METHOD FOR THE PREPARATION OF CHIRAL ALPHA HALOALKANOIC ACIDS

Abstract

What is described herein relates to a method of selectively hydrolyzing an enantiomer of an alpha haloalkanoic acid according to formula I employing a polypeptide having dehalogenase activity comprising an amino acid sequence as set forth in SEQ ID NO. 1 or SEQ ID NO. 4 or a sequence with at least 80% sequence identity to either of said sequences and to the use of said method.

Claims

1. Method of selectively hydrolyzing the S-enantiomer of an alpha haloalkanoic acid according to formula I, ##STR00003## wherein X is a halogen and R is an alkyl chain of 1 to 6 carbon atoms, wherein that said alkyl chain can be straight or branched at carbon atoms or , comprising providing a racemate of the R-enantiomer and the S-enantiomer of said alpha haloalkanoic acid, providing a polypeptide having dehalogenase activity comprising an amino acid sequence as set forth in SEQ ID NO. 1 or SEQ ID NO. 4 or a sequence with at least 80% sequence identity to either of said sequences, reacting the racemate for 1-8 hours, wherein the pH is in the range of 9-10 and the temperature is in the range of 15-35 C. for the polypeptide with dehalogenase activity comprising an amino acid sequence as set forth in SEQ ID NO. 1 or a sequence with at least 80% sequence identity to said sequence or the pH is in the range of 9-10 and the temperature is in the range of 55-65 C. for the polypeptide with dehalogenase activity comprising an amino acid sequence as set forth in SEQ ID NO. 4 or a sequence with at least 80% sequence identity to said sequence. and wherein an enantiomeric excess of the R-enantiomer of between 90.0 and 99.9% is reached after 1-8 hours.

2. Method according to claim 1, wherein the ratio of racemate of the alpha haloalkanoic acid according to Formula I to biomass of whole cells comprising the polypeptide having dehalogenase activity comprising an amino acid sequence as set forth in SEQ ID NO. 1 or SEQ ID NO. 4 or a sequence with at least 80% sequence identity to either of said sequences is between 2:1 to 15:1, optionally between 3:1 to 10:1 optionally 4:1.

3. Method according to claim 1, wherein the halogen of said alpha haloalkanoic acid of formula I is bromide or chloride.

4. Method according to claim 1, wherein moiety R of said alpha haloalkanoic acid of formula I is chosen from the group consisting of ethyl, butyl, 2-methyl-propyl and methyl-cyclopropyl.

5. Method according to claim 1 for selective hydrolysis of the S-enantiomer of said alpha haloalkanoic acid, wherein the enantiomeric excess of the R-enantiomer is between 90.0 and 99.9.

6. Method of selectively hydrolyzing the S-enantiomer of an alpha haloalkanoic acid according to formula II, ##STR00004## wherein R is an alkyl chain of 1 to 6 carbon atoms, wherein that said alkyl chain can be branched at carbon atoms or , Said method comprising using a polypeptide having dehalogenase activity comprising an amino acid sequence as set forth in SEQ ID NO 4 or a sequence with at least 80% sequence identity to said sequences.

7. Method according to claim 5 for selective hydrolysis of the S-enantiomer of said alpha haloalkanoic acid, wherein the enantiomeric excess of the R-enantiomer is between 90.0 and 99.9.

8. Method according to claim 1, wherein said alkyl chain is branched at carbon atoms or , and wherein the carbon atoms following the branch at carbon atoms or are cyclic.

Description

FIGURES

[0114] FIG. 1 shows a reaction carried out under optimized conditions with respect to pH value, i.e. 9.5 and temperature, i.e. 25 C. In this case 100 g/L 2-bromobutyric acid was used as substrate, 20 mM Glycine were present and 2 g/L cell lysate, i.e. E. coli MG1655 harbouring the pKA81a-HADH-PP-AJ plasmid were used to express the dehalogenase enzyme. The cell lysate was added 3 times, every hour.

[0115] FIG. 2 demonstrates that the pH optimum of the reaction of FIG. 1 is between pH 8 and pH 10.

[0116] FIG. 3 demonstrates that instead of using (sterile) cell lysate also whole cells expressing the claimed enzyme can be used for the conversion reaction

[0117] FIG. 4 demonstrates that the efficiency of the conversion reaction is maintained, if the substrate is provided and the enzyme is added to the substrate

[0118] FIG. 5 demonstrates that the efficiency of the conversion reaction is maintained, if the enzyme is provided as fermentation broth and the substrate is added.

EXAMPLES

Example 1 Screening of Activity of Selected Haloacid Dehalogenase

[0119] Different haloacid dehalogenase from P. putida AJ, P. putida 109 and S. tokodaii 7 (Jones et al., 1992 (J Gen Microbiol. 1992 April; 138(4):675-83, Kawasaki et al., 1994 Biosci Biotechnol Biochem. 1994 January; 58(1):160-3 and Bachas-Daunert et al., 2009 Appl Biochem Biotechnol. 2009 November; 159(2):382-93) were cloned into E. coli MG1655 and overexpressed via IPTG treatment. 50 g/L 2-bromobutyric acid was used as substrate and 21.6 g/L cell lysate was added at the beginning of the reaction and after 3.5 h, while the pH was 9 and the temperature was set to 37 C.

[0120] The activity and stereoselectivityi.e. the preferential hydrolysis of the S-enantiomerwas demonstrated for all three enzymes via analytics using gas chromatography with the enzyme from P. putida AJ (SEQ ID NO 1) showing the highest selectivity reaching an ee value of above 90%.

Example 2 Effect of pH on the Reaction Outcome

[0121] In order to test for the optimal pH range the reaction conditions were set to: [0122] 100 g/L 2-bromobutyric acid (substrate) [0123] 1.5 g/L cell lysate of E. coli expressing the P. putida AJ haloacid dehalogenase (SEQ ID NO 1) (6 times, every hour) [0124] 20 mM Glycine [0125] Temperature: 25 C. [0126] pH: kept constant at 8.0, 9.0, 10.0

[0127] As shown in FIG. 2 the best results are achieved with a pH of between 9-10.

Example 3: Further Optimization of the Selective Dehalogenation for Generation of High Enantiomer Excess Using the Haloacid Dehalogenase of P. putida AJ i.e. the Polypeptide Having Dehalogenase Activity Consisting of SEQ ID NO. 1

[0128] In a first case, 100 g/l rac-2-bromobutyric acid in 10 mM Glycine was used as substrate. The reaction was performed at 25 C. even though the optimal temperature for enzyme activity was at 37 C. since the substrate was more stable at 25 C. than at 37 C. and hence an autohydrolysis was prevented (data not shown)

[0129] The enzyme was provided as cell lysate and was added either all at the start of the reaction or every hour at a concentration of 2 mg/ml. The pH was kept constantly at 9.5 via titration with 3 M KOH until completion of reaction after approximately 4 hours. Subsequently, the pH was adjusted to pH 1.5 with concentrated H.sub.2SO.sub.4 and the cell debris was filtered over Celite. Extraction was performed with MTBE and washes to remove the remaining 2-Hydroxybutyric acid were carried out with aq. CuSO.sub.4. Finally, concentration was performed in vacuo.

[0130] It was surprisingly found that the reaction reliably reached enantiomeric excess rate values of above 99% (data not shown).

[0131] These high enantiomeric excess rates i.e. the fact that after the reaction only the R-enantiomer of the alpha haloalkanoic acid and the hydroxylated product of the former S-enantiomer of the alpha haloalkanoic acid are present, render the process attractive for use on large industrial scale.

Example 4: Conversion with Whole Cells

[0132] Surprisingly, it was found that instead of preparing a cell lysate, whole cells could be used for the conversion. This has the advantage that the time-consuming step of preparing a (sterile) cell lysate can be omitted.

[0133] In a first case, racemic 2-bromo butyric acid at a concentration of 100 g/L was used as a substrate. The reaction parameters were set to 150 ml, 25 C., 500 rpm, 20 mM glycine buffer and pH 9.5. The pH was kept constant using 3 M KOH. The reaction vessel was prepared with the substrate mix and the reaction was started by addition of the enzyme, i.e. the addition of whole cells of E. coli MG1655 expressing the dehalogenase enzyme from P. putida AJ i.e. the polypeptide having dehalogenase activity consisting of SEQ ID NO. 1. The cells were added as either at the start of the reaction or stepwise after 1, 2, and 3 hours, at a concentration of 8.3 g/L respectively (cf. FIG. 3). The final concentration of the whole cells containing the enzyme was either 25 g/L or 50 g/L. The reaction was completed after 4 hours. The results presented in FIG. 3 and the table below demonstrate that ideally the reaction is carried out for more than 1 h hour e.g. for 4 hours.

TABLE-US-00001 Sample T[h] % ee 25 g/L biomass 1 62 25 g/L biomass 3 89 25 g/L biomass (feed) 1 52 25 g/L biomass (feed) 3 90

[0134] Subsequently, the reaction was repeated but the biomass consisting of the E coli MG1655 cells expressing the dehalogenase enzyme from P. putida AJ i.e. the polypeptide having dehalogenase activity consisting of SEQ ID NO. 1 was added to the reaction vessel before adding the substrate. The results are shown in FIG. 4 and the Table below. These results demonstrate that it is possible to first provide the enzyme as biomass comprising whole cells and to add the substrate to said biomass.

TABLE-US-00002 t[h] % ee 1 61 2 78 3 88

[0135] Moreover, a conversion was performed using the fermentation broth. Again E. coli MG1655 harboring the pKA81a-HADH-PP-AJ plasmid were used to express the dehalogenase enzyme i.e. the polypeptide having dehalogenase activity consisting of SEQ ID NO. 1 Enzyme production was carried out by fermentation of E. coli in minimal medium using a standard protocol. After gene expression a cell concentration of 100 g/L was reached. The biotransformation was subsequently done with the untreated fermentation broth. The fermentation broth was cooled down to 25 C. and the pH was adjusted to 9.5 by adding 3 M KOH. The dehalogenase reaction was started by adding a substrate mix containing 100 g/L 2-bromobutyric acid, 0.5 Vol. (v/v) glycine buffer at pH 9.5 and 2 Vol (v/v) 5 M KOH. The reaction reached a full conversion i.e. 90% ee after 1 h (cf. FIG. 5).

[0136] From the resulting mix of hydroxybutyric acid and R-2-Bromobutyric acid around 30% pure R-2-Bromobutyric acid can be obtained using standard techniques such as acidification and extraction.

Example 5 Testing of Further Substrates

[0137] In addition to 2-bromobutyric acid further substrates were tested and the results are listed below

TABLE-US-00003 Substrates 2-bromo- 2-bromo-3- 2-fluoro- 2-bromo- 4-methyl- cyclopropyl- 2-chloro- butyric hexanoic pentanoic propanoic Enzymes butyric acid acid acid acid acid P. putida AJ Yes No Yes; Yes; Yes; SEQ ID NO. 1 P. putida 109 Yes No No Yes Yes S. sulfolobus Yes Yes Yes Yes n.a.* SEQ ID NO. 4

Example 6: Upscaling

[0138] A prerequisite for the upscaling of the reaction to 2000 liters was the finding that the enzymes could be used comprised in whole cells which were provided as biomass without the need for preparing a cell lysate. Hence, time consuming and expensive preparation steps such as filtering the cell lysate, which are not economically feasible on large scale, could be omitted. Moreover, the finding that the reaction could be started either by addition of the substrate (racemate) or the biomass comprising the enzyme in whole cells meant that the equipment could be employed with a maximum flexibility. In order to further adapt the process the KOH used for pH titration (cf. above) was exchanged for 50% NaOH, the solvent for product extractions was changed from MTBE to MIBK and the CuSO4 used for removal of side products was exchanged for CaCl2 allowing for an easier and cheaper waste disposal.