WO2008035187A2

WO2008035187A2 - Alcohol dehydrogenase from agromyces sp. and a method of producing a chiral secondary alcohol using same

Info

Publication number: WO2008035187A2
Application number: PCT/IB2007/002731
Authority: WO
Inventors: Richard C. Llyod; Philip A. Keene
Original assignee: Dow Global Technologies LLC
Current assignee: Dow Global Technologies LLC
Priority date: 2006-09-21
Filing date: 2007-09-20
Publication date: 2008-03-27
Anticipated expiration: 2009-03-21
Also published as: WO2008035187A3

Abstract

A method of reducing a ketone to a chiral secondary alcohol. The method includes providing an alcohol dehydrogenase from Agromyces sp. and adding a ketone to the alcohol dehydrogenase to produce a chiral secondary alcohol. An organic solvent, such as isopropanol, is optionally added to the alcohol dehydrogenase and the ketone. A method of producing (2S,5S)-hexanediol is also disclosed, as is an enzyme having alcohol dehydrogenase activity. Additionally methods of producing chiral secondary alcohols from aliphatic methyl ketones, aromatic methyl ketones, and beta ketoesters are provided.

Description

ALCOHOL DEHYDROGENASE FROM AGROMYCES SP. AND A METHOD OF PRODUCING A CHIRAL SECONDARY ALCOHOL USING SAME

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Application 60/846,180, filed September 21, 2006, the disclosure of which is incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0001] The present invention relates to an alcohol dehydrogenase ("ADH") prepared from Agromyces sp. and to a method of stereoselective^ reducing a ketone to a chiral alcohol using the ADH. More specifically, the ADH is prepared from Agromyces mediolanus.

BACKGROUND OF THE INVENTION

[0002] Chiral secondary alcohols are key building blocks used in the pharmaceutical and fine chemicals industries. Enzymatic routes to these compounds are via a kinetic (or dynamic kinetic) resolution of a racemate using a lipase or esterase, or by reducing a ketone to a chiral secondary alcohol. The major advantage of ketone reduction techniques is the prospect of a 100% yield of a single enantiomer product because the ketone is a prochiral starting material. In comparison, conventional resolution techniques provide a theoretical maximum yield of just 50%. In addition, there is a lack of dependable methods available for the asymmetric reduction of aliphatic ketones.

[0003] Alcohol dehydrogenases are ubiquitous in nature, and ADH-mediated biological processes include important reactions, such as the last step of alcoholic fermentation {i.e. conversion of glucose into ethanol in yeasts), the reduction of all-trans retinal to all-trans retinol (vitamin Ai) in the retina, or the degradation of blood alcohol in the liver. The reactions are typically reversible and utilize a cofactor or coenzyme, such as nicotinamide adenine dinucleotide (NADVNADH) or nicotinamide adenine dinucleotide phosphate (N ADP⁺/N ADPH). A large number of ADHs can come from different sources such as yeast, fungal, bacterial and mammalian. ADHs have been studied in biocatalytic applications, and a number have been shown to possess broad substrate specificity.

[0004] ADHs can be used to produce organic compounds, such as alcohols, ketones, or aldehydes. Of particular interest is the enantioselective production of an optically active secondary alcohol by catalytic reduction of the corresponding ketone. However, one obstacle in regard to the use of ADHs is the regeneration of the cofactors, which can be expensive and difficult to use. Previous approaches have used enzymes, such as formate dehydrogenase or glucose dehydrogenase, for the cofactor regeneration. Whole cells have also been used, which bypasses the problems with cofactor regeneration. However, volume efficiencies are often low with the whole cell preparations and many organisms have a low tolerance for cosolvents used in the reaction medium.

BRIEF SUMMARY OF THE INVENTION

[0005] The present invention relates to a method of reducing a ketone to a chiral secondary alcohol that includes providing an alcohol dehydrogenase from Agromyces sp. and adding a ketone to the alcohol dehydrogenase. An organic solvent can be, optionally, added to the alcohol dehydrogenase and/or the ketone or added at the same time the alcohol dehydrogenase is added to the ketone.

[0006] Embodiments of the present invention also include producing chiral secondary alcohols from aliphatic methyl ketones, aromatic methyl ketones, and beta-ketoesters.

[0007] The present invention also relates to a method of producing (ZS^-hexanediol that includes providing an alcohol dehydrogenase from Agromyces sp. and adding acetonylacetone to the alcohol dehydrogenase. An organic solvent can be, optionally, added to the alcohol dehydrogenase and/or the acetonylacetone.

[0008] The present invention also relates to an enzyme having alcohol dehydrogenase activity, wherein the enzyme is obtained from Agromyces sp. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention is described more fully hereinafter, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0010] An ADH from Agromyces sp. is disclosed. The ADH functions as a catalyst to stereoselectively reduce a ketone to a chiral secondary alcohol. Alternatively, the ADH can be used to oxidize an alcohol to a ketone. The reaction can be conducted in the presence of a relatively high concentration of organic solvent and at a relatively high substrate concentration. The ADH can be obtained from Agromyces sp., such as from Agromyces mediolanus also called corynebacterium mediolanum, agromyces mediolanensis, or flavobacterium dehydrogenans. Other Agromyces sp. that can be used include, but are not limited to Agromyces albus, Agromyces aurantiacus, Agromyces brachium, Agromyces cerinus, Agromyces cerinus subsp. cerinus, Agromyces cerinus subsp. nitratus, Agromyces fucosus, Agromyces fucosus subsp. fucosus, Agromyces fucosus subsp. hippuratus, Agromyces hippuratus, Agromyces humatus, Agromyces italicus, Agromyces lapidis, Agromyces luteolus, Agromyces neolithicus, Agromyces ramosus, Agromyces rhizospherae, Agromyces salentinus, Agromyces subbeticus, and Agromyces ulmi.

[0011] A whole cell preparation of Agromyces sp. can be used as a source of the ADH, or the ADH can be in a partially purified or substantially purified form. The term "purified" is used herein to collectively refer to partially purified ADH or substantially purified ADH. Cells of Agromyces sp. may be preserved by lyophilization without loss of activity. Therefore, the cells may be stored for long time periods.

[0012] The ADH from Agromyces sp. has a broad substrate tolerance and is capable of reducing a variety of ketone substrates or of oxidizing a variety of alcohol substrates. As used herein, the term "substrate" refers to a starting material of the reaction, such as the ketone substrate or the alcohol substrate. The term "product" refers to a compound produced by the reaction. For instance, if the ADH is used to reduce a ketone, the product is a corresponding chiral secondary alcohol; if the ADH is used to oxidize a secondary alcohol, the product is a corresponding ketone. The ketone substrate can be an aliphatic ketone, such as an aliphatic methyl ketone. Examples of aliphatic methyl ketones include, but are not limited to, 2-hexanone, acetonylacetone, 2,5- hexanedione, 2-butanone, 2-pentanone, 4-methyl-2-pentanone, 3,3-dimethyl-2-butanone, and 2- heptanone or mixtures thereof. Other ketone substrates can be aromatic methyl ketones. These can include, but are not limited to, acetophenone, 3,5-b/.$(trifluoromethyl)aceotophenone, 2- chloroacetophenoπe, 4-acetylbenzonitrile, 2-(trifluoromethyl)aceotophenone, 4-chloroaceophenone, 1,3-Diacetylbenzene, 2-acetylpyridine, 3-acetylpyridine, and 4-acetylpyridine or mixtures thereof. Other ketone substrates can be cyclic ketones. These can include, but are not limited to 1-tetralone, 2-tetralone, 6-methoxy- 1-tetralone, and 1-indanone and/or mixtures thereof. Other ketone substrates can include beta-ketoesters. Compounds can include, but are not limited to, ethylacetoacetate, ethyl 4-chloro-3-oxobutanoate, ethyl 4-bromo-3-oxobutanoate, ethyl 4-azido-3-oxobutanoate, ethyl 3-oxo- 3-phenylpropanoate, ethyl 4,4,4-trichloro-3-oxobutanoate, ethyl 4,4,4-trifluoro-3-oxobutanoate, ethyl 3-oxo-4-phenylsulfonylbutanoate, ethyl 2-oxo-l-cyclopentanecarboxylate and ethyl 2-oxo-l- cyclohexanecarboxylate.

[0013] If acetonylacetone is the substrate, the chiral secondary alcohol produced by the reaction can be (2S,5S)-hexanediol. (25,55)-Hexanediol is a fundamental building block in the synthesis of (/?,/?)-MeDuPHOS, a key component of one of the world's leading asymmetric hydrogenation catalysts. However, other chiral secondary alcohols may be produced if a different substrate is used.

[0014] Agromyces sp. is tolerant of a high concentration of organic solvent and, therefore, the ADH in the Agromyces sp. cells may catalyze the reaction of the substrate to the product even when the cells are exposed to a high concentration of organic solvent. In other words, the ADH may remain stable and bioactive at a high concentration of organic solvent. A reaction mixture of the ADH, the substrate, a buffer, and the organic solvent may include from approximately 0% by volume to approximately 30% by volume of the organic solvent, such as from approximately 0.1% by volume to approximately 30% by volume of the organic solvent. At high substrate concentrations (ranging from approximately 25 g/L to approximately 50 g/L), the reaction mixture may include approximately 25% by volume of the organic solvent.

[0015] If the ADH is used as a reduction catalyst, the organic solvent may be a secondary alcohol, such as isopropanol, in which the substrate is substantially soluble. Other solvents that can be used include, but are not limited to secondary alcohols such as 2-butanol, 2-pentanol or 2- hexanol, amides such as _V,N-dimethylfoπnamide, and/or oxides such as dimethylsulf oxide. Additionally, cyclohexanol can be used as a cosolvent. When the organic solvent is a secondary alcohol it may also act as a sacrificial co-substrate that is itself oxidized to a ketone such as acetone, thus regenerating the cofactors used in the reduction. Therefore, no additional enzymes are needed to regenerate the cofactors, which can reduce the overall cost of the reaction. Additionally this reaction can allow for the cofactor to be singular which can further reduce costs. If the ADH is used as an oxidation catalyst to catalyze the reverse reaction (from the alcohol to the ketone), the organic solvent can a ketone including but not limited to acetone, methyl ethyl ketone, 2-propanone or 2- hexanone.

[0016] The buffer used in the reaction mixture can be selected to maintain optimal biological activity of the ADH. The selection of a buffer for use with an enzyme, such as the ADH from Agromyces sp., may be determined by a person of ordinary skill in the art. For the sake of example only, the buffer can be 0. IM potassium phosphate buffer having a pH of approximately 7.0. However, the buffer can include a salt other than potassium phosphate, the buffer concentration can be lower or higher than 0. IM, or the pH may be lower or higher than 7.0.

[0017] The substrate can be present in the reaction mixture at a relatively high substrate concentration, such as at a concentration of less than or equal to approximately 50 g/L. For instance, the substrate concentration may range from approximately 1 g/L to approximately 100 g/L, such as from approximately 10 g/L to approximately 50 g/L.

[0018] Cells of Agromyces sp. can be grown by conventional techniques. For example, the cells can be grown in a culture medium in a vessel, such as in a fermenter. An inoculum of a glycerol stock of the Agromyces sp. cells may be introduced into the culture medium. The temperature, pH, or other growth conditions of the culture medium may be maintained to provide optimal growth of the cells. The temperature and pH may be determined by a person of ordinary skill in the art and, therefore, are not described in detail herein. For example, the culture medium may be maintained at a temperature ranging from approximately 20⁰C to approximately 35°C, such as from approximately 25⁰C to approximately 30⁰C. After a desired number of cells have grown, the cells may be harvested by conventional techniques. For instance, the cells may be harvested by centrifugation when the optical density (at 600 nm) of the culture medium has reached approximately 6.0. The cells may then be washed, centrifuged, and the supernatant removed. The cell pellet may be lyophilized to produce a dry form of the Agromyces sp. cells. The lyophilized cells may be stored at a low temperature until needed without substantial loss of activity. For instance, the lyophilized cells may be stored at approximately -20⁰C.

[0019] The lyophilized cells can be rehydrated in the buffer, forming a whole cell preparation of the Agromyces sp. For the sake of example only, the lyophilized cells may be rehydrated in a 0.1 M potassium phosphate buffer having a pH of approximately 7.0. However, additional buffers, additional buffer concentrations, and additional pHs of the buffer are also contemplated and may be selected by a person of ordinary skill in the art. The lyophilized cells may be exposed to the buffer at an appropriate temperature and for an appropriate amount of time before use. For instance, the lyophilized cells may be exposed to the buffer at a temperature that ranges from approximately 20⁰C to approximately 3O⁰C and for a time period that ranges from approximately 10 minutes to approximately 60 minutes.

[0020] The reaction may be performed using the whole cell preparation of Agromyces sp., which includes the ADH. Alternatively, the ADH may be purified from the whole cell preparation. Protein purification techniques are known in the art and, therefore, are not described in detail herein. If a purified ADH is used to perform the reaction, the purified ADH may be dissolved in the buffer before use.

[0021] The ADH, the buffer, and the substrate can be admixed to initiate the reaction of the substrate to the product. If the organic solvent is present, the organic solvent may also be admixed with the ADH, the buffer, and the substrate. For instance, the organic solvent and the substrate may be added to the whole cell preparation or to the purified ADH to catalyze the reaction. The reaction mixture can be maintained under biological reaction conditions of temperature, pH, solvent osmolality, ionic composition, and ambient atmosphere for a period of time sufficient for the reaction to progress. The temperature of the reaction mixture may range from approximately 15°C to approximately 50⁰C, such as from approximately 20⁰C to approximately 40⁰C. In one embodiment, the temperature of the reaction mixture ranges from approximately 22°C to approximately 30⁰C. The pH of the reaction mixture may range from approximately 6.0 to approximately 11.0, such as from approximately 6.0 to approximately 8.5. The reaction may be monitored by spectral analysis to determine whether substantially all of the substrate has been converted to the product. For instance, the reaction may be monitored by gas chromatography ("GC") or liquid chromatography ("LC"). The reaction time may vary depending on the substrate used. The reaction can produce a high enantioselectivity in reducing the ketone substrate to the chiral secondary alcohol.

[0022] In one embodiment, the ADH from Agromyces mediolanus is used to reduce acetonylacetone to (2S,5S)-hexanediol. The (2S,5S)-hexanediol is prepared as a single isomer.

[0023] The nucleic acid sequence of the gene that codes for the ADH obtained from the Agromyces sp. may be determined by conventional techniques. In addition, the amino acid sequence of the ADH may be determined by conventional techniques.

[0024] In addition to isolating the ADH from the whole cell preparation, the ADH may be produced by cloning the gene that codes for the ADH and expressing the gene in an appropriate organism. The coding DNA sequence may be cloned into a vector, which is transfected into host cells and expressed in cell culture. The vector may contain further functional nucleotide sequences for regulating, in particular repressing or inducing, expression of the ADH gene and/or the reporter gene.

[0025] Deposits

[0026] The following sample was deposited in accordance with the Budapest Treaty at the recognized depositary The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, Scotland, United Kingdom, AB21 9YA on 3 March 2006: Agromyces mediolanus NCIMB number NCMB 41380. [0027] The following examples serve to explain embodiments of the present invention in more detail. These examples are not to be construed as being exhaustive or exclusive as to the scope of this invention.

Examples Example 1

Growth of Agromyces mediolanus

[0028] NCIMB 41380, Agromyces mediolanus, was grown in YM media (20 L) in a 30 L BiostatC fermenter at 25°C with no pH control. An inoculom of Agromyces mediolanus was introduced from glycerol stock. The cells were allowed to grow for approximately 43.5 hours until the optical density (at 600 nm) of the broth reached 6.0. The cells were harvested by centrifugation at 12,000 g, yielding approximately 500 g cells. The cells were washed in water (2 L) for 30 minutes and were centrifuged. The supernatant was discarded and the pellet was Iyophilized to produce 51 g of dry cells. The Iyophilized cells were stored at -2O⁰C.

Example 2

10 g/L Substrate Concentration: Small Scale Experiment

[0029] The lyophilized cells (200 mg) described in Example 1 were rehydrated by shaking the cells in 4.75 ml of 0.1 M potassium phosphate buffer having a pH of 7.0. The cells were shaken at 200 rpm and 28⁰C for 30 minutes. The substrate (50 μl or 50 mg) was added in isopropanol (250 μl) and the mixture shaken until GC or LC analysis indicated the reaction proceeded no further. 2- Chloroacetophenone, 1,3-Diacetylbenzene, 2-Acetylpyridme, 3-Acetylpyridine and 4- Acetylpyridine all showed 99% or above e.e. at lOg/1 substrate concentration.

Example 3

50 g/L Substrate Concentration: Small Scale Experiment

[0030] The lyophilized cells (200 mg) described in Example 1 were rehydrated by shaking the cells in 3.75 ml of 0. IM potassium phosphate buffer having a pH of 7.0. The cells were shaken at 200 rpm and 28°C for 30 minutes. The substrate (250 μl or 250 mg) was added in isopropanol (1.25 ml) and the mixture shaken until GC or LC analysis indicated the reaction proceeded no further. 2-hexanone, acetonylacetone acetophenone, 3,5-Z?w-(trifluoromethyl)aceotophenone, 4- acetylbenzonitrile, and 4-chloroacetophenone all showed 97% or above e.e. at 50g/l substrate concentration.

Example 4

50 g/L Substrate Concentration: Large Scale Experiment

[0031] The Iyophilized cells (200 mg) described in Example 1 were rehydrated by shaking the cells in 300 ml of 0. IM potassium phosphate buffer having a pH of 7.0. The cells were shaken at 200 rpm and 28⁰C for 30 minutes. Acetonylacetone (20 ml, 19.4 g, 0.17 mol) was added in isopropanol (100 ml) and the mixture shaken at 200 rpm and 28°C for 25 hours. The mixture was acidified to a pH of 2 with 6M hydrochloric acid, filtered through celite, and the celite plug washed with a 1:1 mixture of wateπisopropanol (1.2 L). The filtrate was concentrated to leave a brown residue, which was suspended in ethyl acetate (400 ml) and heated to 45°C for 10 minutes. After cooling and filtering through celite, the filtrate was concentrated to dryness. The resulting residue ( 14.4 g) was dissolved in methyl tert butyl ether ( 15 ml) and cooled to 2°C. The solution was seeded with a single crystal of (25,55)-hexanediol and placed in the fridge overnight. Filtration yielded 4.33g of off-white crystals of (2S,5S)-hexanediol (>98% de, >98% ee, 22% yield). When further purification of these crystals is desired, recrystallization from ethyl acetate can be performed.

Example 5

[0032] lS-pyridin-4-ylethanol: 4.0 g of lyophilised Agromyces mediolanus NCIMB 41380 cells were rehydrated by shaking in 0.1M potassium phosphate buffer pH 7 (75 mL) at 200 rpm, 28⁰C for 30 mins. 4-Acetylpyridine (5.0 g,41.3 mmol) was added in isopropyl alcohol (25 mL) and the mixture shaken at 200 rpm, 28⁰C for 46 hours, when GC analysis suggested the reaction had proceeded to 90% conversion. The mixture was acidified to pH 2 with 6M HCl, filtered through celite, and the celite plug washed with water (20 mL). The pH of the filtrate was adjusted to 11 with 5M NaOH and extracted with EtOAc (3 x 70 mL). The combined organic extracts were dried (MgSO₄), filtered and concentrated in vacuo to yield an off-white crystalline solid (3.97 g), which was recystallised from EtOAc (4 mL) to yield lS-pyridin-4-ylethanol as white crystals (2.89 g, 58%, >98% e.e.). ¹H NMR (400 MHz, CDCl₃) 8.51 (2H, dd, 75, 2), 7.29 (2H, dd, 75, 2), 4.90 (IH, q, J 6), 2.97 (IH, br s), 1.50 (3H, d, 76); ¹³C NMR (IOO MHZ₁ CDCI₃) 155.3, 149.5, 120.5, 68.6, 25.1.

Example 6

[0033] 4-(lS-hydroxyethyl)benzonitriIe: 1.6 g of lyophilised Agromyces mediolanus NCIMB 41380 cells were rehydrated by shaking in 0. IM potassium phosphate buffer pH 7 (30 mL) at 200 rpm, 28 ⁰C for 30 mins. 4-Acetylbenzenenitrile (2.0 g, 13.8 mmol) was added in isopropyl alcohol (10 mL) and the mixture shaken at 200 rpm, 28 ⁰C for 64 hours, when GC analysis suggested that the reaction was 84% complete. The mixture was acidified to pH 2 with 6M HCl, filtered through celite, and the celite plug washed with water (10 mL). The filtrate was extracted with EtOAc (3 x 35 mL), then the combined organic extracts were washed with sat. brine (30 mL), then dried (MgSO₄), filtered and concentrated in vacuo to yield 4-( 1 S-hydroxyethyl)benzonitrile as a pale yellow oil (1.76 g, 88% recovery, 94% e.e.). ¹H NMR (400 MHz, CDCl₃) 7.64 (2H, m), 7.49 (2H, m), 4.97 (IH, q, 76), 1.95 (IH, br s), 1.50 (3H, d, y 6); ¹³C NMR (IOO MHZ₅ CDCI₃) 151.1, 132.4, 126.1, 118.9, 111.0, 69.7, 25.4.

[0034] While the invention may be susceptible to various modifications and alternative forms, specific embodiments have been shown by way of the Examples and have been described in detail herein. However, it should be understood that the invention is not intended to be limited to the particular forms disclosed. Rather, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the following appended claims.

Claims

CLAIMSWhat is claimed is:

1. A method of reducing a ketone to a chiral secondary alcohol, comprising: providing an alcohol dehydrogenase from Agromyces sp.; and adding a ketone to the alcohol dehydrogenase to produce a chiral secondary alcohol.

2. The method of claim 1, further comprising adding an organic solvent to the alcohol dehydrogenase and the ketone.

3. The method of claim 2, wherein adding an organic solvent to the alcohol dehydrogenase and the ketone comprises forming a reaction mixture that comprises up to approximately 30% by volume of the organic solvent.

4. The method of claim 2, wherein adding an organic solvent to the alcohol dehydrogenase and the ketone comprises adding isopropanol to the alcohol dehydrogenase and the ketone.

5. The method of claim 1, wherein adding the ketone to the alcohol dehydrogenase to produce a chiral secondary alcohol comprises adding a ketone selected from the group consisting of 2-hexanone, cyclohexanone, chloroacetophenone, acetophenone, acetonylacetone, and mixtures thereof.

6. The method of claim 1 , wherein the ketone is present at a concentration of less than or equal to approximately 50 g/L.

7. The method of claim 1 , wherein the ketone is present at a concentration ranging from approximately 10 g/L to approximately 50 g/L.

8. A method of reducing an aliphatic methyl ketone, a cyclic ketone or an aromatic methyl ketone, or a beta-ketoester to a chiral secondary alcohol, comprising: providing an alcohol dehydrogenase from Agromyces sp.; and adding an aliphatic methyl ketone, an aromatic methyl ketone, a cyclic ketone or or a beta- ketoester to the alcohol dehydrogenase to the produce a chiral secondary alcohol.

9. The method of claim 8, further comprising adding an organic solvent to the alcohol dehydrogenase and the aliphatic methyl ketone, aromatic methyl ketone, a cyclic ketone or or beta- ketoester.

10. The method of claim 9, wherein adding an organic solvent to the alcohol dehydrogenase and the aliphatic methyl ketone, aromatic methyl ketone, cyclic ketone or beta- ketoester comprises forming a reaction mixture that comprises up to approximately 30% by volume of the organic solvent.

11. The method of claim 9, wherein adding an organic solvent to the alcohol dehydrogenase and the aliphatic methyl ketone, aromatic methyl ketone, cyclic ketone or beta- ketoester comprises adding isopropanol to the alcohol dehydrogenase and the aliphatic methyl ketone, aromatic methyl ketone, or beta ketoester.

12. The method of claim 8, wherein adding the aliphatic methyl ketone, aromatic methyl ketone, cyclic ketone or beta-ketoester to the alcohol dehydrogenase to produce a chiral secondary alcohol comprises adding the aliphatic methyl ketone, aromatic methyl ketone, a cyclic ketone or beta ketoester selected from the group consisting of 2-hexanone, cyclohexanone, chloroacetophenone, acetophenone, acetonylacetone, and mixtures thereof.

13. The method of claim 8, wherein the aliphatic methyl ketone, aromatic methyl ketone, cyclic ketone or beta-ketoester is present at a concentration of less than or equal to approximately 50 g/L.

14. The method of claim 8, wherein the aliphatic methyl ketone, aromatic methyl ketone, cyclic ketone or beta-ketoester is present at a concentration ranging from approximately 10 g/L to approximately 50 g/L.

15. A method of producing (2S,5S)-hexanediol, comprising providing an alcohol dehydrogenase from Agromyces sp.; and adding acetonylacetone to the alcohol dehydrogenase.

16. The method of claim 15, further comprising adding an organic solvent to the alcohol dehydrogenase and the acetonylacetone.

17. The method of claim 16, wherein adding an organic solvent to the alcohol dehydrogenase and the acetonylacetone comprises adding isopropanol to the alcohol dehydrogenase and the acetonylacetone.

18. An enzyme having alcohol dehydrogenase activity, wherein the enzyme is obtained from Agromyces sp.

19. The enzyme of claim 18, wherein the Agromyces sp. is Agromyces mediolanus.

20. The enzyme of claim 18, wherein the enzyme has biological activity when exposed to from approximately 0% by volume to approximately 30% by volume of an organic solvent.

21. The enzyme of claim 20, wherein the organic solvent is isopropanol.