| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | October 6, 1998 |
| PATENT TITLE |
Enzymatic process for the manufacture of ascorbic acid 2-keto-L-gulonic acid and esters of 2-keto-L-gulonic acid |
| PATENT ABSTRACT | The present invention is directed toward efficient, high-yield processes for making ascorbic acid, 2-keto-L-gulonic acid, and esters of 2-keto-L-gulonic acid. The processes comprise reacting the appropriate starting materials with a hydrolase enzyme catalyst such as a protease, an esterase, a lipase or an amidase |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | April 25, 1997 |
| PATENT REFERENCES CITED |
D. G. Hayes, "The Catalytic Activity of Lipases Toward Hydroxy Fatty Acids-A Review", Journal of the American Oil Chemists' Society, vol. 73, No. 5, pp. 543-549, May 1, 1996. T. Reichstein et al., Helv. Chim. Acta, vol. 17, pp. 311-328 (1934). T. Sonoyama et al., Applied and Envtl. Microbiology, vol. 43, pp. 1064-1069 (1982). M. Shinjoh et al., Applied and Envtl. Microbiology, vol. 61 pp. 413-420 (1995). S. Anderson et al., Science, vol. 230, pp. 144-149 (1985). Yamazaki, J. Agri. Chem. Soc. Japan, vol. 28, pp. 890-894 (1954) (translation not included). Chemical Abstracts, vol. 50, 5992d. F. Thiel, Catalysis Today, pp. 517-536 (1994). A. L. Gutman et al., Tetrahedron Lett., vol. 28, pp.3861-3864 (1987). A. L. Gutman et al., Tetrahedron Lett., vol. 28, pp. 5367-5368 (1987). Enzyme Nomenclature (Academic Press, 1992) (cover pages only). E. L. Smith et al., J. Biol. Chem., vol. 243, pp. 2184-2191 (1968). M. Matsushima et al., FEBS Lett., vol. 293, pp. 37-41 (1991). J. Uppenberg et al., Structure, vol. 2, pp. 293-308, 453 (1994). H. J. Duggleby et al., Nature, vol. 373, pp. 264-268 (1995). Molecular Cloning -A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY.Y. (1989), vol. 1, 2, and 3 (title pages and tables of contents only). Current Protocols in Molecular Biology, F. M. Ausubel et al., editors, Greene Publishing Associates and Wiley-Interscience, N.Y. (1989) (title pages and table of contents only). |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A process for preparing ascorbic acid comprising contacting a compound selected from the group consisting of 2-keto-L-gulonic acid and an ester of 2-keto-L-gulonic acid with a hydrolase enzyme catalyst to form ascorbic acid. 2. The process of claim 1 wherein the hydrolase enzyme catalyst is selected from the group consisting of a protease, an esterase, a lipase and an amidase. 3. The process of claim 2 wherein the protease is obtained from a genera selected from the group consisting of Bacillus or Aspergillus. 4. The process of claim 3 wherein the protease is obtained from a Bacillus licheniformis bacteria. 5. The process of claim 4 wherein the protease is the Subtilisin protease having the sequence as shown in SEQ ID NO: 1. 6. The process of claim 2 wherein the esterase is obtained from pig liver extract. 7. The process of claim 6 wherein the esterase is the pig liver esterase having the sequence as shown in SEQ ID NO: 2. 8. The process of claim 2 wherein the lipase is obtained from a genera selected from the group consisting of Aspergillus, Mucor, Candida, Pseudomonas, Humicola, Rhizopus, Chromobacterium, Alcaligenes, Geotricum and Penicillium. 9. The process of claim 8 wherein the lipase is the Candida Antartica B lipase having the sequence as shown in SEQ ID NO: 3. 10. The process of claim 2 wherein the amidase is obtained from a genus Penicillium. 11. The process of claim 10 wherein the amidase is the Penicillin acylase. 12. The process of claim 1 wherein the hydrolase enzyme catalyst contains an active site serine residue. 13. The process of claim 12 wherein the hydrolase enzyme catalyst contains a catalytic triad of serine, histidine and aspartic acid. 14. The process of claim 1 wherein, prior to contacting the compound with the hydrolase enzyme catalyst, the compound is formed into a solution with a solvent. 15. The process of claim 14 wherein the solvent is selected from the group consisting of water, a C.sub.1 to C.sub.6 alcohol and a mixture thereof. 16. The process of claim 1 wherein contacting the compound with the hydrolase enzyme catalyst occurs at a pH between about 1.5 and 10. 17. The process of claim 1 wherein contacting the compound with the hydrolase enzyme catalyst occurs at a temperature from about 5.degree. C. to about 120.degree. C. 18. The process of claim 1 wherein, prior to contacting the compound with the hydrolase enzyme catalyst, the hydrolase enzyme catalyst is naturally expressed from a host organism in vivo. 19. The process of claim 1 wherein, prior to contacting the compound with the hydrolase enzyme catalyst, a gene sequence encoding the hydrolase enzyme catalyst is inserted into a host organism and the host organism is cultured to express the hydrolase enzyme catalyst in vivo. 20. The process of claim 19 wherein the host organism is Pantoea citrea. 21. The process of claim 18 or claim 19 wherein the host organism produces 2-keto-L-gulonic acid. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION This invention relates to processes for the manufacture of ascorbic acid, 2-keto-L-gulonic acid (KLG), and esters of KLG. More particularly, the present invention relates to the use of enzyme catalysts in the manufacture of ascorbic acid, KLG or esters of KLG. BACKGROUND OF THE INVENTION Ascorbic acid, also known as vitamin C, is a dietary factor which must be present in the human diet to prevent scurvy and which has been identified as an agent that increases resistance to infection. Ascorbic acid is used commercially, for example, as a nutrition supplement, color fixing agent, flavoring and preservative in meats and other foods, oxidant in bread doughs, abscission of citrus fruit in harvesting and reducing agent in analytical chemistry. One current method for the manufacture of ascorbic acid utilizes a modification of the original Reichstein-Grossner synthesis (Reichstein et al., Helv. Chim. Acta, 17:311 (1934); U.S. Pat. No. 2,301,811 to Reichstein; all references cited herein are specifically incorporated by reference). In this process a glucose source is converted to ascorbic acid. During conversion an intermediate of a diacetonide of KLG is produced. Several two stage methods exists for the manufacture of ascorbic acid. In the first stage, glucose is converted via fermentation processes to either an isolated intermediate of KLG (Sonoyama et al., Applied and Envtl. Microbiology, 43:1064-1069 (1982); Anderson et al., Science, 230:144-149 (1985); Shinjoh et al., Applied and Envtl. Microbiology, 61:413-420 (1995)) or the intermediate of the Reichstein-Grossner synthesis, the diacetonide of KLG. The second stage, which converts either of the intermediates to ascorbic acid, proceeds by one of two reported routes. The first route, a modification of the latter steps of the Reichstein-Grossner synthesis, requires a multitude of steps whereby the intermediate is esterified with methanol under strongly acidic conditions to produce methyl-2-keto-L-gulonate (MeKLG). The MeKLG is then reacted with base to produce a metal ascorbate salt. Finally, the metal ascorbate salt is treated with an acidulant to obtain ascorbic acid. The second route is a one-step method comprising acid-catalyzed cyclization of KLG, as originally disclosed in GB Patent No. 466548 to Reichstein) and later modified by Yamazaki (Yamazaki, J. Agri. Chem. Soc. Japan, 28:890-894 (1954), and Chem. Abs., 50:5992d) and again by Yodice (WO 87/00839). The Yodice method is commercially undesirable because it uses large amounts of gaseous hydrogen chloride, requires very expensive process equipment and produces an ascorbic acid product requiring extensive purification. Lipases, a group of hydrolase enzymes, have been used with some success in the synthesis of esters of organic acids. In particular, lipases have been utilized in the transesterification of alcohols in which the esterifying agent is irreversible, such as when vinyl acetate is used as the esterifying agent (Thiel, Catalysis Today, 517-536 (1994)). Gutman et. al., Tetrahedron Lett., 28:3861-3864 (1987), describes a process for preparing simple 5-membered ring lactones from gamma-hydroxy methyl esters using porcine pancreatic lipase as the catalyst. However, Gutman et al., Tetrahedron Lett., 28:5367-5368 (1987), later reported that substituting delta-hydroxy methyl esters for gamma-hydroxy methyl esters and using the same catalyst produced only polymers. In EP 0 515 694 A1 to Sakashita et. al., a synthesis of esters of ascorbic acid, which are acylated on the primary hydroxyl group, comprises reacting ascorbic acid with a variety of fatty acid active esters (i.e., fatty acid vinyl esters) in a polar organic solvent in the presence of a lipase. Thus, there exists a need in the art for methods of producing (a) ascorbic acid or metal salts thereof from KLG or esters of KLG, (b) KLG from esters of KLG and (c) esters of KLG from KLG, which have high yield and high purity with little or no by-product formation and are conducted under mild conditions. Accordingly, it is to the provision of such that the present invention is primarily directed. SUMMARY OF THE INVENTION The present invention discloses an advancement in the chemical and biological arts in which a process for preparing ascorbic acid comprises contacting KLG or an ester of KLG with a hydrolase enzyme catalyst. In another embodiment of the present invention, a process for producing KLG comprises contacting an ester of KLG in an aqueous solution with a hydrolase enzyme catalyst. In still another embodiment of the present invention, a process for producing esters of KLG from KLG comprises contacting an alcoholic solution of KLG with a hydrolase enzyme catalyst. The alcoholic solution contains an alcohol corresponding to an alkyl moiety of the ester of KLG to be prepared. In still another embodiment of the present invention, a process for producing esters of KLG from esters of KLG comprises contacting an alcoholic solution of a first ester of KLG with a hydrolase enzyme catalyst. The alcoholic solution contains an alcohol corresponding to an alkyl moiety of a second ester of KLG which is to be prepared. DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the unexpected discovery that ascorbic acid can be formed from KLG or, more preferably, esters of KLG by inducing ring closure of KLG or esters of KLG using a hydrolase enzyme as a catalyst. The process for producing the ascorbic acid may be performed in the melt or in solution. The process may also be performed in vivo or in vitro. For in vivo processes, the hydrolase enzyme catalyst may be naturally occurring within a host cell or may be introduced into a host cell or organism by recombinant DNA methods. The present invention is also directed to the unexpected discovery that KLG can be prepared in a reversible reaction by reacting an ester of KLG in an aqueous solution using a hydrolase enzyme as a catalyst. Moreover, the present invention is directed to the unexpected discovery that an ester of KLG can be prepared by reacting KLG or another ester of KLG in an alcoholic solution using a hydrolase enzyme as a catalyst. The alcohol used to prepare the solution corresponds to the alkyl moiety of the ester of KLG being prepared. The hydrolase enzymes for use as catalysts in the processes of the present invention may be derived from or isolated from any appropriate source organisms. Examples of which include, but are not limited to, plants, microorganisms, and animals, such as yeast, bacteria, mold, fungus, birds, reptiles, fish, and mammals. Hydrolase enzymes for the purposes of this invention are defined generally by the enzyme class E.C.3.-.-.-, as defined in Enzyme Nomenclature (Academic Press, 1992), and are commercially available. Preferred hydrolase enzymes are those capable of effecting hydrolysis of molecules containing carbonyl or phosphate groups. More specifically, the preferred hydrolases are capable of effecting hydrolysis at a carbonyl carbon bearing a heteroatom single bond. Examples of such carbonyl carbons bearing a heteroatom single bond include, but are not limited to, esters, thioesters, amides, acids, acid halides, and the like. The preferred hydrolases include the enzyme class E.C.3.1.-.-, which includes hydrolases acting on ester bonds, such as esterases and lipases; the enzyme class E.C.3.2-.-, which includes glycosidases; the enzyme class E.C.3.4-.-, which includes peptide hydrolases, such as proteases; and the enzyme class E.C.3.5.-.-, which includes amidases acting on bonds other than peptide bonds. Most preferred hydrolases include proteases, amidases, lipases, and esterases. More preferred hydrolases contain an active site serine residue which is capable of undergoing esterification or transesterification with KLG or esters of KLG. Even more preferred are those hydrolases which contain the catalytic triad of serine, histidine and apartic acid. Preferred proteases include those derived from bacteria of the genera Bacillus or Aspergillus. Particularly preferred proteases are those obtained from the bacteria Bacillus licheniformis. Preferred proteases are those containing at least 70% sequence homology with Subtilisin. Proteases having sequence homology with Subtilisin are used in the detergent industry and, therefore, are readily available. More preferred are proteases having at least 80% sequence homology with Subtilisin, even more preferred are proteases having at least 90% sequence homology with Subtilisin and, in particular, proteases having at least 95% sequence homology to Subtilisin. A highly preferred protease is Subtilisin itself having an amino acid sequence (SEQ ID NO: 1) described by Smith et al., J. Biol. Chem., 243:2184-2191 (1968), and given below: __________________________________________________________________________ MMRKKSFWLG MLTAFMLVFT MAFSDSASAA QPAKNVEKDY IVGFKSGVKT ASVKKDIIKE SGGKVDKQFR IINAAKAKLD KEALKEVKND PDVAYVEEDH VAKALAQTVP YGIPLIKADK VQAQGFKGAN VKVAVLDTGI QASHPDLNVV GGASFVAGEA YNTDGNGHGT HVAGTVAALD NTTGVLGVAP SVSLYAVKVL NSSGSGTYSG IVSGIEWATT NGMDVINMSL GGPSGSTAMK QAVDNAYARG VVVVAAAGNS GSSGNTNTIG YPAKYDSVIA VGAYDSNSNR ASFSSVGAEL EVMAPGAGVY STYPTSTYAT LNGTSMASPH VAGAAALILS KHPNLSASQV RNRLSSTATY LGSSFYYGKG LINVEAAAQ. __________________________________________________________________________ For the convenience of the reader, Table 1 provides a summary of amino acid shorthand used above and in the remainder of the specification. TABLE 1 ______________________________________ Amino Acid Three-Letter Symbol Abbreviation One-Letter ______________________________________ Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic Acid Asp D Cysteine Cys C Glutamine Gln Q Glutamic Acid Glu E Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V ______________________________________ Also encompassed by the scope of the present invention are proteases corresponding to one to six site-specific mutants, sequence additions, and sequence deletions of the sequence given above. Even more preferred are proteases corresponding to zero to two site-specific mutants of the Subtilisin sequence given above. Esterases suitable for the present invention include those obtained from pig liver extract. Preferred esterases are those having at least 70% sequence homology with pig liver esterase having an amino acid sequence (SEQ ID NO: 2) described in Matsushima et al., FEBS Lett., 293:37 (1991), and given below: __________________________________________________________________________ MWLLPLVLTS LASSATWAGQ PASPPVVDTA QGRVLGKYVS LEGLAFTQPV AVFLGVPFAK PPLGSLRFAP PQPAEPWSFV KNTTSYPPMC CQDPVVEQMT SDLFTNFTGK ERLTLEFSED CLYLNIYTPA DLTKRGRLPV MVWIHGGGLV LGGAPMYDGV VLAAHENFTV VVVAIQYRLG IWGFFSTGDE HSRGNWGHLD QVAALHWVQE NIANFGGDPG SVTIFGESFT AGGESVSVLV LSPLAKNLFH RAISESGVAL TVALVRKDMK AAAKQIAVLA GCKTTTSAVF TFVHCLRQKS EDELLDLTLK MKFLTLDFHG DQRESHPFLP TVVDGVLLPK MPEEILAEKD FTFNTVPYIV GINKQEFGWL LPTMMGFPLS EGKLDQKTAT SLLWKSYPIA NIPEELTPVA TFTDKYLGGT DDPVKKKDLF LDLMGDVVFG VPSVTVARQH RDAGAPTYMY EFQYRPSFSS DKFTKPKTVI GDHGDEIFSV FGFPLLKGDA PEEEVSLSKT VMKFWANFAR SGNPNGEGLP HEPFTMYDQE EGYLQIGVNT QAAKRLKGEE VAFWNDLLSK EAAKKPPKIK HAEL. __________________________________________________________________________ Esterases more preferably have at least 80% sequence homology with the sequence of the pig liver esterase given above, even more preferably at least 90% sequence homology, especially preferred at least 95% sequence homology. Highly preferred is the pig liver esterase having the sequence given above. Also encompassed by the scope of the present invention are esterases corresponding to one to six site-specific mutants, sequence additions, and sequence deletions of the sequence given above. Even more preferred are esterases corresponding to zero to two site-specific mutants of the pig liver esterase sequence given above. Preferred lipases include those isolated from pigs and other mammals, microorganisms, and plants. This includes, but is not limited to, lipases obtained from the genera Aspergillus, Mucor, Candida, Pseudomonas, Humicola, Rhizopus, Chromobacterium, Alcaligenes, Geotricum, and Penicillium. Preferred lipases also include extracellular lipases, such as cutinases. More preferred lipases have at least 70% sequence homology with Candida Antartica type B lipase, even more preferred have at least 80% sequence homology, still more preferred have at least 90% sequence homology, and even more preferred have at least 95% sequence homology. A highly preferred lipase is the Candida Antartica type B lipase itself which has an amino acid sequence (SEQ ID NO: 3) described by Uppenberg et al., Structure, 2:293, 453 (1994), and given below: __________________________________________________________________________ MKLLSLTGVA GVLATCVAAT PLVKRLPSGS DPAFSQPKSV LDAGLTCQGA SPSSVSKPIL LVPGTGTTGP QSFDSNWIPL STQLGYTPCW ISPPPFMLND TQVNTEYMVN AITALYAGSG NNKLPVLTWS QGGLVAQWGL TFFPSIRSKV DRLMAFAPDY KGTVLAGPLD ALAVSAPSVW QQTTGSALTT ALRNAGGLTQ IVPTTNLYSA TDEIVQPQVS NSPLDSSYLF NGKNVQAQAV CGPLFVIDHA GSLTSQFSYV VGRSALRSTT GQARSADYGI TDCNPLPAND LTPEQKVAAA ALLAPAAAAI VAGPKQNCEP DLMPYARPFA VGKRTCSGIV TP. __________________________________________________________________________ Also encompassed by the scope of the present invention are lipases corresponding to one to six site-specific mutants, sequence additions, and sequence deletions of the sequence given above. Even more preferred are lipases corresponding to zero to two site-specific mutants of the Candida Antartica type B sequence given above. Preferred amidases include those isolated from bacteria of the genus Penicillium. A more preferred amidase has at least 80% sequence homology with Penicillin acylase. A particularly preferred amidase is Penicillin acylase, which is also referred to as Penicillin amidohydrolase, E.C. 3.5.1.11 (Duggleby et al., Nature, 373:264-268 (1995)). For hydrolases containing serine at their active site, the first step in the reaction of either KLG or esters of KLG is believed to involve formation of a KLG-enzyme ester via acylation by KLG of the active site serine. Intra-molecular ring closure is believed to yield ascorbic acid (or its salts), whereas alcoholysis yields an ester of KLG and hydrolysis yields KLG. The process of the present invention comprises contacting either KLG or an ester of KLG with a hydrolase enzyme to form ascorbic acid. Preferably, this reaction is performed in the presence of an organic solvent system, an aqueous solvent system or a mixture thereof. The organic solvent is preferably a C.sub.1 -C.sub.6 alcohol. The aqueous solvent system or mixed aqueous and organic solvent systems are more preferable because ascorbic acid, KLG, and esters of KLG are generally more soluble in aqueous solvent systems. For the in vitro production of ascorbic acid from esters of KLG, the mixed aqueous and organic solvent systems or organic solvent systems are preferable to minimize competing hydrolysis reactions which can produce KLG as a byproduct. Aqueous solvent systems are especially preferable when utilizing whole cell systems for the production of ascorbic acid in vivo. In one aspect of the present invention, the ascorbic acid is produced from KLG or esters of KLG in in vivo, whole cell, and whole organism production systems in the presence of the hydrolase enzyme catalyst. In one embodiment, the hydrolase enzyme is naturally produced by the host organism. In another embodiment, the hydrolase enzyme is produced by the host organism through recombinant DNA technology. For example, a gene sequence encoding a hydrolase enzyme is inserted in a host organism wherein the host organism may be a microorganism, plant, or animal which is capable of expressing the hydrolase enzyme. The host organism producing the hydrolase enzyme is cultured, i.e. provided with nutrients and a suitable environment for growth, in the presence of KLG or esters of KLG to produce the ascorbic acid. Preferably, the host organism is Pantoea citrea, previously referred to as Erwinia herbicola as disclosed in U.S. Pat. No. 5,008,193 to Anderson et al. Also preferably, the host organism is one that produces KLG in addition to producing the hydrolase enzyme. Representative organisms are from the genera Pantoea or Gluconobacter, such as disclosed in Shinjoh et al.,Applied and Envtl. Microbiology, 61:413-420 (1995), and the genus Corynebacterium as disclosed in Sonoyama et al., Applied and Envtl. Microbiology, 43:1064-1069 (1982). As used herein, recombinant DNA technology includes in vitro recombinant DNA techniques, synthetic techniques and in vivo recombinant/genetic recombination and is well known in the art. See, for example, the techniques described in Maniatis et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience, N.Y. (1989); Anderson et al., Science, 230:144-149 (1985); and U.S. Pat. No. 5,441,882 to Estell et. al. For preparations of KLG from esters of KLG, an aqueous solution of the ester of KLG is reacted with the hydrolase enzyme. A co-solvent may be used in the preparation of KLG and is preferably a C.sub.1 -C.sub.6 alcohol. For preparations of the esters of KLG from KLG or from other esters of KLG, the starting material is in an alcoholic solution wherein the alcohol corresponds to the alkyl moiety of the ester of KLG to be prepared. The alkyl moiety R of the alcohol ROH from which the preferred ester of KLG is derived may be chosen from branched or straight chain, saturated or unsaturated, alkyl, arylalkyls, aryls, and substituted aryls. Preferred R groups include C.sub.1 to C.sub.6 straight or branched chain, saturated or unsaturated alkyls. Even more preferred esters of KLG that are derived for alkyl moieties include MeKLG, ethyl-KLG, n-propyl-KLG, isopropyl-KLG, n-butyl-KLG, isobutyl-KLG, t-butyl-KLG, and n-pentyl-KLG. The most preferred esters of KLG produced are MeKLG due to its ease of manufacture and butyl-KLG due to the advantageous use of the butanol water azeotroph in water removal. A co-solvent may be used in the preparation of the esters of KLG and is preferably water, a C.sub.1 -C.sub.6 alcohol or a mixture thereof. Preferred temperatures for conducting the reactions of the present invention are from about 5.degree. C. to about 120.degree. C. Even more preferred temperatures are from about 25.degree. C. to about 100.degree. C., and especially preferred temperatures are from about 38.degree. C. to about 80.degree. C. The preferred pH for the process of the present invention is between about 1.5 and about 10, and a more preferred pH is between about 3 and about 10. For the preparation of ascorbic acid salts from esters of KLG, a particularly preferred pH range is between about 6 and about 10. For the preparation of ascorbic acid as the free acid, a preferred pH is that under the pKa of ascorbic acid and, more preferred, is that under about 4.2. For the preparation of KLG from esters of KLG, a particularly preferred pH range is between about 5 and about 10 due to the generally enhanced rates of enzyme assisted hydrolysis in this pH range. Alternatively, a pH of between about 1.5 and about 2.5 is particularly desirable for the generation of KLG in protonated form. Finally, for the preparation of esters of KLG from KLG, a particularly preferred pH range is between about 3 and about 6. Each hydrolase has a temperature optimum, a pH optimum, and a pH and temperature range associated with activity. Thus, the appropriate pH and temperature range for a given hydrolase is that which allows for activity of the hydrolase and avoids conditions which are denaturing or inactivating to the hydrolase. For conditions which may be denaturing, such as high temperature or the use of denaturing solvents such as methanol or the like, a minimal amount of testing may be required to define those hydrolases which remain active under a given set of conditions. The following examples are offered by way of illustration and are not intended to limit the scope of the claimed invention. EXAMPLES Proton and carbon nuclear magnetic resonance (NMR) spectra were recorded on a Varian Gemini 300 NMR instrument operating at 300 MHZ in proton mode and 75 MHZ in carbon mode. All NMR spectra were referenced to tetramethylsilane (TMS) at 0 parts per million (ppm) and peak frequencies were recorded in ppm unless otherwise specified. HPLC (high-performance liquid chromatography) analysis was carried out using ultraviolet (UV) detection. Mass spectra (MS) were obtained using a Fisons VG Analytical Ltd. Autospec Mass Spectrometer in FD (field desorption) mode. The KLG used in the experiments was obtained by fermentation according to the method of Lazarus et. al., Anderson et al., Science, 230:144-149 (1985), and was purified by concentration and crystallization. KLG may alternatively be prepared by chemical conversion from L-sorbose according to methods well known in the art (see e.g., U.S. Pat. No. 2,301,811 to Reichstein). A standard of methyl-2-keto-L-gulonate was purchased from Aldrich Chemical Company (Rare and Specialty Chemicals Catalog), in addition to being prepared by esterification of KLG by methods similar to the procedure used for the preparation of butyl-KLG, described below. Enzyme hydrolase samples were obtained from commercial sources, including Sigma Chemical Company, Altus Biologics, Recombinant Biocatalysis, Boehringer Mannheim, Novo Nordisk, Genencor International, Thermogen, and Fluka |
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