid construct advantageously additionally com understood as meaning in particular the natural alpha-hy prises 3'- and/or 5'-terminal regulatory sequences for enhanc droxycarboxylic acid racemase genes and the derivatives and 55 ing expression, and Such sequences are selected for optimal homologs thereof, which have been linked operably or func expression as a function of the chosen host organism and the tionally with one or more regulatory signals, advantageously gene, or genes. for controlling, for example increasing, gene expression. These regulatory sequences are intended to make possible In addition to these regulatory sequences, the natural regu the specific expression of the genes and protein expression. lation of these sequences may still be present before the actual 60 Depending on the host organism, this may mean, for example, structural genes and, if appropriate, may have been geneti that the gene is expressed, or overexpressed, only after induc cally modified so that the natural regulation has been tion, or that it is immediately expressed and/or overexpressed. Switched off and expression of the genes increased. However, The regulatory sequences or factors may preferably have a the nucleic acid construct may also be simpler in construc positive effect on, and thus enhance, the gene expression of tion, that is to say that no additional regulatory signals have 65 the gene introduced. Thus, enhancement of the regulatory been inserted before the coding sequence and the natural elements can take place advantageously at the transcriptional promoter together with its regulation has not been removed. level by using strong transcription signals such as promoters US 7,829,324 B2 13 14 and/or enhancers. However, it is also possible to enhance homolog from a related microorganism or else derived from a translation, for example by improving the stability of the mammalian, yeast or insect source. However, the vector used mRNA. for homologous recombination may also be designed in such In a further embodiment of the vector, the vector compris a way that the endogenous gene is mutated or otherwise ing the nucleic acid construct according to the invention or the 5 modified upon homologous recombination, while still encod nucleic acid according to the invention may advantageously ing the functional protein (for example, the upstream regula also be introduced into the microorganisms in the form of a tory region can be modified in such a way that the expression linear DNA and integrated into the genome of the host organ of the endogenous protein is thus modified). The modified ism via heterologous or homologous recombination. This segment of the gene according to the invention is present in linear DNA may consist of a linearized vector, such as a 10 the homologous recombination vector. The construction of plasmid, or only of the nucleic acid construct or the nucleic Suitable vectors for homologous recombination is described, acid according to the invention. for example, in Thomas, K. R. and Capecchi, M. R. (1987) For the optimal expression of heterologous genes in organ Cell 51:503. isms, it is advantageous to modify the nucleic acid sequences Suitable recombinant host organisms for the nucleic acid in accordance with the specific codon usage used in the organ 15 according to the invention or the nucleic acid construct are, in ism. Codon usage can be determined readily with the aid of principle, all prokaryotic or eukaryotic organisms. Microor computer evaluations of other, known genes of the organism ganisms such as bacteria, fungi or yeasts are advantageously in question. used as host organisms. Gram-positive or Gram-negative bac An expression cassette according to the invention is pre teria are advantageously used. The genus and species pared by fusing a suitable promoter to a suitable codon nucle Escherichia coli is very especially preferred. otide sequence and a terminator signal or polyadenylation The host organism, or host organisms, according to the signal. Conventional recombination and cloning techniques invention preferably comprise at least one of the nucleic acid as are described, for example, in T. Maniatis, E. F. Fritsch and sequences, nucleic acid constructs or vectors which are J. Sambrook, Molecular Cloning: A Laboratory Manual, described in the present invention and which encode an Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 25 enzyme with alpha-hydroxycarboxylic acid racemase activ (1989) and in T. J. Silhavy, M. L. Berman and L. W. Enquist, 1ty. Experiments with Gene Fusions, Cold Spring Harbor Labo Depending on the host organism, the organisms used in the ratory, Cold Spring Harbor, N.Y. (1984) and in Ausubel, F. M. method according to the invention are cultured or grown in et al. Current Protocols in Molecular Biology, Greene Pub the manner with which the skilled worker is familiar. As a lishing Assoc. and Wiley Interscience (1987) are used for this 30 rule, microorganisms are grown in a liquid medium compris purpose. ing a carbon source, usually in the form of sugars, a nitrogen For expressionina suitable host organism, the recombinant Source, usually in the form of organic nitrogen sources such as nucleic acid construct or gene construct is advantageously yeast extract or salts such as ammonium sulfate, trace ele inserted into a host-specific vector which makes possible the ments such as salts of iron, manganese, magnesium, and, if optimal expression of the genes in the host. Vectors are well 35 appropriate, vitamins, at temperatures of from 0° C. to 100° knownto the skilled worker and can be foundin, for example, C., preferably from 10°C. to 60°C., while passing in oxygen. "Cloning Vectors” (Pouwels P. H. et al., Ed., Elsevier, The pH of the liquid nutrient medium can be kept constant, Amsterdam-New York-Oxford, 1985). i.e. regulated during the culture period, or not. The microor ganisms can be cultured batchwise, semibatchwise or con F. Hosts which are Useful in Accordance with the Invention 40 tinuously. Nutrients can be provided at the beginning of the The Vectors according to the invention allow the generation fermentation or fed in semicontinuously or continuously. The of recombinant microorganisms which are transformed for ketone can be added directly during the culturing period or example with at least one vector according to the invention advantageously thereafter. The enzymes can be isolated from and which can be employed in the production of the enzymes the organisms by the methods described in the examples or according to the invention. The above-described recombinant 45 else used for the reaction in the form of a crude extract. constructs according to the invention are advantageously introduced into a suitable host system, where they are G. Recombinant Production of the Enzymes According to the expressed. In this context, cloning and transfection methods Invention: with which the skilled worker is familiar, such as coprecipi The invention furthermore relates to methods for the tation, protoplast fusion, electroporation, retroviral transfec 50 recombinant production of polypeptides according to the tion and the like, are used in order to express the abovemen invention or of functional, biologically active fragments tioned nucleic acids in the expression system in question. thereof, in which method a polypeptide-producing microor Suitable systems are described, for example, in Current Pro ganism is cultured, expression of the polypeptides is induced, tocols in Molecular Biology, F. Ausubel et al., Ed., Wiley if appropriate, and the polypeptides are isolated from the Interscience, New York 1997, or Sambrook et al. Molecular 55 culture. If desired, the polypeptides can also be produced on Cloning: A Laboratory Manual. 2. ed., Cold Spring Harbor an industrial scale in this manner. Laboratory, Cold Spring Harbor Laboratory Press, Cold The recombinant microorganism can be cultured and fer Spring Harbor, N.Y., 1989. mented by known methods. Bacteria can be multiplied for Microorganisms which have undergone homologous example in TB or LB medium and at a temperature of from 20 recombination can also be prepared in accordance with the 60 to 40° C. and a pH value of 6 to 9. The individual cultivation invention. To this end, a vector is prepared which contains at conditions which are suitable are described, for example, in T. least one segment of a gene according to the invention or of a Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: coding sequence, into which, if appropriate, at least one A Laboratory Manual, Cold Spring Harbor Laboratory, Cold amino acid deletion, addition or substitution has been intro Spring Harbor, N.Y. (1989). duced in order to modify, for example functionally to disrupt, 65 Unless the polypeptides are secreted into the culture the sequence according to the invention ("knockout' vector). medium, the cells are then disrupted and the product is The sequence introduced may, for example, also be a obtained from the lysate by known protein isolation methods. US 7,829,324 B2 15 16 The cells can be disrupted either by high-frequency ultra I. Carrying Out the Method According to the Invention for the Sound, by high pressure. Such as, for example, in a French Isomerization of Alpha-Hydroxycarboxylic Acids pressure cell, by osmolysis, by allowing detergents, lytic The enzymes with alpha-hydroxycarboxylic acid race enzymes or organic solvents to act on them, by homogenizers mase activity can be used in the method according to the or by combining several of the abovementioned methods. invention as the free enzyme or as an immobilized enzyme. The polypeptides can be purified with known chromato The method according to the invention is advantageously graphic methods, such as molecular sieve chromatography carried out at a temperature of from 0°C. to 95°C., preferably (gel filtration). Such as Q-Sepharose chromatography, ion from 10° C. to 85°C., especially preferably from 15° C. to 75° exchange chromatography and hydrophobic chromatogra C., in particular 25 to 40°C. phy, or else by other customary methods such as ultrafiltra 10 The pH value in the method according to the invention is tion, crystallization, salting out, dialysis and native gel elec advantageously maintained at from pH 4 to 12, preferably trophoresis. Suitable methods are described, for example, in from pH 4.5 to 9, especially preferably from pH 5 to 8. Cooper, F. G., Biochemische Arbeitsmethoden Methods in The method according to the invention may employ grow Biochemistry, Verlag Walter de Gruyter, Berlin, N.Y. or in ing cells which comprise nucleic acids, nucleic acid con Scopes, R., Protein Purification, Springer Verlag, New York, 15 structs or vectors according to the invention. Alternatively, Heidelberg, Berlin. quiescent or disruptive cells may be used. Disruptive cells are To isolate the recombinant protein, it may be advantageous understood as meaning, for example, cells which have been to employ vector systems or oligonucleotides which extend made permeable via treatment with, for example, solvents, or the cDNA by specific nucleotide sequences and thus encode else cells which have been disrupted via enzyme treatment, modified polypeptides or fusion proteins which serve, for via methanical treatment (for example French press or ultra example, for simpler purification. Such Suitable modifica sound) or via any other method. The crude extracts thus tions are, for example, what are known as "Tags' which act as obtained are advantageously suitable for the method accord anchors, such as, for example, the modification known as ing to the invention. Fully or partially purified enzymes may hexa-histidine anchor, or epitopes which can be recognized also be used for the method. Immobilized microorganisms or by antibodies as antigens (described for example in Harlow, 25 enzymes are likewise Suitable, and these can be employed E. and Lane, D., 1988, Antibodies: A Laboratory Manual. advantageously reaction. Cold Spring Harbor (N.Y.) Press). These anchors can serve If free organisms or immobilized enzymes are used for the for attaching proteins to a solid Support, such as, for example, method according to the invention, they are expediently sepa a polymer matrix, which, for example, may be used for pack rated off before the extraction step, for example via filtration ing a chromatography column, or else to a microtiter plate or 30 or centrifugation. any other Support. The product prepared in the method according to the inven These anchors can simultaneously also be used for identi tion can be recovered advantageously from the aqueous reac fying the proteins. Moreover, customary tags, such as fluo tion solution by extraction or distillation. An extraction step rescent dyes, enzyme tags which, after reaction with a Sub can be repeated several times in order to increase yield. strate, form a detectable reaction product, or radiolabels, 35 Examples of suitable extractants are customary solvents with alone or in combination with the anchors, may additionally be which the skilled worker is familiar, such as toluene, meth used for identifying the proteins in order to derivatize the ylene chloride, diisopropyl ether, benzene, MTBE or ethyl proteins. acetate, without imposing any limitation. After concentration of the organic phase, the products can, H. Nonrecombinant Isolation of the Enzymes According to 40 as a rule, be recovered in good chemical purity, i.e. a chemical the Invention purity of more than 80%. However, the organic phase con Enzymes according to the invention are expediently iso taining the product may, after extraction, also be only par lated from one of the above-described natural sources (Lac tially concentrated and the product can be crystallized out. To tobacillus strains) or from another microorganism which this end, the solution is advantageously cooled down to a expresses alpha-hydroxycarboxylic acid racemase activity 45 temperature of from 0°C. to 10°C. However, crystallization and which has been identified with the aid of a screening can also be effected directly from the organic solution or from method according to the invention. an aqueous solution. The product which has crystallized out The microorganism can be cultured and fermented by can be taken up again in the same or a different solvent for known methods. Unless the polypeptides are secreted into the recrystallization and be recrystallized. culture medium, the cells are then disrupted and the product is 50 The method according to the invention can be operated obtained from the lysate by known protein isolation methods. batchwise, semibatchwise or continuously. The cells can be disrupted either by high-frequency ultra The method can advantageously be carried out in bioreac Sound, by high pressure. Such as, for example, in a French tors such as, for example, described in Biotechnology, Vol pressure cell, by osmolysis, by allowing detergents, lytic ume 3, 2" Edition, Rehm et al. (ed.), (1993), in particular enzymes or organic solvents to act on them, by homogenizers 55 chapter II. or by combining several of the abovementioned methods. The polypeptides can be purified with known chromato K. Further Applications of the Invention graphic methods, such as molecular sieve chromatography The present invention opens up a multiplicity of further (gel filtration). Such as Q-Sepharose chromatography, ion possible applications of the isomerization reactions accord exchange chromatography and hydrophobic chromatogra 60 ing to the invention. The following examples may be men phy, or else by other customary methods such as ultrafiltra tioned with imposing any limitation: tion, crystallization, salting out, dialysis and native gel elec a) Racemization of alpha-hydroxycarboxylic acids for trophoresis. Suitable methods are described, for example, in “recycling the undesired stereoisomer in traditional race Cooper, F. G., Biochemische Arbeitsmethoden Methods in mate separations after removal of the desired product. The Biochemistry, Verlag Walter de Gruyter, Berlin, N.Y. or in 65 advantage is the neat enzymatic racemization reaction; in Scopes, R., Protein Purification, Springer Verlag, New York, contrast, chemically catalyzed racemization reactions usually Heidelberg, Berlin. proceed under drastic reaction conditions which lead to US 7,829,324 B2 17 18 decomposition, to the formation of secondary products and to side reactions (elimination and the like). (Compare also: Kon TABLE 1 trollierte Racemisierung Von organischen Verbindungen Controlled racemization of organic compounds: E. J. Separation of enantioners using chiral GC Ebbers, G. J. A. Ariaans, J. P. M. Houbiers, A. Bruggink, B. 5 Inlet Temperature Retention Zwanenburg, Tetrahedron, 1997, 53,9417-9476) Compound pressure psi program C. time min b) Step deracemization of alpha-hydroxycarboxylic acids. Lactate 12 45° C. 4.6 (R) If the racemase cannot be employed directly in a "one-pot 6.0 (S) process in a (chemically or enzymatically catalyzed) enan Phenyl lactate 12 125° C. 7.5 (R) 10 8.5 (S) tioselective step (for example owing to the reaction condi 4-Fluorophenyl lactate 12 80° C.O-5°C. min 12.0 (R) tions required), the enzymatic racemization is carried out 170° C. 12.5 (S) after the enantioselective step without separating the enantio meric product (cf. analogous example using mandelate race NMR mase: U.T. Strauss, K. Faber, Tetrahedron: Asymmetry, 1999, 15 10, 4079-4081). 'H- and 'C NMR spectra were recorded using a Bruker 360 or 500 MHz; the chemical shifts are shown in ppm (Ö c) The ideal case of an application corresponds to a direct scale); tetramethylsilane (TMS) was used as the internal stan combination of the racemase with a chemo- or biocatalytic dard. step in a "one-pot process', which is known as "dynamic racemate separation'. Such methods are sophisticated and HPLC extremely effective. The e.e. values were determined via a JASCO HPLC sys The following may be mentioned as examples of an enan tem type PU-980 pumps and an MD 910 UV detector. A DAICEL Chiralpack AD (0.46 cmx25 cm) column was used tioselective step which can be combined with a racemization for the separation. according to the invention: 25 (i) lipase-catalyzed esterification, for example acyltransfer Melting Points reaction (U. T. Strauss, K. Faber, Tetrahedron.: Asymmetry, The melting points were determined using an MPD 1999, 10, 4079-4081). 350BML5 from GALLENKAMP. (ii) lipase- or protease-catalyzed amide formation (F. van 2. Microbiological Methods and Materials Rantwijk, M. A. P. J. Hacking, R. A. Sheldon, Monatsh. Chem./Chem. Monthly, 2000, 131,549-569.) 2.1 Growing the Cultures The above description and the examples which follow are Bacterial Strains merely intended to illustrate the invention. The large number Table 2 contains a list of the strains studied, their culture of possible modifications which are obvious to the skilled 35 conditions and their deposit numbers. worker also come within the scope of the invention. TABLE 2 Experimental Part Growth conditions of bacterial strains studied A. General Information Strain DSM number Medium Temperature 1. Equipment and Methods Used 40 L. haloferax volcanii 5176 372 37o C. Thin-Layer Chromatography L. haloarctia 3756 372 37o C. valismortis The reactions are monitored, and the purity of the products L. paracasei 2O008 30° C. is examined, by thin-layer chromatography. Silica gel 60s L. paracasei 20207 30° C. on aluminum foil, from Merck, was used for this purpose. 45 L. paracase 2649 30° C. Detection was carried out both by UV light (254mm) and by L. acidophilus 20079 30° C. L. brevis 20054 30° C. spraying with molybdate solution (NH4)Mo.OX4H2O L. piscicola 2O722 92 30° C. (100 g/l) and Ce(SO)x4H2O (4 g/1) in HSO (10%), fol L. halotolerans 2O190 30° C. lowed by heating. L. Confistis 2O196 30° C. 50 L. acetotolerans 2O749 23 30° C. Column Chromatography L. delbrueckii ssp. 20074 37o C. delbruecki The racemization products are purified by column chroma L. kandieri 20593 30° C. tography. Silica gel with a particle size of 43-63 um from L. filiciosus 2O349 30° C. Merck was used as Stationary phase for this purpose. Mix L. farciminis 20184 30° C. 55 L. gasseri 2O243 37o C. tures of petroleum ether (PE) and ethyl acetate (EA) were L. alimentarius 2O249 30° C. employed as eluents, the composition being adapted to Suit L.iensenii 20557 37o C. the separation problem in question. Separation was carried L. curvatus 20010 30° C. out under elevated pressure. L. Sakei ssp. Sakei 20017 30° C. L. Oris 4864 37o C. Gas Chromatography 60 The gas-chromatographic separation of the racemization products was carried out using a Varian gas chromotograph Strain Maintenance 3800-split/splitless injector (FID). The column used was a Strains were maintained by freezing the cells in specific Chirasil-DEX, Chrompack, permethyl-3-cyclodextrin, cryosolutions. To this end, the culture was transferred into chemically bound, 25mx0.32 mmx0.25um, H. Examples of 65 sterile centrifuge tubes and centrifuged for 30 minutes at suitable separation conditions are shown in Table 1 which 8000 rpm and 4° C., and the supernatant solution was follows decanted off. Thereafter, the cells were suspended in cryoso US 7,829,324 B2 19 20 lution, transferred into sterile vials and maintained at -70° C. Medium 231 The cryosolution used was either a solution of 20% by vol Medium 231, which differs from medium 11 only in terms ume DMSO and 0.7% by weight of NaClin HO (Sölkner), or of the pH value, was used for L. acetotolerans. The compo glycerol with 5% by volume of H2O (Weigers). nents listed in Table 3 were combined and only then sterilized, Growing the Strains and the pH was adjusted to 5.2. The cultures were grown in 1000 ml Erlenmeyer flasks in a drying oven at 30° C. or 37° C. (depending on the bacterial Medium 92 strain). For each bacterial strain, 1 liter of the sterile medium Medium 92 was used for L. piscicola. Its composition is in question was first divided into four flasks and inoculated 10 shown in Table 4. with in each case 500 ul of the cell solution which had previ ously been defrosted. All sterile steps of the procedure were TABLE 4 carried out in a laminar-flow clean bench (Heraeus “Herasafe', type HS9). Composition of medium 92 Harvesting the Cells 15 Substance Concentration gll After a growth phase of 3 to 15 days, depending on the Soya extract, tryptic digest (Oxoid) 30.00 bacterial strain, the cells were harvested. To this end, the Yeast extract (Oxoid) 3.00 culture was transferred into centrifuge tubes and centrifuged for 20 minutes at 8000 rpm and 4°C., and the supernatant was decanted off. To wash the cells, they were suspended in Medium 372 approximately 40 ml of buffer (50 mM Bis-Tris, 0.01 M Medium 372 was used for the strains Haloferax volcanii MgCl, pH-6) and centrifuged for another 20 minutes. The and Haloarcula vallismortis. washing procedure was performed twice. The cells were sub sequently resuspended in a little buffer, transferred into a 25 TABLE 5 round-bottom flask and shock-frozen. This was done by swirling the flaskin a Dewar vessel filled with liquid nitrogen. Composition of medium 372 Freeze-drying was carried out in a lyophilizer from Braun (“Christ Alpha 1-4). The lyophilized cells were transferred Substance Concentration gll into glass flasks and stored in the fridge until used. 30 Yeast extract (Oxoid) S.OO Casamino acids (Oxoid) S.OO 2.2. Nutrient Media and Sterilization Sodium glutamate (Oxoid) 1.00 KC 2.00 The nutrient media recommended for the bacterial strains Sodium citrate 3.00 in question were used for growing the microorganisms. The MgSO x 7H2O 2O.OO media were sterilized prior to inoculation by autoclaving NaCl 2OOOO (Varioklav steam sterilizer, H+P Labortechnik GmbH, 35 FeCl2 x 4H2O O.036 Munich) at 121°C. and a superatmospheric pressure of 1 bar. MnCl2 x 4H2O O.OOO36 Medium 11 Medium 11 was used as nutrient medium for the following B. Experiments Carried Out bacterial Strains: L. paracasei, L. acidophilus, L. brevis, L. 40 halotolerans, L. confusus, L. farciminis, L. gasseri, L. alimen EXAMPLE 1. tarius, L. jensenli, L. delbrueckii, L. curvatus, L. Sakei ssp. sakei, L. kandeleri and L. fructosus. Synthesis of Substrates and Reference Material Table 3 shows the composition of the medium. To prevent 45 a possible Maillard reaction and precipitation of the salts, the a) Synthesis of Enantiomerically Pure components of the media were sterilized separately and com 2-hydroxy-3-phenylpropionate bined after sterilization only. A Maillard reaction occurs when compounds such as reducing Sugars react with amino Reaction mixture: 1 g of enantiomerically pure phenylala acids and proteins, and it causes a dark coloration of the 50 medium. nine (6.05 mmol) is dissolved in 12 ml of HSO (1M). 1.66 g (24 mmol) of NaNO, are added portionwise with ice-cool TABLE 3 ing. After the addition, the cooling system is removed and stirring of the reaction mixture is continued overnight at room Composition of medium 11 55 temperature. Substance Concentration gll Work-up: The aqueous solution is extracted three times Casein peptone (Oxoid) 10.00 using 4 ml of ethyl ether in each case, the organic phase is Peptone (Oxoid) 10.00 washed with saturated sodium chloride solution, and the Yeast extract (Oxoid) S.OO Glucose 2O.OO 60 sodium chloride solution is reextracted once with ethyl ether. KHPO 2.00 The combined organic phase is dried over NaSO and the Sodium acetate trihydrate 8.00 solvent is evaporated on a Rotavapor. The yellow oily residue Ammonium citrate 2.00 MgSO4 x 7H2O O.20 is treated with 2 ml of hexane and crystallized by scraping MnSOx H2O O.OS with a glass rod. A precipitate of white crystals forms. The Tween 80 1.00 65 crystals are washed three times using approximately 2 ml of hexane, and the remainder of the solvent is evaporated on a rotary evaporator. US 7,829,324 B2 21 22 Optical rotation (literature): (S)-phenyl lactate:IC)--- were combined and dried over NaSO, and the solvent was 20.8 (c=2, HO) evaporated on a rotary evaporator. To achieve separation of (R)-phenyl lactate: CF-420.9 (c=2.3 H2O) the two enantiomers with a chiral column, all of the com pounds studied required derivatization. To this end, each b) Synthesis of rac-4-fluorophenyl lactate rac-3 sample was treated with 500 ul of BF-methanol, shaken for 10 minutes at 60° C. and extracted by shaking with 1 ml of Reaction mixture: 400 mg (2.2 mmol) of rac-4-fluorophe CHCl2. The organic phase was dried over NaSO and con nylalanine, 670 mg (9.7 mmol) of NaNO in 4.8 ml HSO centrated to a volume of approximately 100 ul. (1M). A racemization reaction was considered negative (-) when Yield: 197 mg (49.0%), white crystals 10 the enantiomeric excess after 2 days amounted to >95%; "H NMR: (360 MHz, CDC1,) 8–2.97 (1H, dd, J =7.2 Hz, enantiomeric excesses of <20% were assessed as positive (+). J–14.4 HZ, CH); 3.17 (1H, dd, J=3.6 Hz, J-14.4 Hz, Any other result received the score (-). The results of the CH); 4.49 (1H, dd, J=3.6 Hz, J-6.1 HZ, CH OH); 6.98 screenings are compiled in Table 6. 7.02 (2H, m, Ar): 7.20-7.28 (2H, m, Ar). 'C NMR: (90 MHz, CDC1,) 8–39.2 (CH): 70.9 (CH 15 TABLE 6 OH): 115.3 (d. J=21.6 Hz, Ar-m): 130.9 (d. J–21.6 Hz, Ar-o): 131.6 (d. J=3.6 Hz, Ar-i); 162.1 (d. J–243.9 Hz, Ar-p); 178.1 Results of the activity Screening (COOH). TLC data: solvent: EA: R-0.35 DSM Substrate Melting point: 73° C. Microorganism Number (S)-1 (S)-2 (S)-3 L. paracasei 2O008 ------c) Synthesis of (S)-4-fluorophenyl lactate (S)-3 L. paracasei 20207 -- -- L. paracasei 2649 -- -- Reaction mixture: 452 mg (2.5 mmol) (S)-4-fluoropheny L. acidophilus 20079 n.d. 25 L. brevis 20054 -- lalanine, 750 mg (10.9 mmol) of NaNO, in 5.4 ml HSO L. piscicola (L. Carnis) 2O722 n.d. (1M). L. halotolerans 2O190 -- Yield: 330 mg (72.5%), white crystals L. Confistis 2O196 H NMR: see rac-4-fluorophenyl lactate L. acetotolerans 2O749 -- -- L. deilbruecki 20074 ------'C NMR: see rac-4-fluorophenyl lactate L. kandieri 20593 n.b. TLC data: solvent: EA, R-0.35 30 L. filiciosus 2O349 Melting point 69° C. L. farciminis 20184 -- L. gaSSeri 2O243 n.d. The compounds L. alimentarius 2O249 -- (S)-(+)-2-hydroxy-3-methylbutanoic acid L.iensenii 20557 -- (S)-(–)-2-hydroxy-4-methylpentanoic acid L. halofax volcanii 5176 n.d. (S)-2-hydroxy-4-phenylbutyric acid 35 L. haloarctia 3756 n.d. (S)-2-hydroxy-3-phenylpropanoic acid vaismortis += good activity were synthesized analogously to Example 1, starting from the -= no activity corresponding commercially available enantiomerically pure ~= moderate activity amino acids. 40 n.d.= not determined

EXAMPLE 2 EXAMPLE 3 Screening for Racemase Activity Determination of the Enantiomeric Excesses with 45 To obtain a first indication regarding the racemization Various Substrates activity of the microorganisms, they were observed for race mization of the (natural) substrate (S)-lactate 1 and/or the For determining the enantiomeric excesses, the procedure (non-natural) substrates (S)-phenyl lactate 2 and (S)-4-fluo of Example 2 was followed, except that the amount of cells rophenyl lactate 3. 50 employed was reduced to 50 mg. For the screening, approximately 10 mg of lyophilized cells were rehydrated in an Eppendorf vial in 900 ul of Bis a) Experiments with L-(-)-3-phenylactic acid as Tris buffer (50mMBis-Tris, pH-6) for one hour at 42°C. and Substrate 150 rpm. Then, in each case 10 mg substrate which had previously been dissolved in 100 ul of buffer and brought to 55 First, 50 mg of lyophilized cells were treated with 1 ml of pH 6-7 with NaOH were added. The reaction mixtures were Bis-Tris buffer (50 mM, pH6) and rehydrated for 1 hour in a incubated in a shaker-incubator at 42° C. and 150 rpm. A shaker-incubator at 42°C. and 150 rpm. Thereafter, in each blank value, where identical reaction conditions were main case 5 mg of substrate, dissolved in 100 ul of buffer, were tained without addition of cells, was measured in parallel with added and the mixtures were incubated in a shaker-incubator each reaction mixture. Since all the control experiments were 60 (30°C., 150 rpm). All samples were measured by means of negative, spontaneous racemization under the given reaction chiral GC after in each case 24, or 48, hours. To achieve conditions can be ruled out. separation of the enantiomers by GC, the samples required After one or two days in the shaker-incubator, the Eppen derivatization. dorf vials were centrifuged for 5 minutes at 13000 rmp and in The derivatization was carried out with BF-methanol (see each case 400 ul of the Supernatant solution were subse 65 above) or with trifluoroacetic anhydride. For the derivatiza quently removed. The solution was extracted twice by shak tion with trifluoroacetic anhydride, 5 mg of 3-phenylactic ing with in each case 600 ul of EA and the organic phases acid were dissolved in 500 ul of CHCl, treated with 5 drops US 7,829,324 B2 23 24 of trifluoroacetic anhydride, shaken for 1 minute at room The two substrates were reacted with all the strains. The temperature and Subsequently treated with approximately degree of racemization was measured by GC after in each 500 ul of HO. The organic phase is dried over NaSO, and case 24 hours and 48 hours. the samples were then measured by GC on a chiral column. In the case of (S)-(+)-2-hydroxy-3-methylbutanoic acid, The results were calculated using the following formulae: 5 only strain DSM 20207 reveals activity. A total of 5 strains revealed racemization toward the substrate (S)-(–)-2-hy conversion rate%:R/(R+S)x100 droxyisocaproic acid. The results are compiled in Tables 8 and 9. racemization%:2R (R+S)x100 10 In addition to the strain Lactobacillus paracasei Subsp. TABLE 8 paracasei DSM 20008, the following four further strains with Screening with (S)-(+)-2-hydroxy-3-methylbutanoic acid good or excellent racemase activity with regard to L-(-)-3- phenylactic acid were found: 24 hours 48 hours Lactobacillus paracasei Subsp. paracasei DSM 2649 15 Lactobacillus paracasei subsp. paracasei DSM 20207 COW. COW. Lactobacillus delbrueckii subsp. delbrueckii DSM 2649 Strain f . ". f E. ". Lactobacillus Oris DSM 4864 The results for the above strains are compiled in Table 7 DSM 20207 86 7 14 73 14 27 and shown as culture condition groups (shaker or drying 2O oven, argon or natural atmosphere) and culture time groups.

TABLE 7 Results of the screening with L(-)-phenylactic acid Drying oven (argon) Shaker (air) DSM 24h 48 h. 24h

number harvest COW. COW. COW. of the Oil ee rate racen. ee rate racen. ee rate racen. strain day 9.6 % 9.6 % 9.6 % 9.6 % (96) 2649 3 8O 10 2O 49 26 51 69 15 31 4 14 43 86 O 50 1OO 20207 3 8 46 92 O 50 1OO 4 65 17 35 20074 3 83 8 16 64 18 36 4864 4 23 38 77 20008 3 O 50 1OO O 50 1OO Shaker (air) Drying oven (ar) DSM 48 h. 24h 48 h.

number harvest COW. COW. COW. of the Oil ee rate racen. ee rate racen. ee rate racen. strain day 9.6 % 9% 9.6 % 9.6 % 9.6 % 2649 3 51 24 49 77 12 23 O SO 100 4 20207 3 4 46 26 S4 17 41 83 O SO 100 20074 3 13 43 87 O SO 100 4864 4 O 50 1OO 70 15 30 45 27 55 20008 3 O 50 1OO O SO 100

To determine whether the racemase activity observed cor relates with a lactate racemase activity, the natural Substrate 55 TABLE 9 L-(+)-lactic acid was reacted with the strains which revealed - - - good to very good activity with L-(-)-3-phenylactic acid. Screening with (S)-(-)-2-hydroxvisocaproic acid It emerged that 100% racemization was detectable after as 24 hours 48 hours little as 24 hours when the strains DSM 20207, 20074 and 20008 were allowed to convert the natural substrate L-(+)- 60 COW. COW. lactic acid. ee rate 806. ee rate 806. No conversion was obtained with strain DSM 2649. Strai8. %9. %9. %9. %9. %9. %9. DSM 2649 88 6 12 75 12 25 b) Experiments with Various Other Alkyl Substrates DSM 20008 85 7 15 82 9 18 The following were used as substrates: 65 DSM 20207 94 3 6 73 14 17 (S)-(+)-2-hydroxy-3-methylbutanoic acid (CHO) DSM 20074 94 3 6 93 4 7 (S)-(–)-2-hydroxyisocaproic acid (CH2O) US 7,829,324 B2 25 26 Cyc is an unsubstituted or mono- or poly Substituted, TABLE 9-continued mono- or binuclear carbo- or heterocyclic ring, wherein said method comprises isomerizing a substrate Screening with (S)-(–)-2-hydroxyisocaproic acid consisting essentially of a first stereoisomer of an alpha 24 hours 48 hours hydroxycarboxylic acid of the formula (I) with an enzyme in a reaction medium to obtain a second stere COW. COW. oisomer oran isomer mixture comprising the first stere ee rate 806. ee rate 806. oisomer and the second stereoisomer, Strain % %) % % % wherein the enzyme is contained in an extract of a micro DSM 2001.7 86 7 14 89 10 2O 10 organism or present in intact cells of a microorganism which express the enzyme, wherein said microorganism In Summary, it can be stated that, again, the same Strains is selected from the group consisting of the Strains L. which had been active toward L-(-)-3-phenylactic acid also paracasei DSM 15755, L. paracasei DSM 15751, L. revealed an activity here. delbrueckii DSM 15754, L. Sakei DSM 15753 and L. 15 Oris DSM 15752, and wherein said microorganism is c) Experiments with (S)-2-hydroxy-4-phenylbutyric capable of racemizing at least one compound selected from the group consisting of (R) and/or (S) form of acid phenyl lactate, 4-fluorophenyl lactate, 2-hydroxy-4- phenylbutyric acid, 2-hydroxy-4-methylpentanecar The degree of racemization was measured by HPLC after boxylic acid and 2-hydroxy-3-methylbutyric acid. in each case 24 hours and 48 hours. The results are compiled 2. The method of claim 1, wherein the second stereoisomer in Table 10. is removed from the isomer mixture and the remaining part of the isomer mixture is subjected to a further isomerization TABLE 10 step. Screening with (S)-2-hydroxy-4-phenylbutyric acid 25 3. The method of claim 1, wherein the isomer mixture is Subjected to a Subsequent chemical or enzymatic stereoselec 24 hours tive reaction and a reaction mixture is obtained, wherein the ee conv. rate 806. reaction mixture obtained is subjected to a further isomeriza Strain % % % tion step. 30 4. The method of claim 1, wherein the isomerization reac DSM 2649 6 47 94 DSM 20054 90 5 10 tion is coupled with a Subsequent chemical or enzymatic, DSM 20190 70 15 30 enantioselective reaction, during which the second stereoiso DSM 20008 25 38 75 mer is removed from the reaction medium. DSM 20207 3 48 97 5. The method of claim3, wherein the subsequent chemical DSM 20749 48 26 52 35 or enzymatic, enantioselective reaction is an esterification or an amidation of the alpha-hydroxycarboxylic acid. In connection with the present invention, the following 6. A method for the microbiological isomerization of microorganisms of the DSMZ, which are available to the alpha-hydroxycarboxylic acids of the formula I public, were redeposited on 11 Jul. 2003 in accordance with the rules of the Budapest Treaty with DSMZ (Deutsche Sam 40 mlung von Mikroorganismen and Zellkulturen GmbH, Ger (I) man Collection of Microorganisms and Cell Cultures, Inhof HOyer" fenstra?e 7 B, 38.124 Braunschweig, Germany). R CO2H

45 where DSMNumber Deposit Number R is straight-chain or branched C-C alkyl or C-C alk 20207 DSM 15755 enyl or—(CH2)-Cyc, where n is an integer of 0 to 4, and 20074 DSM 15754 Cyc is an unsubstituted or mono- or poly Substituted, 20017 DSM 15753 50 mono- or binuclear carbo- or heterocyclic ring, 4864 DSM 15752 wherein said method comprises 2649 DSM 15751 (i) screening and obtaining a microorganism of the genus Lactobacillus that is capable of racemizing at least one compound selected from the group consisting of (R) We claim: 55 and/or (S) form of phenyl lactate, 4-fluorophenyl lactate, 1. A method for the microbiological isomerization of 2-hydroxy-4-phenylbutyric acid, 2-hydroxy-4-methyl alpha-hydroxycarboxylic acids of the formula I pentanecarboxylic acid and 2-hydroxy-3-methylbutyric acid, (ii) isomerizing a Substrate consisting essentially of a first (I) 60 stereoisomer of an alpha-hydroxycarboxylic acid of the HOyer formula (I) with an enzyme in a reaction medium to R CO2H obtain a second stereoisomer oran isomer mixture com prising the first stereoisomer and the second stereoiso mer, wherein the enzyme is contained in an extract of where 65 said microorganism or present in intact cells of said R is straight-chain or branched C-C alkyl or C-C alk microorganism which express the enzyme, wherein said enyl or—(CH2)-Cyc, where n is an integer of 0 to 4, and microorganism is selected from the group consisting of US 7,829,324 B2 27 28 the strains L. paracasei DSM 15755, L. paracasei DSM 9. The method of claim 6, wherein the isomerization reac 15751, L. delbrueckii DSM 15754, L. Sakei DSM 15753 tion is coupled with a Subsequent chemical or enzymatic, and L. Oris DSM 15752. enantioselective reaction, during which the second stereoiso 7. The method of claim 6, wherein the second stereoisomer mer is removed from the reaction medium. is removed from the isomer mixture and the remaining part of 5 the isomer mixture is subjected to a further isomerization step. 10. The method of claim 8, wherein the subsequent chemi 8. The method of claim 6, wherein the isomer mixture is calorenzymatic, enantioselective reaction is an esterification Subjected to a Subsequent chemical or enzymatic stereoselec or an amidation of the alpha-hydroxycarboxylic acid. tive reaction and a reaction mixture is obtained, wherein the 10 reaction mixture obtained is subjected to a further isomeriza tion step.