# Patent

## Title
Method of production of optically active halohydrocarbons and alcohols using hydrolytic dehalogenation catalysed by haloalkane dehalogenases

## Abstract

A method of production of optically active compounds, particularly halohydrocarbons, haloalcohols, alcohols, halopolyols and polyols using hydrolytic dehalogenation of racemic or prochiral halegenhydrocarbons by dehalohenation catalysed by haloalkane dehalogenases (the enzyme code number EC 3.8.1.5) where at least one wild type or modified haloalkane dehalogenase is applied to at least one racemic or prochiral chlorinated, brominated or iodinated compound at the temperature ranged between +10 and +70° C. and pH value between 4.0 and 12.0, in aqueous system or in a monophasic organic solution or in a monophasic organic/aqueous solution or in organic/aqueous biphasic systems.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This is a continuation-in-part of application Ser. No. 11/793,635 filed Jun. 20, 2007 and incorporated here by reference.


FIELD OF THE INVENTION
This invention relates to method of production of optically active haloalkanes and alcohols using hydrolytic dehalogenation. The Sequence Listing which is incorporated here by reference, appears after the Abstract.
STATE OF THE ART
Enzymes are catalysts of biological systems that determine the patterns of chemical transformations. The most striking characteristics of enzymes are their catalytic power and specificity. They are highly effective catalysts for an enormous diversity of chemical reactions because of their capacity to specifically bind a very wide range of molecules. The enzymes catalyse reactions by destabilizing substrate or by stabilizing transition state and determining which one of several potential chemical reactions actually will take place.
The manufacture of enantiomerically pure compounds has become an expanding area of fine chemical industry. When pharmaceuticals, agrochemicals, food additives and their synthetic intermediates are marketed as single enantiomers, high enantiomeric purities, typically enantiomeric excess (e.e.)>98%, are required (enantiomeric excess is derived from the concentration of the two enantiomenrs cR and cS; Equation 1).




  
    
      
        
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Enzyme-catalyzed reactions have become popular alternatives to classical chemistry for their high selectivity and activity under mild reaction conditions, and several industrial processes using enzymes as catalysts are already in use. Clearly, the enantioselective performance of the catalyst is the single most important factor for the success of such a process (evaluation of this property is facilitated by the use of enantiomeric ratio (E); E-values can be expressed as ratio kcat/Km of the rate constants kcat for catalysis and the Michaelis-Menten constants Km of the two enantiomers; Equation 2).
Chemical transformation of halogenated compounds is important from both the environmental and synthetic point of view. Six major pathways for enzymatic transformation of halogenated compounds have been described: (i) oxidation, (ii) reduction, (iii) dehydrohalogenation, (iv) hydration, (v) methyl transfer and (vi) hydrolytic, glutathione-dependent and intramolecular substitution. Redox enzymes are responsible for the replacement of the halogen by a hydrogen atom and for oxidative degradation. Elimination of hydrogen halide leads to the formation of an alkene, which is further degraded by oxidation. The enzyme-catalysed formation of an epoxide from a halohydrin and the hydrolytic replacement of a halide by hydroxyl functionality take place in a stereospecific manner and are therefore of high synthetic interest [Falber, K. (2000) Biotransformations in Organic Chemistry, Springer-Verlag, Heidelberg, 450].
Haloalkane dehalogenases (the enzyme code number EC 3.8.1.5) are enzymes able to remove halogen from halogenated aliphatic compounds by a hydrolytic replacement, forming the corresponding alcohols [Janssen, D. B., Pries, F., and Van der Ploeg, J. R. (1994) Annual Review of Microbiology 48, 163-191]. Hydrolytic dehalogenation proceeds by formal nucleophilic substitution of the halogen atom with a hydroxyl ion. The mechanism of hydrolytic dehalogenation catalysed by the haloalkane dehalogenase enzymes (EC 3.8.1.5) is shown in Eq. 3. A co-factor or a metal ion is not required for the enzymatic activity of haloalkane dehalogenases. The reaction is initiated by binding of the substrate in the active site with the halogen in the halide-binding site. The binding step is followed by a nucleophilic attack of aspartic acid (Asp) on the carbon atom to which the halogen is bound, leading to cleavage of the carbon-halogen bond and formation of alkyl-enzyme intermediate. The intermediate is subsequently hydrolysed by activated water, with histidine (His) acting as a base catalyst, with formation of enzyme-product complex.
Asp or glutamic acid (Glu) keeps H is in proper orientation and stabilises a positive charge that develops on H is imidazole ring during the reaction. The final step is release of the products.




    Eq. 3—Reaction mechanism for the hydrolytic dehalogenation by haloalkane dehalogenases (EC 3.8.1.5)


The first haloalkane dehalogenase has been isolated from the bacterium Xanthobacter autotrophicus GJ10 in 1985 [Janssen, D. B., Scheper, A., Dijkhuizen, L., and Witholt, B. (1985) Applied and Environmental Microbiology 49, 673-677; Keuning, S., Janssen, D. B., and Witholt, B. (1985) Journal of Bacteriology 163, 635-639]. Since then, a large number of haloalkane dehalogenases has been isolated from contaminated environments [Scholtz, R., Leisinger, T., Suter, F., and Cook, A. M. (1987) Journal of Bacteriology 169, 5016-5021; Yokota, T., Omori, T., and Kodama, T. (1987) Journal of Bacteriology 169, 4049-4054; Janssen, D. B., Gerritse, J., Brackman, J., Kalk, C., Jager, D., and Witholt, B. (1988) European Journal of Biochemistry 171, 67-92; Sallis, P. J., Armfield, S. J., Bull, A. T., and Hardman, D. J. (1990) Journal of General Microbiology 136, 115-120; Nagata, Y., Miyauchi, K., Damborsky, J., Manova, K., Ansorgova, A., and Takagi, M. (1997) Applied and Environmental Microbiology 63, 3707-3710; Poelarends, G. J., Wilkens, M., Larkin, M. J., van Elsas, J. D., and Janssen, D. B. (1998) Applied and Environmental Microbiology 64, 2931-2936]. More recently, hydrolytic dehalogenating activity of several species of genus Mycobacterium isolated from clinical material [Jesenska, A., Sedlacek, I., and Damborsky, J. (2000) Applied and Environmental Microbiology 66, 219-222] have been reported, and haloalkane dehalogenases have been subsequently isolated from pathogenic bacteria [Jesenska, A., Bartos, M., Czernekova, V., Rychlik, I., Pavlik, I., and Damborsky, J. (2002) Applied and Environmental Microbiology 68, 3724-3730]. Haloalkane dehalogenases can be also found also the rhizobial strains colonizing roots of plants [Sato, Y., Monincova, M., Chaloupkova, R., Prokop, Z., Ohtsubo, Y., Minamisawa, K., Tsuda, M., Damborsky, J., Nagata, Y. Applied and Environmental Microbiology, (2005) 71: 4372-4379].
Structurally, haloalkane dehalogenases belong to the α/β-hydrolase fold superfamily [Ollis, D. L., Cheah, E., Cygler, M., Dijkstra, B., Frolow, F., Franken, S. M., Harel, M., Remington, S. J., Silman, I., Schrag, J., Sussman, J. L., Verschueren, K. H. G., and Goldman, A. (1992) Protein Engineering 5, 197-211; Nardini, M., and Dijkstra, B. W. (1999) Current Opinion in Structural Biology 9, 732-737]. Without exception, haloalkane dehalogenases contain a nucleophile elbow [Damborsky, J. (1998) Pure and Applied Chemistry 70, 1375-1383; Damborsky, J., and Koca, J. (1999) Protein Engineering 12, 989-998], which is the most conserved structural feature within the α/β-hydrolase fold. The other highly conserved region in haloalkane dehalogenases is the central β-sheet. Its strands, flanked on both sides by α-helices, form the hydrophobic core of the main domain that carries the catalytic triad Asp-His-Asp/Glu. The second domain, consisting solely of α-helices, lies like a cap on top of the main domain. Residues on the interface of the two domains form the active site. Whereas there is significant similarity in the catalytic core, the sequence and structure of the cap domain diverge considerably among different dehalogenase. The cap domain is proposed to play a prominent role in determining substrate specificity [Pries, F., Van den Wijngaard, A. J., Bos, R., Pentenga, M., and Janssen, D. B. (1994) Journal of Biological Chemistry 269, 17490-17494; Kmunicek, J., Luengo, S., Gago, F., Ortiz, A. R., Wade, R. C., and Damborsky, J. (2001) Biochemistry 40, 8905-8917].
A number of haloalkane dehalogenases from different bacteria have been biochemically characterised. A principal component analysis of activity data indicated the presence of three specificity classes within this family of enzymes [Nagata, Y., Miyauchi, K., Damborsky, J., Manova, K., Ansorgova, A., and Takagi, M. (1997) Applied and Environmental Microbiology 63, 3707-3710; Damborsky, J., and Koca, J. (1999) Protein Engineering 12, 989-998; Damborsky, J., Nyandoroh, M. G., Nemec, M., Holoubek, I., Bull, A. T., and Hardman, D. J. (1997) Biotechnology and Applied Biochemistry 26, 19-25]. Three haloalkane dehalogenases representing these different classes have been isolated and structurally characterised in atomic detail so far: the haloalkane dehalogenase DhlA from Xantobacter autotrophicus GJ10 [Keuning, S., Janssen, D. B., and Witholt, B. (1985) Journal of Bacteriology 163, 635-639; Franken, S. M., Rozeboom, H. J., Kalk, K. H., and Dijkstra, B. W. (1991) The EMBO Journal 10, 1297-1302], the haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 [Kulakova, A. N., Larkin, M. J., and Kulakov, L. A. (1997) Microbiology 143, 109-115; Newman, J., Peat, T. S., Richard, R., Kan, L., Swanson, P. E., Affholter, J. A., Holmes, I. H., Schindler, J. F., Unkefer, C. J., and Terwilliger, T. C. (1999) Biochemistry 38, 16105-16114] and the haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 [Nagata, Y., Miyauchi, K., Damborsky, J., Manova, K., Ansorgova, A., and Takagi, M. (1997) Applied and Environmental Microbiology 63, 3707-3710; Marek, J., Vevodova, J., Kuta-Smatanova, I., Nagata, Y., Svensson, L. A., Newman, J., Takagi, M., and Damborsky, J. (2000) Biochemistry 39, 14082-14086]. The size, geometry and physico-chemical properties of active sites and entrance tunnels, as well as nature and spatial arrangement of the catalytic residues (catalytic triad, primary and secondary halide-stabilizing residues [Bohac, M., Nagata, Y., Prokop, Z., Prokop, M., Monincova, M., Koca, J., Tsuda, M., and Damborsky, J. (2002) Biochemistry 41, 14272-14280] can be related to the substrate specificity, which is different for enzymes representing different classes [Damborsky, J., Rorije, E., Jesenska, A., Nagata, Y., Klopman, G., and Peijnenburg, W. J. G. M. (2001) Environmental Toxicology and Chemistry 20, 2681-2689].
Several patent applications concern the dehalogenation methods using dehalogenase enzymes. For instance, the application WO 98/36080 A1 relates to dehalogenases capable of converting the halogenated aliphatic compounds to vicinal halohydrines and DNA sequences encoding polypeptides of enzymes as well as to DNA sequences and the methods of producing the enzymes by placing the expression constructs into host cells. The patent document WO 01/46476 A1 relates to methods of dehalogenation of alkylhalogenes catalyzed by altered hydrolase enzymes under formation of stereoselective or stereospecific reaction products as alcohols, polyols and epoxides. This patent document includes also method of providing altered nucleic acids that encode altered dehalogenase or other hydrolase enzymes. The patent document WO 02/068583 A2 relates to haloalkane dehalogenases and to polynucleotides encoding the haloalkane dehalogenases. In addition, methods of designing new dehalogenases and method of use thereof are also provided. The dehalogenases have increased activity and stability at increased pH and temperature.
Although several patent applications relate to enzymatically catalysed dehalogenation, there has been no report that the specific family of hydrolytic enzymes, haloalkane dehalogenases (EC 3.8.1.5), shows sufficient enantioselectivity or regioselectivity for large-scale production of optically active alcohols. In 2001, Pieters and co-workers [Pieters, R. J., Spelberg, J. H. L., Kellogg, R. M., and Janssen, D. B. (2001) Tetrahedron Letters 42, 469-471] have investigated chiral recognition of haloalkane dehalogenases DhlA and DhaA. The magnitude of the chiral recognition was low; a maximum E-value of 9 could be reached after some structural optimization of the substrate. In the beginning of 2004, twenty years after discovery of the first haloalkane dehalogenase, the development of enantioselective dehalogenases for use in industrial biocatalysis was defined as one of the major challenges of the field [Janssen, D. B. (2004) Current Opinion in Chemical Biology 8, 150-159].
All the reactions conducted by Pieters and co-workers [Pieters, R. J., Spelberg, J. H. L., Kellogg, R. M., and Janssen, D. B. (2001) Tetrahedron Letters 42, 469-471] exhibited low enantioselectivity which is not sufficient for practical applications. Common characteristic of these reactions was the site for nucleophilic attack of dehalogenating enzyme on the chiral molecule. In all cases, the nucleophilic attack took place on the carbon next to the chiral carbon and not directly on the chiral carbon. This fact is obvious from the rules for preferential attack at alpha-carbon atom over beta-carbon atom defined for haloalkane dehalogenases by Damborsky and co-workers [Damborsky, J., Rorije, E., Jesenska, A., Nagata, Y., Klopman, G., Peijnenburg, W. J. G. M. (2001) Environmental Toxicology and Chemistry 20: 2681-2689].
DESCRIPTION OF THE INVENTION
Object of the invention is a method of production of optically active halohydrocarbons and alcohols using hydrolytic dehalogenation catalysed by a haloalkane dehalogenase, wherein at least one wild type or modified haloalkane dehalogenase selected from the group of the haloalkane dehalogenases (EC 3.8.1.5) or their mixtures is applied to at least one racemic or prochiral chlorinated, brominated or iodinated compound at the temperature ranged between +10 and +70° C. and pH value between 4.0 and 12.0, in aqueous system or in a monophasic organic solution or in a monophasic organic/aqueous solution or in organic/aqueous biphasic systems.
Another aspect of the invention is the method of production of optically active halohydrocarbons and alcohols, wherein the chlorinated, brominated or iodinated compounds has at least one halogen atom bound directly to the chiral or prochiral carbon and the nucleophilic attack takes place directly on the chiral or prochiral carbon.
A further aspect of the invention is the method of production of optically active halohydrocarbons and alcohols, wherein the haloalkane dehalogenase is at least one wild type or modified haloalkane dehalogenase selected from the group consisting of:

    
    
        haloalkane dehalogenase DbjA SEQ ID NO: 1, 2,
        haloalkane dehalogenase LinB SEQ ID NO: 3, 4,
        haloalkane dehalogenase DhaA SEQ ID NO: 5, 6,
        haloalkane dehalogenase DmbA SEQ ID NO: 7, 8,
        haloalkane dehalogenase DmbB SEQ ID NO: 9, 10,
        haloalkane dehalogenase DmbC SEQ ID NO: 11, 12,
        haloalkane dehalogenase DrbA SEQ ID NO: 13, 14,
        haloalkane dehalogenase DhmA SEQ ID NO: 15, 16,
        haloalkane dehalogenase DbeA SEQ ID NO: 17, 18.
    
    


Another aspect of the invention is the method of production of optically active halohydrocarbons and alcohols, wherein the haloalkane dehalogenase is at least one wild type or modified polypeptide with haloalkane dehalogenase activity having an amino acid sequence that corresponds at least in 95% to the sequence SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18.
Another aspect of the invention is the method of production of optically active halohydrocarbons and alcohols, wherein the haloalkane dehalogenase is at least one wild type or modified polypeptide with haloalkane dehalogenase activity having an amino acid sequence that corresponds at least in 90% to the sequence SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18.
Another aspect of the invention is the method of production of optically active halohydrocarbons and alcohols, wherein the haloalkane dehalogenase is at least one wild type or modified polypeptide with haloalkane dehalogenase activity having an amino acid sequence that corresponds at least in 85% to the sequence SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18.
A further aspect of the invention is the method of production of optically active halohydrocarbons and alcohols, wherein the haloalkane dehalogenase is at least one wild type or modified polypeptide with haloalkane dehalogenase activity having the amino acid sequence that corresponds at least in 80% to the sequence SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18.
Another aspect of the invention is the method of production of optically active halohydrocarbons and alcohols, wherein the haloalkane dehalogenase is at least one wild type or modified polypeptide with haloalkane dehalogenase activity having an amino acid sequence that corresponds at least in 75% to the sequence SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18.
Another aspect of the invention is the method of production of optically active halohydrocarbons and alcohols, wherein the haloalkane dehalogenase is at least one wild type or modified polypeptide with haloalkane dehalogenase activity having an amino acid sequence that corresponds at least in 70% to the sequence SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18.
Another aspect of the invention is the method of production of optically active halohydrocarbons and alcohols, wherein the haloalkane dehalogenase is at least one wild type or modified polypeptide with haloalkane dehalogenase activity having an amino acid sequence that corresponds at least in 65% to the sequence SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18.
Another aspect of the invention is the method of production of optically active halohydrocarbons and alcohols, wherein the haloalkane dehalogenase is at least one wild type or modified polypeptide with haloalkane dehalogenase activity having an amino acid sequence that corresponds at least in 60% to the sequence SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18.
Another aspect of the invention is the method of production of optically active halohydrocarbons and alcohols, wherein the haloalkane dehalogenase is at least one wild type or modified polypeptide with haloalkane dehalogenase activity having an amino acid sequence that corresponds at least in 55% to the sequence SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18.
Another aspect of the invention is the method of production of optically active halohydrocarbons and alcohols, wherein the haloalkane dehalogenase is at least one wild type or modified polypeptide with haloalkane dehalogenase activity having an amino acid sequence that corresponds at least in 50% to the sequence SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18.
Another aspect of the invention is the method of production of optically active halohydrocarbons and alcohols, wherein the haloalkane dehalogenase is at least one wild type or modified polypeptide with haloalkane dehalogenase activity having an amino acid sequence that corresponds at least in 45% to the sequence SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18.
Another aspect of the invention is the method of production of optically active halohydrocarbons and alcohols, wherein the haloalkane dehalogenase is at least one wild type or modified polypeptide with haloalkane dehalogenase activity having an amino acid sequence that corresponds at least in 40% to the sequence SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18.
Another aspect of the invention is the method of production of optically active halohydrocarbons and alcohols, wherein the haloalkane dehalogenase is at least one wild type or modified polypeptide with haloalkane dehalogenase activity having an amino acid sequence that corresponds at least in 35% to the sequence SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18.
Another aspect of the invention is the method of production of optically active halohydrocarbons and alcohols, wherein the haloalkane dehalogenase is at least one wild type or modified polypeptide with haloalkane dehalogenase activity having an amino acid sequence that corresponds at least in 30% to the sequence SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18.
Another aspect of the invention is the method of production of optically active halohydrocarbons and alcohols, wherein the haloalkane dehalogenase is at least one wild type or modified polypeptide with haloalkane dehalogenase activity having an amino acid sequence that corresponds at least in 25% to the sequence SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18.
Another aspect of the invention is the method of production of optically active halohydrocarbons and alcohols, wherein it is performed at presence of surfactants to allow using of enhanced reagent concentration.
A further aspect of the invention is the method of production of optically active halohydrocarbons and alcohols, wherein the enzyme halolkane dehalogenase is in soluble or crystalline or lyophilized or precipitated form.
Another aspect of the invention is the method of production of optically active halohydrocarbons and alcohols, wherein the enzyme haloalkane dehalogenase is immobilized by adsorption or ionic binding or covalent attachment onto the surface of a macroscopic carrier material.
A further aspect of the invention is the method of production of optically active halohydrocarbons and alcohols, wherein the enzyme haloalkane dehalogenase is immobilized by cross-linking or confined to a solid matrix or membrane-restricted compartments.
Hydrolytic dehalogenation of broad series of racemic substrates catalysed by the series of enzymes: haloalkane dehalogenases DmbA, DmbB, DmbC from bacterium Mycobacterium bovis, DhlA from Xanthobacter autotrophicus, DhaA from Rhodococcus rhodochrous, DrbA from Rhodopirellula baltica, LinB from Sphingomonas paucimobilis, DhmA from Mycobacterium avium, DbjA from Bradyrhizobium japonicum and DbeA from Bradyrhizobium elkanii have been conducted. The dehalogenation reactions were performed with both: (i) substrates carrying halogens on non-chiral carbon and (ii) newly also the substrates carrying halogen substituent directly on the chiral carbon. The results showed that the magnitude of the chiral recognition was low for all the substrates where the dehalogenation took place on a non-chiral carbon (Table 1), which was in correspondence with the previous observations of Pieters and co-workers [Pieters, R. J., Spelberg, J. H. L., Kellogg, R. M., and Janssen, D. B. (2001) Tetrahedron Letters 42, 469-471]. Newly, excellent enantioselectivity with haloalkane dehalogenases DmbA, DmbB, DmbC, DbjA, DhaA, DrbA, DhmA and LinB was observed in the reactions taking place directly on the chiral carbon, e.g., methyl-2-bromopropionate, ethyl-2-bromopropionate, ethyl-2-bromobutyrate, 3-bromo-2-butanone and 2-bromopropiophenone.
This observation demonstrated for the first time that the proteins from the family of haloalkane dehalogenases (EC 3.8.1.5) can possess high enantioselectivity sufficient for practical industrial application. We discovered that an essential precondition for high enantioselectivity of haloalkane dehalogenases is the nucleophilic attack taking place directly on a chiral carbon (chiral centre) of converted molecule.







TABLE 1







Examples of chiral recognition, hydrolytic dehalogenation


of selected racemic substrates catalyzed by haloalkane


dehalogenases DhlA, DhaA, LinB and DbjA.









E-values











Substrate
DhlA
DhaA
LinB
DbjA














methyl 2-bromopropionate
n.a.
68
52
>200


ethyl 2-bromopropionate
n.a.
59
97
194


ethyl 2-bromobutyrate
n.a.
>200
>200
>200


3-bromo-2-butanone
n.a.
>200
3
>200


2-bromopropiophenone
n.a.
>200
13
>200


methyl 2,4-dibromobutyrate
1.6
1
1.9
1.3


2-bromopentane
5.5
7
16
145


2-bromohexane
n.d.
4
12
35


2-bromoheptane
2.4
2.9
2.8
28


1,2-dichloropropane
2
n.d.
n.d.
n.d.


1,2-dibromopropane
3
1.3
1.3
2.6


1,2-dibromobutane
1.1
2
10
2.7


1,2-dichlorobutane
n.d.
n.d.
n.d.
n.d.


1,3-dibromobutane
2
1.3
4.6
1.4


1,3-dichlorobutane
2.8
1.6
2.6
1.0


1-bromo-3-chloro-2-methylpropane
1.8
1.7
1.8
1.4


1,2-dibromo-3,3-dimethylbutane
n.d.
n.d.
1.1
n.d.


epibromohydrine
1.4
1.2
1.1
1.9





n.d. . . . not detected (activity < 0.2 nM · s−1 · mg−1 of enzyme)


n.a. . . . not analyzed






These results indicate that hydrolytic dehalogenation catalysed by the enzymes haloalkane dehalogenases (EC 3.8.1.5) has high potential to produce optically active haloalkanes, haloalcohols, alcohols or diols with high optical purity.
By the method of the present invention, racemic reagents or prochiral compounds, e.g., haloalkanes, haloalcohols, halopolyols, are converted enantioselectively or enantiospecifically by hydrolytic dehalogenation taking place directly on chiral or prochiral carbon during the reaction catalysed by the enzyme haloalkane dehalogenase to provide optically active compounds with high purity, which can be used as medicaments, agrochemicals, food additives, cosmetics or ferroelectric liquid crystals or as an intermediate thereof. In general, the method includes hydrolytic dehalogenation of one or more chiral or prochiral reagents yielding one or more chiral products (e.g., haloalkanes, haloalcohols, alcohols, halopolyols, polyols) by incubating the reagent or reagents with one or more wild type or modified haloalkane dehalogenase. The hydrolytic dehalogenation of the reagent catalysed by the enzyme haloalkane dehalogenase is performed in an aqueous buffer system (e.g., potassium phosphate buffer, Tris-sulfate buffer, glycine buffer, acetate buffer, citrate buffer) at pH being close to the optimum of the haloalkane dehalogenase (pH=7.0-8.5). The pH-activity profile is broader and allows pH variations from 4 to 12 while maintaining a reasonable activity. The variation of pH and buffer type may influence the selectivity of the reaction since the conformation of the enzyme depends on its ionization state. The hydrolytic dehalogenation catalysed by the enzyme can be performed at the temperature range 10-70° C. with reaction optimum around 40° C. The concentration of the enzyme is set with respect to the reaction rate. The concentration of the reagent is dependent on the solubility of the reagent in reaction medium. The methods of the present invention can employ many different halohydrocarbon and haloalkane reagents (e.g., molecules, molecular appendages or substituent groups, etc.) that typically include from about one to about 100 carbon atoms. The carbon atoms or one or more subsets of the carbon atoms can include a straight chain structure, a branched structure, a ring structure, a double bond, a triple bond, and the like. For example, a preferred general class of reactants can include essentially any haloalkane whether cyclic or acyclic (e.g., haloalkanes, haloalkenes, haloalkynes, haloalkyl nitriles, haloalkyl amides, haloalkyl carboxylic acids, haloalkyl carboxylic acid esters, haloalcohols, halopolyols, haloepoxides, haloalkylethers). The reactant can be a xenobiotic or a naturally occurring compound, which can also be a component of a mixture derived from various chemical manufacturing operations or from other processes. Additionally, reaction pathways can involve various intermediates and reactants (e.g., with at least one prochiral or chiral center) that can be enantioselectivelly or enantiospecifically converted to products.
The hydrolytic dehalogenation of the reagent can be catalysed by the enzyme expressed in the natural producer or in a heterologous host organism, in non-living or living cells, crude extract or purified, immobilized on a carrier material, free in aqueous solution, in a monophasic organic/aqueous solution or in organic/aqueous biphasic systems, under atmospheric or elevated pressure and the like. Organic solvents can be utilized to allow the use of a high reagent concentration, to increase the productivity of the reaction and to favour enzymatic stereoselective reaction over spontaneous hydrolysis. Addition of water-miscible organic co-solvents, e.g., methanol, tert-butanol, acetone, dioxane, acetonitrile, dimethyl formamide, dimethyl sulfoxide, tetrahydrofurane, 3-methyl-3-pentanol and pyridine, can be used at a concentration of up to 70% of the total volume depending on the enzyme stability.
Reaction systems consisting of two macroscopic phases, namely, the aqueous phase containing the dissolved enzyme and the second phase of organic solvents, e.g., ethyl acetate, diethyl ether, methyl tert-butyl ether, cyclohexanol, n-propylacetate, ethyl chloroacetate, bis(2-chloroethyl)ether, isopropyl acetate, butyl acetate, isobutyl acetate, hexanol, isoamyl acetate, n-amyl acetate, toluene, octanol, isoheptane, butyl ether, cyclohexane, 2-methylpentane, n-hexane, methylcyclohexane or n-octane, can be used to achieve a spatial separation of the enzyme from the organic phase. The reaction takes place in aqueous phase where the enzyme is in favourable environment and not in direct contact with the organic solvent, where majority of the substrate and/or product is located. A sufficient mass transfer of the reagent to the enzyme, the product from the enzyme and between the two phases can be obtained by shaking or stirring. The bulk water can be replaced by water immiscible organic solvent and the solid enzyme is then suspended in a monophasic organic solution. Optimum catalytic activity of the enzyme in the organic solvent can be reached by adjustment and maintenance of water-content. This can be conventionally achieved by a pair of salt-hydrates, e.g., CaCl2.H2O/2H2O, NaI anh./2H2O, Na2HPO4 anh./2H2O, NaOAc anh./3H2O, NaBr anh./2H2O, Na4P2O7 anh./7 H2O, Na2HPO4.2H2O/7H2O, Na2SO4 anh./10H2O, Na2HPO4. 7H2O/12H2O, added to the solvent by functioning as a water-buffer. Alternatively, a saturated salt solution, e.g., LiBr, LiCl, MgCl2, K2CO3, Mg(NO3)2, NaBr, NaCl, KCl, K2SO4, being in equilibrium with a sufficient amount of undissolved salt, can be circulated through the reaction compartment via a silicone tubing that is submerged in the reaction medium. Any water produced or consumed during the reaction is equilibrated by diffusion through the tube walls, maintaining an equilibrium water activity set by the salt solution.
Surfactants (e.g., Bio-Terge AS-40, Standapol ES-1, Chemal LA-9, Tetronic 1307, Geropon T-77, Rhodasurf ON-870, Trition X45, BRIJ 35, TWEEN, Igepal CA210, Ninate 411, Trition X305, Surfactant 10G, Silwet L7600, BioTerge AS-40, Brij 96, Aerosol OT, or anionactive surfactant of general formula CnH2n+1OSO3Me, where n ranges from 10 to 16 and Me means opposite ion [Na+, K+ or monoethanolamonium]), can be added to the reaction mixture to achieve a lower surface tension, a better solubility of lipophilic reactants in reaction environment and for the formation of stable emulsions of two or more immiscible liquids. By addition of the surfactant to the reaction mixture, composed from the water phase with soluble protein and the second phase which is the organic solvent, we can get the emulsion wherein the hydrophilic parts of the surfactant molecules are oriented into the small droplets of water phase closing the enzyme and the lipophilic parts of the molecules are oriented to the second phase, to the organic solvent.
The enzyme solubility in lipophilic organic solvents can be modified by covalent attachment of the amphipathic polymer polyethylene glycol (PEG) to the surface of the enzyme. Linkage of the polymer chain onto the enzyme surface is achieved by reaction of ε-amino groups of lysine residues with a ‘linker’, e.g., cyanuric chloride. Protein stabilizers such as polyalcohols, e.g., sugar alcohols or glycerol, inactive proteins, e.g., bovine serum albumin, or polymers, which have a certain structural resemblance to that of water, e.g., polyethylene glycol, polyvinyl alcohol, can be added to the reaction medium to increase stability of the enzyme.
The physical state of the enzyme may be crystalline, lyophilized or precipitated. The enzymes can be immobilized by adsorption, e.g., inorganic and organic material such as diatomaceous earth (Celite), activated charcoal, aluminium oxide, cellulose, synthetic resins, ionic binding, e.g., cation exchange resins such as carboxymethyl cellulose or Amberlite IRA or anion exchange resins such as N,N-diethyl-aminoethylcellulose or Sephadex, or covalent attachment onto the surface of a macroscopic organic or inorganic carrier material. In general, covalent immobilization involves two steps: (i) activation of the carrier with a reactive ‘spacer’ group and (ii) enzyme attachment. The functional groups of the enzyme, which are commonly involved in covalent binding, are nucleophilic, e.g., N-terminal and ε-amino groups of lysine or carboxy-, sulfhydryl-, hydroxyl-, and phenolic functions. Inorganic, e.g., porous glass, or organic, e.g., cellulose, dextran, starch, chitin, agarose, carrier and synthetic co-polymers, e.g., VA-Epoxy Biosynth, Eupergit, can be used for covalent immobilization. The enzyme molecules can be immobilized by a cross-linking (linkage to each other) by bifunctional reagent, e.g., glutardialdehyde, dimethyladipimidate, dimethyl suberimidate, hexamethylenediisocyanate.
The enzyme can be confined to a restricted area where it remains catalytically active—entrapment into a solid matrix or membrane-restricted compartments. The enzyme in non-living or living cells can be entrapped into a biological matrix, e.g., agar gel, alginate gel, κ-carragenane. The gel-formation may be initiated by a variation of the temperature or by changing the ionotropic environment of the system. An agar gel is obtained by dropping a mixture of cells in warm (40° C.) solution of agar into well-stirred ice-cold (0-5° C.) aqueous buffer. Calcium-alginate or κ-carragenane gels are prepared by dropping the cell containing sodium alginate solution to a CaCl2- or KCl-solution, respectively.
The enzyme can be entrapped to inorganic stable matrices, e.g., silica gel. The sol-gel process is initiated by the hydrolysis of a tetraalkoxysilane of the type Si(OR)4, where R is a short chain alkyl group, e.g., n-propyl, n-butyl, in the presence of the enzyme. Hydrolysis and condensation of the Si(OR)4 monomers, catalysed by a weak acid or base, triggers the cross-linking and simultaneous formation of amorphous SiO2. A tight network, which is able to carry isolated enzyme, can be obtained by polymerization of synthetic monomers, e.g., polyacrylamide, in the presence of the enzyme. Depending on the immobilization technique, the properties of the enzyme such as stability, selectivity, catalytic rate, binding affinity and temperature characteristics may be significantly altered.
Enzyme can be separated from the rest of the reaction medium by a membrane. Small substrate and/or product molecule can freely diffuse through the membrane, but the large enzyme cannot. Mixture of an aqueous buffer, an organic solvent and a detergent, e.g., Triton, bis(2-ethylhexyl)sodium sulfosuccinate, cetyltrimethyl ammonium bromide, give ‘reverse micelles’ in arrangement where the organic solvent constitutes the bulk phase. A double layer ‘vesicles’ (liposomes) can be formed when water is the bulk phase. The aqueous environment entrapped inside these micro-cells contains the enzyme. The enzyme can be detained in a reaction compartment by a synthetic membrane, based on polyamide or polyethersulfone, of defined pore size (1000-10 000 Dalton). A variety of shapes of the synthetic membrane can be used (e.g., foils, hollow fibres). In simple form, the enzyme solution can be enclosed in dialysis tubing, like a tea bag, mounted on a gently rotating magnetic stirring bar.
The substrate specificity, stereo- or regio-selectivity of the hydrolytic dehalogenation catalysed by haloalkane dehalogenase can be improved by an alteration of the enzyme using the rational design based on structural analysis, e.g., protein crystallography, nuclear magnetic resonance and circular dichroism spectroscopy, and biochemical characterization, e.g., steady-state kinetics, transient kinetics, stability and thermostability assays, spectroscopic analyses and the like, followed by computer modelling, e.g., sequence comparisons, phylogenetic analysis, homology modelling, molecular docking, molecular mechanics, molecular dynamics, quantum mechanics and multivariate statistics, and DNA mutagenesis, e.g., cassette mutagenesis, site-directed mutagenesis, chemical mutagenesis, error-prone PCR, site saturation mutagenesis, ensemble mutagenesis, recursive ensemble mutagenesis, scanning saturation mutagenesis, mutator strains, etc. The procedure includes altering at least one amino acid residue of the haloalkane dehalogenases (EC 3.8.1.5) or recombining two or more members of the haloalkane dehalogenases (EC 3.8.1.5) to obtain an enzyme with improved substrate specificity, stereo- or regio-selectivity. Thereafter, the altered haloalkane dehalogenase nucleic acids can be expressed to provide an altered haloalkane dehalogenase. Optionally, the altered haloalkane dehalogenase nucleic acids can be introduced into a cell, in which the introduced altered haloalkane dehalogenase nucleic acids can be expressed to provide an altered haloalkane dehalogenase.



BRIEF DESCRIPTION OF FIGURES
FIG. 1 represents the enantiomeric discrimination of 2-bromopentane by using haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110. Racemic 2-bromopentane (A) was converted enantioselectively to 2-pentanol (B). Concentration of both enantiomers (S-enantiomer, black circles) and (R-enantiomer, empty circles) of 2-bromopentane in time.
FIG. 2 represents the time course of the synthesis of optically pure methyl (S)-lactate by stereoselective hydrolytic dehalogenation of methyl 2-bromopropionate catalysed by haloalkane dehalogenase DhaA isolated from Rhodococcus rhodochrous. Concentration of methyl (S)-lactate (empty circles) and methyl (R)-lactate (black circles) in time.
FIG. 3 represents the enantiomeric discrimination of ethyl 2-bromopropionate by modified protein MUT01 with haloalkane dehalogenase activity. Concentration of ethyl (R)-2-bromopropionate (empty circles) and ethyl (S)-2-bromopropionate (black circles) in time.
FIG. 4 represents the enantiomeric discrimination of 2-bromopropiophenone by the haloalkane dehalogenases DhaA (A) and DbjA (B). Concentration of both enantiomers (black and empty circles) of methyl lactate in time.



EXAMPLES
Example 1
Preparation of optically pure (S)-2-pentanol by stereoselective hydrolytic dehalogenation of 2-bromopentane catalysed by the haloalkane dehalogenase DbjA (SEQ ID No. 1, 2) isolated from Bradyrhizobium japonicum USDA110
To overproduce DbjA wild type enzyme, the corresponding gene was cloned in the pYBJA2 vector and transcribed by the tac promoter (Ptac) under the control of lacIq. Escherichia coli BL21 containing the pAQN plasmid was cultured in 0.25 L of Luria broth at 37° C. The induction of the enzyme synthesis was initiated by the addition of isopropyl-β-D-thiogalactopyranoside to a final concentration of 0.5 mM when the culture reached an optical density of 0.6 at 600 nm. After induction, the culture was incubated at 30° C. for 4 h and then harvested. The cells were disrupted by sonication using Soniprep 150 (Sanyo, UK). The supernatant was used after centrifugation at 100,000×g for 1 h. The dehalogenase was purified on a Ni-NTA Sepharose column HR 16/10 (Qiagen, Germany). The His-tagged DbjA was bound to the resin in the equilibrating buffer, which contained 20 mM potassium phosphate buffer pH 7.5, 0.5 M sodium chloride and 10 mM imidazole. Unbound and weakly bound proteins were washed off by buffer containing 60 mM imidazole. The His-tagged DbjA enzyme was then eluted by buffer with 160 mM imidazole. The active fractions were pooled and dialysed overnight against 50 mM potassium phosphate buffer, pH 7.5. The enzyme was stored at 4° C. in 50 mM potassium phosphate buffer, pH 7.5, containing 10% glycerol and 1 mM 2-mercaptoethanol. The hydrolytic dehalogenation of racemic 2-bromopentane was catalysed by haloalkane dehalogenase DbjA at a room temperature (21° C.) in 20 ml of buffer containing 50 mM tris(hydroxymethyl)aminomethane (pH 8.2, adjusted by addition of H2SO4). The reaction was initiated by addition of purified haloalkane dehalogenase DbjA to a final enzyme concentration 1 μM. The method uses a high magnitude of chiral recognition of 2-bromopentane by the haloalkane dehalogenase DbjA (E-value>145). The reaction was stopped after the complete conversion of the preferred enantiomer. Optically pure (S)-2-bromopentane with enantiomeric excess of 99% and yield 48% and optically pure (S)-2-pentanol with enantiomeric excess of 96% and yield 48% were obtained (FIG. 1).
Example 2
Preparation of Optically Pure Methyl (S)-Lactate by Stereoselective Hydrolytic dehalogenation of methyl 2-bromopropionate catalysed by the haloalkane dehalogenase DhaA (SEQ ID No. 5, 6) isolated from Rhodococcus rhodochrous NCIMB 13064.
To overproduce DhaA wild type enzyme, the corresponding gene was cloned in the pAQN vector and transcribed by the tac promoter (Ptac) under the control of lacIq. Escherichia coli BL21 containing the pAQN plasmid was cultured in 0.25 L of Luria broth at 37° C. The isopropyl-β-D-thiogalactopyranoside to a final concentration of 0.5 mM was added when the culture reached an optical density of 0.6 at 600 nm. The culture was incubated at 30° C. for 4 h and then harvested. The cells were disrupted by sonication using Soniprep 150 (Sanyo, UK). The supernatant was used after centrifugation at 100,000×g for 1 h. The His-tagged DhaA was purified on a Ni-NTA Sepharose column HR 16/10 (Qiagen, Germany). The enzyme was stored at 4° C. in 50 mM potassium phosphate buffer, pH 7.5, containing 10% glycerol and 1 mM 2-mercaptoethanol. Hydrolytic dehalogenation of racemic ethyl 2-bromobutyrate was catalysed by DhaA enzyme at room temperature (21° C.) in 1 L batch stirred reactor in 50 mM Tris(hydroxymethyl)aminomethane (pH=8.2; by H2SO4). The reaction was initiated by addition of purified haloalkane dehalogenase DhaA to final enzyme concentration 5 μM. The method uses a high magnitude of the chiral recognition of methyl 2-bromopropionate by the haloalkane dehalogenase DhaA (E-value >200). After complete conversion of the preferred enantiomer, high enantiomeric purity of methyl (S)-lactate was reached (e.e.>99.9%). The optically pure methyl (S)-lactate can be easily separated from the reaction mixture (FIG. 2).
Example 3
Preparation of optically pure ethyl (S)-2-hydroxybutyrate by stereoselective hydrolytic dehalogenation of ethyl 2-brombutyrate catalysed by the haloalkane dehalogenases DbjA, LinB, DhaA, DmbA, DmbB, DmbC, DrbA, DhmA and DbeA
The wild type enzymes DbjA, LinB, DhaA, DmbA, DmbB, DmbC, DrbA, DhmA and DbeA were prepared by overproduction in Escherichia coli BL21. Every corresponding gene was cloned in the pAQN vector and transcribed by the tac promoter (Ptac) under the control of lacIq. Escherichia coli BL21 containing the pAQN plasmid was cultured in 0.25 L of Luria broth at 37° C. The isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 0.5 mM when the culture reached the optical density of 0.6 at 600 nm. The culture was incubated at 30° C. for 4 h and then harvested. The cells were disrupted by sonication using Soniprep 150 (Sanyo, UK). The supernatant was used after centrifugation at 100,000×g for 1 h. His-tagged haloalkane dehalogenases were purified on a Ni-NTA Sepharose column HR 16/10 (Qiagen, Germany). The enzyme was stored at 4° C. in 50 mM potassium phosphate buffer, pH 7.5, containing 10% glycerol and 1 mM 2-mercaptoethanol. Chiral recognition of ethyl 2-bromobutyrate was tested with the following haloalkane dehalogenases: DbjA, LinB, DhaA, DmbA, DmbB, DmbC, DrbA, DhmA and DbeA. The reactions were performed at room temperature (21° C.) in 20 ml of reaction mixture containing 50 mM Tris(hydroxymethyl)aminomethane (pH=8.2; by H2SO4). The reactions were initiated by the addition of purified haloalkane dehalogenases to the final enzyme concentration of 1 μM. The method uses a high magnitude of the chiral recognition of ethyl 2-bromobutyrate by haloalkane dehalogenases (Table 2). In all cases, the conversion of ethyl 2-bromobutyrate by the haloalkane dehalogenases DbjA, LinB, DhaA, DmbA, DmbB, DmbC, DrbA, DhmA and DbeA, respectively, proceeds to the entire conversion of the preferred enantiomer and 99.9% enantiomeric excess of ethyl (S)-2-hydroxybutyrate is reached.







TABLE 2







Chiral recognition of ethyl 2-bromobutyrate by hydrolytic


dehalogenation catalysed by the haloalkane dehalogenases


DbjA, LinB, DhaA, DmbA, DmbB, DmbC, DrbA, DhmA and DbeA.









E-values
















Substrate
DbjA
LinB
DhaA
DmbA
DmbB
DmbC
DrbA
DhmA
DbeA





ethyl-2-bromo-butyrate
>200
>200
>200
>200
>200
>200
>200
>200
>200









Example 4
Preparation of optically pure ethyl (S)-2-bromopropionate and ethyl (S)-lactate by stereoselective hydrolytic dehalogenation of racemic ethyl 2-bromopropionate catalysed by modified protein MUT01 (SEQ ID No. 19, 20) with haloalkane dehalogenase activity
Modified protein MUT01 with haloalkane dehalogenase activity having an amino acid sequence showing between 21.1 and 93.7% identity with amino acid sequences of haloalkane dehalogenases DbjA, LinB, DhaA, DmbA, DmbB, DmbC, DrbA, DhmA and DbeA (Table 3) was used for preparation of optically pure (S)-2-hexanol and (S)-ethyl lactate. Modified protein MUT01 is coded by the gene with DNA sequence showing between 32.4 and 94.8% identity to DNA sequences of DbjA, LinB, DhaA, DmbA, DmbB, DmbC, DrbA DhmA and DbeA (Table 3).







TABLE 3







Sequence identity of modified protein MUT01 (SEQ ID No.


19, 20) to sequences of haloalkane dehalogenases DbjA,


LinB, DhaA, DmbA, DmbB, DmbC, DrbA, DhmA and DbeA.











Amino acid sequence



DNA sequence identity:
identity: haloalkane


Haloalkane
haloalkane dehalogenases vs.
dehalogenases vs. MUT01


dehalogenase
MUT01 (SEQ ID No. 19)
(SEQ ID No. 20)





DbjA
58.3%
52.5%


LinB
51.7%
46.9%


DhaA
94.8%
93.7%


DmbA
51.4%
42.4%


DmbB
38.7%
26.5%


DmbC
37.5%
24.6%


DrbA
32.4%
21.1%


DhmA
39.7%
26.4%


DbeA
54.7%
47.9%









Recombinant gene of the modified protein was obtained by using QuikChangeTM Site-Directed Mutagenesis Kit (Stratagene, USA). Plasmid pUC19 with recombinant gene was used as the first template. Mutant recombinant gene was recloned into pET21b vector. Resulting plasmid containing the recombinant gene MUT01 was transformed to host organism E. coli BL21 (DE3). The host organism E. coli BL21 (DE3) was cultivated in 250 ml Luria broth at 37° C. Isopropyl-β-D-thiogalactopyranoside was added to final concentration of 0.5 mM when the culture reached an optical density of 0.6 at 600 nm. The culture was incubated at 30° C. for 4 h and then harvested. The cells were disrupted by sonication using Soniprep 150 (Sanyo, UK). The supernatant was used after centrifugation at 100,000×g for 1 h. The modified protein MUT01 with his-tag was purified on a Ni-NTA Sepharose column HR 16/10 (Qiagen, Germany). The enzyme was stored at 4° C. in 50 mM potassium phosphate buffer, pH 7.5, containing 10% glycerol and 1 mM 2-mercaptoethanol. The hydrolytic dehalogenation of racemic 2-bromohexane and racemic ethyl 2-bromopropionate was catalysed by modified protein MUT01 at a room temperature (21° C.) in 20 ml of buffer containing 50 mM tris(hydroxymethyl)aminomethane (pH 8.2, adjusted by addition of H2SO4). The reaction was initiated by addition of purified protein MUTO to a final concentration 1 μM. The method uses high magnitude of the chiral recognition of ethyl 2-bromopropionate (E-value>200) by protein MUT01 with haloalkane dehalogenase activity. After entire conversion of preferred enantiomer of ethyl 2-bromopropionate with high enantiomer excess is reached and the resulting optically pure ethyl (S)-lactate with e.e. 99.9% and optically pure ethyl (S)-2-bromopropionate (e.e. =99.9%) can be easily separated (FIG. 3).
Example 5
Preparation of optically pure 2-bromopropiophenone by stereoselective hydrolytic dehalogenation of racemic mixture by the haloalkane dehalogenase DbjA (SEQ ID No. 1, 2) from Bradyrhizobium japonicum and DhaA (SEQ ID No. 5, 6) from Rhodococcus rhodochrous 
To overproduce DbjA and DhaA wild type enzymes, the corresponding genes were cloned in the pYBJA2 vector and transcribed by the tac promoter (Ptac) under the control of lacIq. Escherichia coli BL21 containing the pAQN plasmid was cultured in 0.25 L of Luria broth at 37° C. The induction of the enzyme synthesis was initiated by the addition of isopropyl-β-D-thiogalactopyranoside to a final concentration of 0.5 mM when the culture reached an optical density of 0.6 at 600 nm. After induction, the culture was incubated at 30° C. for 4 h and then harvested. The cells were disrupted by sonication using Soniprep 150 (Sanyo, UK). The supernatant was used after centrifugation at 100,000×g for 1 h. The dehalogenase was purified on a Ni-NTA Sepharose column HR 16/10 (Qiagen, Germany). The His-tagged DbjA was bound to the resin in the equilibrating buffer, which contained 20 mM potassium phosphate buffer pH 7.5, 0.5 M sodium chloride and 10 mM imidazole. Unbound and weakly bound proteins were washed off by buffer containing 60 mM imidazole. The His-tagged DbjA enzyme was then eluted by buffer with 160 mM imidazole. The active fractions were pooled and dialysed overnight against 50 mM potassium phosphate buffer, pH 7.5. The enzyme was stored at 4° C. in 50 mM potassium phosphate buffer, pH 7.5, containing 10% glycerol and 1 mM 2-mercaptoethanol. The hydrolytic dehalogenation of racemic 2-bromopropiophenone was catalysed by the haloalkane dehalogenases DbjA and DhaA, respectively, at a room temperature (21° C.) in 20 ml of buffer containing 50 mM tris(hydroxymethyl)aminomethane (pH 8.2, adjusted by addition of H2SO4). The reaction was initiated by addition of the purified haloalkane dehalogenase DbjA or DhaA to a final enzyme concentration 1 μM. The method uses a high magnitude of the chiral recognition of 2-bromopropiophenone by the haloalkane dehalogenase DbjA (E-value>200). The reaction was stopped after complete conversion of preferred enantiomer. Final enantiomeric excess>99.5% was reached for optically pure non-preferred enantiomer of 2-bromopropiophenone in reactions catalysed by both DbjA and DhaA (FIG. 4).
Example 6
Rational Engineering of Specificity of the Haloalkane Dehalogenase LinB (SEQ ID No. 3, 4) Using Phylogenetic Analysis and Computer Modelling
The amino acid Leucine (Leu) in position 177 was identified as a determinant of the substrate specificity of haloalkane dehalogenase LinB by structural analysis and comparison of the primary sequence of LinB (SEQ ID NO: 4) with protein sequences of other haloalkane dehalogenase family members. Leu 177 is positioned at the mouth of the largest entrance tunnel leading to the enzyme active site and is pointing directly into the tunnel. At the same time it is the most variable pocket residue of the haloalkane dehalogenase-like proteins showing 9 different substitutions in 14 proteins. Saturated mutagenesis in position 177 of LinB was performed using site-directed mutagenesis. The plasmid pULBH6 was used as a template. To overproduce LinB mutants in E. coli, His-tagged mutant LinB genes were cloned in pAQN vector and the genes were transcribed by the tac promoter (P tac) under the control of lacIq. E. coli BL21 containing these plasmids were cultured in 1 L of Luria broth. When the culture reached an optical density of 0.6 at 600 nm the induction of enzyme expression (at 30° C.) was initiated by the addition of isopropyl-β-D-thiogalactopyranoside to a final concentration of 1 mM. The cells were harvested and disrupted by sonication using a Soniprep 150 (Sanyo Gallenkamp PLC, Loughborough, UK). The supernatant was used after centrifugation at 100,000×g for 1 hr. The crude extract was further purified on a Ni-NTA Sepharose column HR 16/10 (QIAGEN, Hilden, Germany). The His-tagged LinB mutants were bound to the resin in the equilibrating 20 mM potassium phosphate buffer (pH 7.5) containing 0.5 M sodium chloride and 10 mM imidazole. Unbound and weakly bound proteins were washed out by the buffer containing 45 mM imidazole. The His-tagged enzyme was then eluted by the buffer containing 160 mM imidazole. The active fractions were pooled and dialysed against 50 mM potassium phosphate buffer (pH 7.5) overnight. The enzyme was stored in 50 mM potassium phosphate buffer (pH 7.5) containing 10% glycerol and 1 mM 2-mercaptoethanol. Specific activities of LinB with twelve different halogenated substrates representing different chemical groups (mono-, di- and tri-halogenated, chlorinated, brominated and iodinated; α- and β-substituted, aliphatic and cyclic, saturated and unsaturated compounds) were assessed (Table 4).







TABLE 4





Substrate specificity of purified wild type and mutant haloalkane dehalogenases.

















Relative activities (% of wt)

















Proteins
wt1a
L177A
L177C
L177G
L177F
L177K
L177T
L177W
wt2b
L177D





1-chlorobutane
100
142
 37
 94
229
 61
—c
138 
100
104 


1-chlorohexane
100
106
179
125
215
100
143
89
100
44


1-bromobutane
100
356
243
380
243
201
553
60
100
347 


1-iodobutane
100
133
210
344
126
131
424
58
100
259 


1,2-dichloroethane
—c
—c
—c
—c
—c
—c
—c
—c
—c
—c


1,2-dibromoethane
100
155
 52
 78
 70
 97
107
 7
100
77


1,3-diidopropane
100
360
192
164
130
132
108
117 
100
209 


1,2-dichloropropane
—c
—c
—c
—c
—c
—c
—c
—c
—c
—c


1,2,3-trichloropropane
—c
—c
—c
—c
—c
—c
—c
—c
—c
—c


chlorocyclohexane
100
—c
—c
 33
—c
—c
—c
139 
100
38


bromocyclohexane
100
115
298
398
267
117
198
80
100
63


3-chloro-2-
100
199
157
200
 98
110
 96
91
100
25


methylpropene












Relative activities (% of wt)


















Proteins
L177H
L177I
L177M
L177P
L177Q
L177R
L177S
L177V
L177Y







1-chlorobutane
56
—c
144
—c
 54
46
 60
74
55



1-chlorohexane
75
—c
162
—c
165
31
104
80
80



1-bromobutane
224 
—c
227
—c
458
165 
381
132 
112 



1-iodobutane
208 
—c
187
—c
373
161 
363
101 
97



1,2-dichloroethane
—c
—c
—c
—c
—c
—c
—c
—c
—c



1,2-dibromoethane
55
—c
126
—c
123
68
 84
84
16



1,3-diidopropane
140 
—c
202
—c
159
127 
123
206 
102 



1,2-dichloropropane
—c
—c
—c
—c
—c
—c
—c
—c
—c



1,2,3-trichloropropane
—c
—c
—c
—c
—c
—c
—c
—c
—c



chlorocyclohexane
—c
—c
 79
—c
—c
—c
 21
91
— 



bromocyclohexane
43
—c
104
—c
120
37
156
41
41



3-chloro-2-
82
—c
100
—c
138
83
 65
79
78



methylpropene







aspecific activities (in μmol · s−1 · mg−1 of enzyme) of the wild type enzyme in the first set of mutants are 0.0338 (1-chlorobutane), 0.0208 (1-chlorohexane), 0.0633 (1-bromobutane), 0.0104 (1-iodobutane), 0.2200 (1,2-dibromoethane), 0.0463 (1,3-diidopropane), 0.0018 (chlorocyclohexane), 0.0201 (bromocyclohexane) and 0.1366 (3-chloro-2-methylpropene).



bspecific activities (in μmol · s−1 · mg−1 of enzyme) of the wild type enzyme in the second set of mutants are 0.0287 (1-chlorobutane), 0.0315 (1-chlorohexane), 0.0477 (1-bromobutane), 0.0408 (1-iodobutane), 0.1894 (1,2-dibromoethane), 0.0262 (1,3-diiodopropane), 0.0034 (chlorocyclohexane), 0.0336 (bromocyclohexane) and 0.1312 (3-chloro-2-methylpropene).



cactivity not detectable






Without exception, all mutants exhibited modified activities compared to the wild type enzyme. In general, activity of LinB enzyme increases with the introduction of small and non-polar amino acid to the position 177. This residue is partially blocking the entrance tunnel and its size and polarity influence binding of the substrate molecules to the active site. Especially poor binding is observed when negative charge is introduced in position 177 (Km for mutant where Leu 177 was substituted by Asp is 21.9 mM with 1-chlorobutane and 14 mM with 1,2-dibromoethane). The activity and substrate specificity of haloalkane dehalogenase can obviously be modulated by the residues positioned far from the active site if they are a part of the entrance tunnel. Modification of the catalytic properties of haloalkane dehalogenases using site-directed mutagenesis by specifically targeting such distant residues (identified using rational design) provides functional enzymes at much higher rate compared to mutagenesis of the active site residues.
Example 7
Hydrolytic dehalogenation of 1-iodohexane catalysed by haloalkane dehalogenase DbjA (SEQ ID No. 1, 2) isolated from Bradyrhizobium japonicum USDA110 in the presence of organic solvents
Hydrolytic dehalogenation of 1-iodohexane catalysed by haloalkane dehalogenase DbjA isolated from Bradyrhizobium japonicum USDA110 was performed in presence of organic solvents. Reaction mixture was prepared by mixing glycine buffer (7.5 g glycine in 1 L distilled water, pH was adjusted to value 8.6 by addition of NaOH solution) with appropriate volumes of organic solvents to reach specified final concentrations (5, 10, 20% v/v; volume concentration of organic solvent in water buffer). The substrate 1-iodohexane was added to the reaction mixture to final concentration 1 mM. The reaction was initiated by addition of enzyme DbjA to a final enzyme concentration 1 μM. The reaction was monitored by colorimetric detection of iodide ion concentration increase by using Iwasaki method [Iwasaki, I., Utsumi, S., Ozawa, T. Bulletin of the Chemical Society of Japan (1952) 25, 226].
The enzyme DbjA has shown high tolerance to the presence of all the tested organic solvents in concentration 5 and 10% (v/v). The DbjA enzyme has shown an increasing activity in the presence of acetone, 1,4-dioxane and methanol in the reaction medium. The hydrolytic dehalogenation of 1-iodohexane catalysed by haloalkane dehalogenase DbjA reached 1.68 times increase of reaction rate in the 20% (v/v) acetone compared to that in the glycine buffer.
Haloalkane dehalogenases can catalyse hydrolytic dehalogenation not only in aqueous solution but also in the environment of organic solvents, where the higher solubility of desired substrate and higher reaction rate of enzyme are achieved at the same time.
The undesired side reactions are suppressed and enantioselectivity can be improved in the presence of organic solvents as well.







TABLE 5







Relative activities of DbjA in the presence of organic solvents.









Relative enzyme activity (% )













5% (b)
10% (b)
20% (b)


Organic solvent
log P (a)
of solvent
of solvent
of solvent












glycine buffer
—
100.0 ± 6.8 (c)











Formamide
−1.57
73.4 ± 7.6
76.3 ± 5.0
 16.3 ± 0.6


Formamide
−1.57
73.4 ± 7.6
76.3 ± 5.0
 16.3 ± 0.6


ethyleneglycol
−1.23
87.1 ± 9.8
80.5 ± 8.9
109.8 ± 4.0


N,N-dimethylformamide
−1.04
110.5 ± 11.6
112.6 ± 3.8 
101.9 ± 1.0


Methanol
−0.82
93.5 ± 0.9
128.7 ± 3.5 
160.7 ± 2.0


1,4-dioxane
−0.50
121.4 ± 3.9 
139.9 ± 7.8 
152.0 ± 9.5


acetonitrile
−0.39
117.5 ± 8.9 
124.1 ± 1.0 
 0.0 ± 0.0


ethanol
−0.32
106.8 ± 8.9 
91.8 ± 7.3
102.2 ± 2.2


acetone
−0.24
113.8 ± 7.3 
140.3 ± 2.1 
168.2 ± 3.1


tetrahydrofurane
0.46
111.9 ± 13.4
96.9 ± 9.0
 0.0 ± 0.0





(a) logarithm of n-octanol-water partition coefficient


(b) volume concentration of organic solvent in water buffer; (v/v)


(c) relative activity in glycine buffer






Example 8
Preparation of optically pure (S)-2-pentanol by stereoselective hydrolytic dehalogenation of 2-bromopentane catalysed by the haloalkane dehalogenase DbeA (SEQ ID No. 17, 18) isolated from Bradyrhizobium elkanii 
To overproduce DbeA wild type enzyme, the corresponding gene was cloned in the pYBJA2 vector and transcribed by the tac promoter (Ptac) under the control of lacIq. Escherichia coli BL21 containing the pAQN plasmid was cultured in 0.25 L of Luria broth at 37° C. The induction of the enzyme synthesis was initiated by the addition of isopropyl-β-D-thiogalactopyranoside to a final concentration of 0.5 mM when the culture reached an optical density of 0.6 at 600 nm. After induction, the culture was incubated at 30° C. for 4 h and then harvested. The cells were disrupted by sonication using Soniprep 150 (Sanyo, UK). The supernatant was used after centrifugation at 100,000×g for 1 h. The dehalogenase was purified on a Ni-NTA Sepharose column HR 16/10 (Qiagen, Germany). The His-tagged DbeA was bound to the resin in the equilibrating buffer, which contained 20 mM potassium phosphate buffer pH 7.5, 0.5 M sodium chloride and 10 mM imidazole. Unbound and weakly bound proteins were washed off by buffer containing 60 mM imidazole. The His-tagged DbeA enzyme was then eluted by buffer with 160 mM imidazole. The active fractions were pooled and dialysed overnight against 50 mM potassium phosphate buffer, pH 7.5. The enzyme was stored at 4° C. in 50 mM potassium phosphate buffer, pH 7.5, containing 10% glycerol and 1 mM 2-mercaptoethanol. The hydrolytic dehalogenation of racemic 2-bromopentane was catalysed by haloalkane dehalogenase DbeA at a room temperature (21° C.) in 20 ml of buffer containing 50 mM tris(hydroxymethyl)aminomethane (pH 8.2, adjusted by addition of H2SO4). The reaction was initiated by addition of purified haloalkane dehalogenase DbeA to a final enzyme concentration 1 μM. The method uses a high magnitude of chiral recognition of 2-bromopentane by the haloalkane dehalogenase DbeA (E-value=98). The reaction was stopped after the complete conversion of the preferred enantiomer. Optically pure (S)-2-bromopentane with enantiomeric excess of 97% and yield 47% and optically pure (S)-2-pentanol with enantiomeric excess of 94% and yield 47% were obtained.
INDUSTRIAL APPLICABILITY
The invention can be applied for industrial production of optically active compounds, particularly haloalkanes, haloalcohols, alcohols, halopolyols, polyols, hydroxyketones and hydroxyesters using hydrolytic dehalogenation of racemic or prochiral halogenhydrocarbons by dehalogenation catalysed by haloalkane dehalogenases (EC 3.8.1.5).

## Claims

1. A method of production of optically active halohydrocarbons and alcohols using hydrolytic dehalogenation catalysed by a haloalkane dehalogenase wherein haloalkane dehalogenase DbjA having the amino acid sequence SEQ ID NO: 2 is applied to at least one racemic or prochiral chlorinated, brominated or iodinated compound at a temperature range between +10 and +70° C. and pH value between 4.0 and 12.0, in an aqueous system or in a monophasic organic solution or in a monophasic organic/aqueous solution or in organic/aqueous biphasic systems, wherein the chlorinated, brominated or iodinated compounds have at least one halogen atom bound directly to the chiral or prochiral carbon and the nucleophilic attack takes place directly on the chiral or prochiral carbon.

2. The method according to claim 1, wherein it is performed in the presence of surfactants to allow using of enhanced reagent concentration.

3. The method according to claim 1, wherein the haloalkane dehalogenase DbjA is in soluble or crystalline or lyophilized or precipitated form.

4. The method according to claim 1, wherein the haloalkane dehalogenase DbjA is immobilized by adsorption or ionic binding or covalent attachment onto the surface of a macroscopic carrier material.

5. The method according to claim 1, wherein the haloalkane dehalogenase DbjA is immobilized by cross-linking or confined to a solid matrix or membrane-restricted compartments.