ENZYMATIC REACTION MEDIUM CONTAINING SURFACTANT

Abstract

The present invention is directed to aqueous reaction mixtures for enzymatic synthesis reactions comprising a surfactant. The surfactant in the reaction mixture increases stability and yield of the enzymatic reaction. Furthermore, method for performing an enzymatic reaction using said aqueous reaction mixtures are provided.

Claims

1. An aqueous reaction mixture comprising an enzyme, a substrate of the enzyme and a surfactant, wherein the surfactant is selected from the group consisting of (i) surfactants having the following formula I: ##STR00027## (ii) surfactants having the following formula II: ##STR00028## and (iii) surfactants having the following formula III: ##STR00029## wherein R1 comprises a poly(C.sub.1-4-alkylene glycol) group; R2, R3, R4 and R5 are independently hydrogen or C.sub.1-4-alkyl; and R6 is C.sub.5-80-alkyl or C.sub.5-80-alkenyl.

2. The reaction mixture according to claim 1, wherein R1 is a poly(C.sub.1-4-alkylene glycol) group, which is attached to the oxygen atom of the core structure via a covalent bond or a linking group, and which optionally comprises a terminal C.sub.1-4-alkyl group.

3. The reaction mixture according to claim 2, wherein the linking group is a dicarboxylic acid which forms ester linkages to the core structure and the poly(C.sub.1-4-alkylene glycol) group.

4. The reaction mixture according to claim 3, wherein the linking group is a dicarboxylic acid having 2 to 20 carbon atoms, such as succinic acid or sebacic acid.

5. The reaction mixture according to claim 2, wherein the terminal C.sub.1-4-alkyl group is methyl.

6. The reaction mixture according to claim 1, wherein the poly(C.sub.1-4-alkylene glycol) group is a poly(ethylene glycol) group.

7. The reaction mixture according to claim 1, wherein the poly(C.sub.1-4-alkylene glycol) group has an average molecular weight of about 250 to 2500 g/mol, in particular about 500 to 1500 g/mol, such as about 550, about 750 or about 1000 g/mol.

8. The reaction mixture according to claim 1, wherein R5 is methyl.

9. The reaction mixture according to claim 1, wherein R4 is methyl.

10. The reaction mixture according to claim 1, wherein R2 and R3 are independently hydrogen or methyl.

11. The reaction mixture according to claim 1, wherein (i) the surfactant has formula (I) and R2, R3, R4 and R5 are all methyl; or (ii) the surfactant has formula (II) and R4 and R5 are methyl; or (iii) the surfactant has formula (III) and R2 is hydrogen and R3, R4 and R5 are methyl.

12. The reaction mixture according to claim 1, wherein R6 is branched or linear, in particular branched.

13. The reaction mixture according to claim 1, wherein (i) the surfactant has formula (I) and R6 is C.sub.8-25-alkyl or C.sub.8-25-alkenyl, in particular C.sub.14-18-alkyl or C.sub.14-18-alkenyl; or (ii) the surfactant has formula (II) and R6 is C.sub.5-20-alkyl or C.sub.5-20-alkenyl, in particular C.sub.8-12-alkyl or C.sub.8-12-alkenyl; or (iii) the surfactant has formula (III) and R6 is C.sub.30-80-alkyl or C.sub.30-80-alkenyl, in particular C.sub.45-55-alkyl or C.sub.45-55-alkenyl.

14. The reaction mixture according to claim 13, wherein (i) the surfactant has formula (I) and R6 is C.sub.16-alkyl or C.sub.16-alkenyl, in particular 4,8,12-trimethyltridecyl or 4,8,12-trimethyl-3,7,11-tridecatrienyl (ii) the surfactant has formula (II) and R6 is C.sub.10-alkyl, in particular 5-ethyl-6-methylheptan-2-yl; or (iii) the surfactant has formula (III) and R6 is C.sub.50-alkenyl, in particular 3,7,11,15,19,23,27,31,35,39-decamethyltetraconta-2,6,10,14,18,22,26,30,34,38-decanyl.

15. The reaction mixture according to claim 1, wherein the surfactant has formula (I) and in particular is a vitamin E-derived compound.

16. The reaction mixture according to claim 15, wherein the surfactant is a D--tocopherol poly(ethylene glycol) succinate, in particular TPGS-750-M, TPGS-1000, PTS (PEG-600/alpha-tocopherol-based diester of sebacic acid).

17. The reaction mixture according to claim 1, wherein the surfactant has formula (II) and in particular is an ubiquinol-derived compound.

18. The reaction mixture according to claim 17, wherein the surfactant is polyethyleneglycol ubiquinol succinate (PQS), in particular PQS comprising mPEG such as mPEG2000.

19. The reaction mixture according to claim 1, wherein the surfactant has formula (II) and in particular is a -sitosterol-derived compound.

20. The reaction mixture according to claim 19, wherein the surfactant is -sitosterol methoxyethyleneglycol succinate (Nok), in particular Nok comprising mPEG such as mPEG550.

21. The reaction mixture according to claim 1, wherein the enzyme is selected from the group consisting of reductases, transferases, dehydrogenases, lipases, and esterases.

22. The reaction mixture according to claim 1, wherein the enzyme is selected from the group consisting of ketoreductases, ene reductases, transaminases, alcohol dehydrogenases, amino acid dehydrogenases, and phenylalanine ammonia lyases.

23. The reaction mixture according to claim 1, further comprising a co-enzyme or co-factor which is necessary to perform the enzymatic reaction.

24. The reaction mixture according to claim 23, wherein the co-enzyme or co-factor is selected from the group consisting of alcohol dehydrogenases, NAD, NADP, FAD and pyidoxal monophosphate.

25. The reaction mixture according to claim 23, wherein the enzyme is a ketoreductase, the substrate is a ketone and the co-factor is NADP.

26. The reaction mixture according to claim 23, wherein the enzyme is an ene reductase, the substrate is a compound comprising a carbon-carbon double bond and the co-factor is NAD.

27. The reaction mixture according to claim 23, wherein the enzyme is a transaminase, the substrate is a ketone and the co-factor is pyridoxal monophosphate.

28. The reaction mixture according to claim 1, wherein the concentration of the surfactant in the reaction mixture is above its critical micellar concentration.

29. The reaction mixture according to claim 1, wherein the concentration of the surfactant in the reaction mixture is at least 0.01% (w/w), in particular at least 0.02% (w/w), especially at least 0.5% (w/w) or at least 1% (w/w).

30. The reaction mixture according to claim 1, wherein the concentration of the surfactant in the reaction mixture is in the range of 0.1% to 10% (w/w), in particular 0.5% to 5% (w/w), especially 0.75% to 3% (w/w), such as about 2% (w/w).

31. The reaction mixture according to claim 1, wherein the enzyme is present in an amount in the range of 0.1% to 50% of the amount of the substrate, in particular 0.5% to 35%, especially 1% to 20%.

32. The reaction mixture according to claim 1, further comprising a buffer.

33. The reaction mixture according to claim 32, wherein the buffer is selected from the group consisting of TRIS, phosphate, citrate, acetate and ammonia.

34. The reaction mixture according to claim 1, wherein the reaction mixture has a pH value suitable for the enzymatic reaction.

35. The reaction mixture according to claim 34, wherein the pH value is in the range of from 4.0 to 10.0, in particular from 6.0 to 8.0, especially from 6.5 to 7.5, such as about 7.0.

36. The reaction mixture according to claim 1, wherein the reaction mixture is of industrial scale.

37. The reaction mixture according to claim 36, wherein the volume of the reaction mixture is at least 10 l, preferably at least 100 lor at least 1000 l.

38. A method of performing an enzymatic reaction, comprising the steps of (a) providing a reaction mixture according to claim 1, and (b) allowing the enzymatic reaction to proceed.

39. The method according to claim 38, wherein the reaction is performed at a temperature of 80 C. or less.

40. The method according to claim 39, wherein the reaction is performed at a temperature in the range of 10 C. to 50 C., in particular 20 C. to 45 C.

41. The method according to claim 38, wherein the pH in the reaction mixture varies by at least 1.0 during the reaction, in particular at least 2.0.

42. A method for increasing the yield of an enzymatic reaction comprising adding a surfactant to an aqueous mixture comprising an enzyme and a substrate of the enzyme, wherein the surfactant is selected from the group consisting of (i) surfactants having the following formula I: ##STR00030## (ii) surfactants having the following formula II: ##STR00031## and (iii) surfactants having the following formula III: ##STR00032## wherein R1 comprises a poly(C.sub.1-4-alkylene glycol) group; R2, R3, R4 and R5 are independently hydrogen or C.sub.1-4-alkyl; and R6 is C.sub.5-80-alkyl or C.sub.5-80-alkenyl.

43. A method for increasing the yield of an enzymatic reaction comprising adding a surfactant to an aqueous mixture comprising an enzyme and a substrate of the enzyme, wherein the surfactant is selected from the group consisting of (i) surfactants having the following formula I: ##STR00033## (ii) surfactants having the following formula II: ##STR00034## and (iii) surfactants having the following formula III: ##STR00035## wherein R1 comprises a poly(C.sub.1-4-alkylene glycol) group; R2, R3, R4 and R5 are independently hydrogen or C.sub.1-4-alkyl; and R6 is C.sub.5-80-alkyl or C.sub.5-80-alkenyl.

44. The method according to claim 42, wherein the reaction mixture is a reaction mixture as defined in any one of claims 1 to 37.

Description

EXAMPLES

Example 1: Synthesis of benzyl (R)-4-hydroxy-2-(4-(methoxycarbonyl)phenyl)piperidine-1-carboxylate using ketoreductase

[0060] Surfactants have been applied in biocatalysis in modulating enzyme activities with beneficial effects, such as increasing the solubility of reactants and enhancing reaction selectivity. Although a number of surfactants including ionic liquids, SDS and Triton X have been utilized in a wide range of biotransformations, TPGS-750-M as an alternative surfactant developed in the Lipshutz lab has not been investigated in biocatalysis. During the investigations of a ketoreductase (KRED) mediated reaction as shown in scheme 1, the low solubility of both starting ketone 1 and product alcohol 2 necessitated significant efforts to search for a suitable reaction media for this heterogeneous reaction.

##STR00022##

[0061] First, a number of co-solvent systems including TPGS, PEG400 and DMSO in aqueous solutions were employed under parallel screening conditions for the above reaction. As shown in FIG. 1, during the first phase of the reaction within 2.5 h, TPGS performed better than PEG400 and DMSO with a 10% conversion difference. As the reaction proceeded further, the reaction is significantly faster in TPGS and a conversion difference of around 40% was observed as compared to the reaction in PEG400 and DMSO within 6 h. After 22 h, the reaction came to essentially complete conversion in TPGS (100%) while the reaction in PEG400 and DMSO stopped at 82% and 86% conversions, respectively. This phenomenon reflected the less stability of the ketoreductase in the organic solvent additives of both PEG400 and DMSO system.

[0062] With the screening results in hand, we further optimized the reaction in gram scale. Especially we compared the performance of TPGS and DMSO system. Thus the reactions were carried out in 2% TPGS and 15% DMSO aqueous solution respectively at 40 C. using a 5 w % of enzyme loading. The reaction reached 93% conversion after 18 h in TPGS system while a lower 80% conversion after 20 h in DMSO system. More importantly, the reaction continued and reached 98.8% conversion in TPGS after additional 25 h. But an additional 2 wt % enzyme has to be added to push the reaction to 98.6% conversion in DMSO (entry 1, table 1). This observation clearly demonstrated the more active and stable nature of the enzyme in TPGS as compared to DMSO. Another interesting observation was that after an accidental exposure of the enzyme under pH 4.7 for 24 h in the reaction system of TPGS and readjustment of pH to 7, the enzyme is still active enough to catalyze the reaction further with the same activity (entry 2, table 1). The superior stability could be afforded by the molecular interactions between the TPGS and protein and the catalytic active site of the enzyme is protected from the aqueous reaction media.

TABLE-US-00001 TABLE 1 Comparison of ketoreductase in TPGS and DMSO Conditions 2% TPGS-750M DMSO/water Conversion @ 93% for 18 h; up to 98.8% for 80% for 20 h; up to 40 C. (with 5% additional 25 h without 98.6% for additional enzyme) additional enzymes 32 h, but 2% more enzyme necessary Enzyme activity Still active for 6 d, even pH pH should be drop to 4.7 for 24 h @ rt constant 6.9-7.1 for active catalysis

[0063] Finally the reaction in TPGS is more amenable to scale up, and compound 1 can be added in solid form without preformation of a solution or milling to reduce the particle size. A typical experimental procedure is as follows: to a degassed 2% weight solution of TPGS-750-M in buffer water (34 mL, 10 v; stock solution prepared from 74 mL of 2 wt % TPGS-750-M water solution, 1.6 g of Na.sub.2HPO.sub.4.12H.sub.2O and 0.5 g of NaH.sub.2PO.sub.4.2H.sub.2O) was added glucose (3.2 g, 18 mmol, 2.0 eq) in a mechanically stirred reactor equipped with pH/ORP controller at rt. The suspension was stirred at rt for 20 minutes, and to the resulting mixture was sequentially added NADP (59 mg, 68% purity), GDH (33 mg) and ketoreductase (0.16 g, 5 wt % of substrate 1). Then substrate 1 (3.3 g, 9 mmol, 1.0 eq) was added and the pH of the reaction mixture was adjusted to 6.8-7.2 by addition of 1M aqueous NaOH at rt. The resulting reaction mixture was heated to 40 C. and stirred at 40 C. for 43 hr until completion of the reaction as determined by HPLC. As the reaction proceeded, the product 2 precipitated out from the reaction mixture and formed a suspension. The resulting suspension was filtered at 40 C., and the resulting wet cake was washed with water and dried to give product 2 as an off-white solid (2.8 g, purity 97%, yield 85%).

##STR00023##

[0064] In conclusion, a beneficial effect of TPGS as an additive in a ketoreductase mediated biotransformation was shown, including superior reaction kinetics and process robustness. The underlying enzyme stability in TPGS will find similar benefits when applied to a wide range of biocatalytic transformations.

Example 2: Synthesis of (R)-5-fluoro-3-(3-fluorophenyl)-2-(1-hydroxyethyl-4H-chromen-4-one using ketoreductase

[0065] ##STR00024##

Reaction Mixture:

[0066] 20 mg substrate 3
enzyme ketoreductase KRED-EW-109 (amount: see table 2)

20 mg D-glucose

[0067] 0.4 mg (2% (m/m)) glucose dehydrogenase (GDH)
0.2 mg (1% (m/m)) NADP
0.1M PBS (amount: see table 2)
2% TPGS-750-M (m/m) in 0.1M PBS (amount: see table 2)
DMSO (amount: see table 2)

Reaction Conditions: pH 7.0, 30 C.

[0068]

TABLE-US-00002 TABLE 2 2% TPGS DMSO Conversion No. enzyme 0.1M PBS in PBS (v/v) 17.5 h 24 h 1 1 mg 0.9 ml N.A. 0.1 ml 72.6% 67.5% 5.0% (m/m) 10% (v/v) 2 N.A. 1 ml N.A. 87.2% 87.2% 3 0.2 mg 0.9 ml N.A. 0.1 ml 3.9% 4.1% 1.0% (m/m) 10% (v/v) 4 N.A. 1 ml N.A. 11.7% 13.2% 5 0.1 mg 0.9 ml N.A. 0.1 ml 1.5% 1.5% 0.5% (m/m) 10% (v/v) 6 N.A. 1 ml N.A. 7.9% 9.3% 7 1 mg 0.9 ml N.A. 0.1 ml 63.2% 61.0% 5.0% (m/m) 10% (v/v) 8 0.5 ml 0.5 ml N.A. 89.9% 90.4% 9 0.2 mg 0.9 ml N.A. 0.1 ml 2.1% 2.5% 1.0% (m/m) 10% (v/v) 10 0.5 ml 0.5 ml N.A. 12.6% 14.4% 11 0.1 mg 0.9 ml N.A. 0.1 ml 1.2% 1.6% 0.5% (m/m) 10% (v/v) 12 0.5 ml 0.5 ml N.A. 7.6% 8.4%

Example 3: Synthesis of 1-phenylethanol using ketoreductase

[0069] ##STR00025##

Reaction Mixture:

[0070] 20 l acetophenone
enzyme ketoreductase KRED-EW-124 (amount: see table 3)

20 mg D-glucose

[0071] 0.4 mg (2% (m/m)) glucose dehydrogenase (GDH)
0.2 mg (1% (m/m)) NADP
0.1M PBS (amount: see table 3)
2% TPGS-750-M (m/m) in 0.1M PBS (amount: see table 3)
DMSO (amount: see table 3)

Reaction Conditions: pH 7.0, 30 C.

[0072]

TABLE-US-00003 TABLE 3 2% TPGS DMSO Conversion No. enzyme 0.1M PBS in PBS (v/v) 18 h 1 8 mg 0.9 ml N.A. 0.1 ml 53.5% 40% (m/m) 10%(v/v) 2 N.A. 1 ml N.A. 55.0% 3 4 mg 0.9 ml N.A. 0.1 ml 48.7% 20% (m/m) 10%(v/v) 4 N.A. 1 ml N.A. 52.4% 5 2 mg 0.9 ml N.A. 0.1 ml 45.2% 10% (m/m) 10%(v/v) 6 N.A. 1 ml N.A. 47.5% 7 8 mg 0.9 ml N.A. 0.1 ml 53.2% 40% (m/m) 10%(v/v) 8 0.5 ml 0.5 ml N.A. 52.5% 9 4 mg 0.9 ml N.A. 0.1 ml 47.9% 20% (m/m) 10%(v/v) 10 0.5 ml 0.5 ml N.A. 50.6% 11 2 mg 0.9 ml N.A. 0.1 ml 43.3% 10% (m/m) 10%(v/v) 12 0.5 ml 0.5 ml N.A. 45.2%

Example 4: Synthesis of (S)-3-(3-bromopyridin-4-yl)-methylcyclohexan-1-one Using ene Reductase

[0073] ##STR00026##

Reaction Mixture:

[0074] 20 l substrate 5
enzyme ene reductase ENE012 (amount: see table 4)

20 mg D-glucose

[0075] 0.4 mg (2% (m/m)) glucose dehydrogenase (GDH)
0.2 mg (1% (m/m)) NAD
0.1M PBS (amount: see table 4)
2% TPGS-750-M (m/m) in 0.1M PBS (amount: see table 4)
toluene (amount: see table 4)

Reaction Conditions: pH 7.0, 30 C.

[0076]

TABLE-US-00004 TABLE 4 2% TPGS Toluene Conversion No. enzyme 0.1M PBS in PBS (v/v) 19.5 h 43.5 h 1 4 mg 0.8 ml N.A. 0.2 ml 45.8% 36.7% 20% (m/m) 20% (v/v) 2 N.A. 1 ml N.A. 82.7% 79.9% 3 2 mg 0.8 ml N.A. 0.2 ml 30.9% 33.8% 10% (m/m) 20% (v/v) 4 N.A. 1 ml N.A. 42.3% 69.3% 5 4 mg 0.8 ml N.A. 0.2 ml 47.8% 44.4% 20% (m/m) 20% (v/v) 6 0.5 ml 0.5 ml N.A. 62.3% 59.0% 7 2 mg 0.8 ml N.A. 0.2 ml 28.1% 28.2% 10% (m/m) 20% (v/v) 8 0.5 ml 0.5 ml N.A. 47.6% 75.2%