JP2012228221A - Improved halohydrin epoxidase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an improved halohydrin epoxidase in which stereoselectivity, product inhibition resistance, and the like are improved, and to provide a method, by using the same, for efficiently producing optically active epihalohydrin or 4-halo-3-hydroxybutyronitrile by using them.SOLUTION: In the amino acid sequence of a wild type halohydrin epoxidase shown by a specific sequence, an improved halohydrin epoxidase consisting of the amino acid sequence into which the amino acid mutations substituting Ala at the second position with Lys, and also Ala at the 128th position with Cys, Gln, Ile, Met or Val are introduced is used.

Description

  The present invention relates to an improved halohydrin epoxidase and a method for producing the same.

  Halohydrin epoxidase, also called halohydrin hydrogen halide lyase, halohydrin dehalogenase or haloalcohol dehalogenase, catalyzes the activity of converting 1,3-dihalo-2-propanol to epihalohydrin and its reverse reaction It is an enzyme (EC number: 4.5. It is known that halohydrin epoxidase has three isozymes (A type, B type, and C type) from the homology of amino acid sequences and the like (Non-patent Document 1: J. Bacteriology, 183 (17). ), 5058-5066, 2001). As type A halohydrin epoxidase, HheA derived from Corynebacterium sp. N-1074 strain (Non-patent Document 2: Biosci. Biotechnol. Biochem., 58 (8), 1451-1457, 1994), HheA AD2 derived from Arthrobacter sp. AD2 strain (Non-patent document 1), Deh-PY1 derived from Arthrobacter sp. Strain PY1 (Non-patent document 3: J. Health. Sci. , 50 (6), 605-612, 2004) and the like. Further, as B-type halohydrin epoxidase, HheB (Non-patent Document 2) derived from Corynebacterium sp. N-1074 strain, and HheB GP1 (Mycobacterium sp.) GP1 strain ( Non-Patent Document 1), DehA (Non-Patent Document 4: Enz. Microbiol. Technol., 22, 568-574, 1998) and the like derived from Arthrobacter erythii strain H10a are known. As C-type halohydrin epoxidase, HheC derived from Agrobacterium radiobacter DH094 (Patent Document 1: JP-A-10-210981), Agrobacterium radiobacter AD1 strain HheC derived from non-patent document 1, HalB derived from Agrobacterium tumefaciens (non-patent document 5: Thesis (1996) University of Wales, Cardiff, United Kingdom), etc. are known.

  Epihalohydrin is a useful substance as a raw material for synthesizing various pharmaceuticals and physiologically active substances. For example, (R) -4-halo-3-hydroxybutyronitrile obtained by ring-opening cyanation of (R) -epihalohydrin is known to be useful as a raw material for the synthesis of L-carnitine (Patent Literature). 2: JP-A-57-165352). For at least some of the halohydrin epoxidases, in addition to the activity of converting 1,3-dihalo-2-propanol to epihalohydrin and the activity of catalyzing the reverse reaction, the epihalohydrin can be opened in the presence of a cyanide compound. It has been clarified that it catalyzes a reaction of cyanation to form 4-halo-3-hydroxybutyronitrile. As an example using this reaction, 1,3-dihalo-2-propanol is optically active. Process for producing halo-3-hydroxybutyronitrile (Patent Document 3: JP-A-3-053889, Patent Document 4: JP-A-2001-25397) and optically active 4-halo-3-hydroxybutyrate from epihalohydrin A method for producing ronitrile (Patent Document 5: JP-A-3-053890) is known.

  By the way, recent advances in genetic engineering technology have made it possible to intentionally create mutants in which one or more of the constituent amino acids of the enzyme protein are deleted, added or inserted, or substituted with other amino acids. ing. Depending on the type of mutation, these mutants may have activity, stability, resistance to organic solvents, heat resistance, acid resistance, alkali resistance, substrate specificity, stereoselectivity, etc. It is known that the performance of the system is improved. These performance improvements can significantly reduce production costs in industrial production using enzyme reactions by reducing enzyme catalyst production costs per activity, stabilizing enzyme catalysts, simplifying reaction processes, and improving reaction yields. May bring. Therefore, creation of useful improved enzymes having various performances improved in many enzymes. A halohydrin epoxidase has also been reported as a mutant in which one or more constituent amino acids are deleted, added, inserted, or substituted with other amino acids. For example, Patent Document 6 (US Patent Application Publication No. 2005/0272064) includes 570 types of HheC mutants derived from Agrobacterium radiobacter strain AD1 and Corynebacterium sp. One variant of HheB from N-1074 strain is described. Patent Document 7 (US Patent Application Publication No. 2006/0099700) discloses 1422 types of mutants of HheC derived from Agrobacterium radiobacter AD1 strain and Corynebacterium sp. One variant of HheB from N-1074 strain is described. Some of these mutants have improved activity in the conversion reaction of ethyl (R) -4-chloro-3-hydroxybutyrate to ethyl (S) -4-cyano-3-hydroxybutyrate. It has been shown. Further, Patent Document 8 (WO 2008/108466 pamphlet) describes 22 mutants of HheB derived from Corynebacterium sp. N-1074 strain. Several of these mutants have been shown to have improved halohydrin epoxidase activity per transformant.

Japanese Patent Laid-Open No. 10-210981 JP-A-57-165352 Japanese Patent Laid-Open No. 3-053889 Japanese Patent Laid-Open No. 2001-25397 JP-A-3-053890 US Patent Application Publication No. 2005/0272064 US Patent Application Publication No. 2006/0099700 International Publication No. 2008/108466 Pamphlet

J. et al. Bacteriol. , 183 (17), 5058-5066, 2001 Biosci. Biotechnol. Biochem. 58 (8), 1451-1457, 1994. J. et al. Health. Sci. , 50 (6), 605-612, 2004 Enz. Microbiol. Technol. , 22, 568-574, 1998 Thesis (1996) University of Wales, Cardiff, United Kingdom

  When halohydrin epoxidase is used industrially, further advancement as an enzyme catalyst is desired. For example, when the purpose is to produce optically active 4-halo-3-hydroxybutyronitrile, the optical purity of 4-halo-3-hydroxybutyronitrile produced by a known halohydrin epoxidase is not always sufficient. It is not expensive. Therefore, a halohydrin epoxidase with improved stereoselectivity that produces 4-halo-3-hydroxybutyronitrile with higher optical purity is desired. When 4-halo-3-hydroxybutyronitrile is produced from 1,3-dihalo-2-propanol as a starting material by halohydrin epoxidase, the product (halide ion and 4-halo-3- The reaction rate may decrease due to inhibition of (hydroxybutyronitrile). Accordingly, halohydrin epoxidases that are less susceptible to product inhibition are desired.

  Accordingly, an object of the present invention is to provide a halohydrin epoxidase capable of solving these problems, a method for producing the same, and a method for producing epihalohydrin or 4-halo-3-hydroxybutyronitrile using the same. is there.

The inventors of the present invention have intensively studied to solve the above problems. As a result, the amino acid sequence of the wild-type halohydrin epoxidase, particularly the amino acid sequence of the wild-type halohydrin epoxidase classified into the B-type isozyme, was analyzed. Among them, by substituting any or all of the amino acid residues at a predetermined position with other amino acids, halohydrin epoxidase activity per transformant, stereoselectivity, product inhibition resistance, product accumulation ability, etc. The present inventors have found that an improved halohydrin epoxidase having improved attributes can be created, and the present invention has been completed.
That is, the present invention is as follows.
(1) The following amino acid sequence I:
S-α1-α2-α3-α4-α5-α6-α7-α8-α9-α10-α11-α12-Y-α13-α14-A-R-α15 (SEQ ID NO: 15)
(S represents a Ser residue, Y represents a Tyr residue, A represents an Ala residue, R represents an Arg residue, and α1 to α15 each independently represent an identical or different arbitrary amino acid residue.)
In the amino acid sequence of the wild-type halohydrin epoxidase containing the amino acid residue 1 residue C-terminal from the starting amino acid residue (usually Met residue) is replaced with Lys, and the α10 residue is An improved halohydrin epoxidase consisting of an amino acid sequence into which an amino acid mutation substituted by another amino acid is introduced.
(2) The improved halohydrin epoxidase according to (1), wherein the amino acid mutation at the α10 residue is Cys, Gln, Ile, Met or Val.
(3) The improved halohydrin epoxidase according to (1) or (2), wherein the wild-type halohydrin epoxidase is classified into group B.
(4) The improved type according to any one of (1) to (3), wherein the wild-type halohydrin epoxidase is derived from a bacterium belonging to the genus Corynebacterium or a genus Mycobacterium. Halohydrin epoxidase.
(5) A gene encoding the improved halohydrin epoxidase according to any one of (1) to (4).
(6) A recombinant vector comprising the gene according to (5).
(7) A transformant or a transductant obtained by introducing the recombinant vector according to (6) into a host.
(8) A culture obtained by culturing the transformant or transductant according to (7).
(9) A method for producing an improved halohydrin epoxidase, comprising collecting the improved halohydrin epoxidase from the culture according to (8).
(10) By contacting the improved halohydrin epoxidase according to any one of (1) to (4) and / or the culture according to (8) with 1,3-dihalo-2-propanol. A method for producing epihalohydrin, characterized by producing epihalohydrin.
(11) The improved halohydrin epoxidase according to any one of (1) to (4) and / or the culture according to (8), in the presence of a cyanide compound, 1,3-dihalo-2-propanol Alternatively, 4-halo-3-hydroxybutyronitrile is produced by contacting with epihalohydrin to produce 4-halo-3-hydroxybutyronitrile.
(12) An amino acid mutation in which the second Ala is substituted with Lys and the 128th Ala is substituted with another amino acid in the amino acid sequence of the wild-type halohydrin epoxidase represented by SEQ ID NO: 1 An improved halohydrin epoxidase consisting of an amino acid sequence into which is introduced.
(13) An improved halohydride comprising an amino acid sequence in which an amino acid mutation in which the 136th Ala is substituted with another amino acid in the amino acid sequence of the wild-type halohydrin epoxidase represented by SEQ ID NO: 2 is introduced Phosphoepoxidase.
(14) In the amino acid sequence of the wild-type halohydrin epoxidase represented by SEQ ID NO: 1, the second Ala is substituted with Lys, and the 128th Ala is Cys, Gln, Ile, Met or Val. An improved halohydrin epoxidase consisting of an amino acid sequence into which an amino acid mutation substituted in is introduced.
(15) In the amino acid sequence of the wild-type halohydrin epoxidase represented by SEQ ID NO: 2, from the amino acid sequence introduced with an amino acid mutation in which the 136th Ala is replaced with Cys, Gln, Ile, Met or Val An improved halohydrin epoxidase.

  According to the present invention, an improved halohydrin epoxidase having improved stereoselectivity, product inhibition resistance and the like can be obtained, and optically active epihalohydrin or 4-halo-3-hydroxybutyroyl can be obtained using these. Nitrile can be produced efficiently.

It is a figure which shows plasmid pSTT002.

  Embodiments of the present invention will be described below. However, the present embodiments are examples for explaining the present invention, and the present invention is not intended to be limited to these embodiments. The present invention can be implemented in various forms without departing from the gist thereof.

1. Halohydrin epoxidase (1) Wild-type halohydrin epoxidase “Wild-type halohydrin epoxidase” refers to a halohydrin epoxidase that can be isolated from natural organisms, and its origin is not limited. . Also, it means a halohydrin epoxidase that retains its natural attribute without any intentional or unintentional deletion of amino acids or substitution or insertion with other amino acids in the amino acid sequence constituting the enzyme. .

Furthermore, the wild-type halohydrin epoxidase used in the present invention has the following amino acid sequence I (19 amino acid residues):
S-α1-α2-α3-α4-α5-α6-α7-α8-α9-α10-α11-α12-Y-α13-α14-A-R-α15 (SEQ ID NO: 15)
(S represents a Ser residue, Y represents a Tyr residue, A represents an Ala residue, R represents an Arg residue, and α1 to α15 each independently represent the same or different arbitrary amino acid residues). It consists of an amino acid sequence. Whether or not the wild-type halohydrin epoxidase consists of an amino acid sequence comprising amino acid sequence I is provided by public sequence databases such as the National Center for Biotechnology Information (NCBI). GenBank database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=protein) is searched for those registered as halohydrin epoxidase, and the corresponding amino acids What is necessary is just to investigate whether the said amino acid sequence I is contained in a sequence. If necessary, a search can be performed using gene information analysis software. Further, if there is a known document describing the amino acid sequence of wild-type halohydrin epoxidase, it may be referred to. For example, J. et al. Bacteriology, 183 (17), 5058-5066, 2001, by referring to the alignment method of various wild-type halohydrin epoxidase amino acid sequences, whether amino acid sequence I is included, and all amino acids It is possible to know which position in the array is included. The Ser residue, Tyr residue, Arg residue and Ala residue in amino acid sequence I are highly conserved in the amino acid sequences of various known wild-type halohydrin epoxidases.

  Wild-type halohydrin epoxidases are known to be roughly classified into three isozymes (A-type, B-type, and C-type) based on amino acid sequence homology and the like (J. Bacteriology, 183 (17)). , 5058-5066, 2001). The wild-type halohydrin epoxidase used in the present invention preferably belongs to the B type.

  Examples of halohydrin epoxidases (also referred to as “HheB”) classified as type B include HheB (Biosci. Biotechnol. Biochem., 58 (8), derived from Corynebacterium sp. N-1074 strain. 1451, 1994), HheB GP1 (J. Bacteriology, 183 (17), 5058-5066, 2001) derived from Mycobacterium sp. GP1 strain, Deh derived from Arthrobacter eriiii H10A strain (Enz. Microbiol. Technol., 22, 568-574, 1998). Of these, those of the genus Corynebacterium and Mycobacterium are preferred.

Among these halohydrin epoxidases, those whose amino acid sequences have been clarified are described in the GenBank database (http://www.ncbi.net) provided by the National Center for Biotechnology Information (NCBI). nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=protein), the following Accession No. It is registered by.
“Accession No. BAA14362”: amino acid sequence of HheB derived from Corynebacterium sp. N-1074 strain,
“Accession No. AAK73175”: amino acid sequence of HheB GP1 derived from Mycobacterium sp. GP1 strain.

  In the present invention, the amino acid sequence (HheB) of halohydrin epoxidase derived from the Corynebacterium sp. Strain N-1074 has two initiation codons, so that , It has been reported that there are two types of halohydrin epoxidases that differ only in the presence or absence of 8 amino acid residues near the N-terminus (Biosci. Biotechnol. Biochem., 58 (8), 1451-1457, 1994). . In the present invention, when referring to both (the above two types of halohydrin epoxidases), HheB consisting of the amino acid sequence (SEQ ID NO: 2) translated from the first initiation codon is referred to as “HheB (1st)”. HheB consisting of the amino acid sequence (SEQ ID NO: 1) translated from the second start codon (next methionine) will be referred to as “HheB (2nd)”, both of which are wild-type halohydrin epoxidases Suppose that

  In this specification, the wild-type halohydrin epoxidase HheB derived from the Corynebacterium sp. Strain N-1074 will be mainly described as an example, but as described above, the halohydrin epoxidase is described. The origin of is not limited, and even if it is a halohydrin epoxidase other than HheB, the same explanation can be applied as long as it consists of an amino acid sequence including the amino acid sequence I. In addition, any improved halohydrin epoxidase can be obtained by manipulating the position of mutation or the amino acid species or base sequence to be mutated in the present invention. In the present invention, the term “high homology” refers to, for example, 60% or more homology, preferably 75% or more, particularly preferably 90% or more.

(2) Improved halohydrin epoxidase The present inventors analyzed the crystal structure of wild-type halohydrin epoxidase consisting of the amino acid sequence shown in SEQ ID NO: 1, and based on the results, the amino acid sequence shown in SEQ ID NO: 1 Of these, the α10 amino acid residue (Ala) in the aforementioned amino acid sequence I (SEQ ID NO: 15) was found to be important for the stereoselectivity and substrate recognition of HheB, and the mutation of the improved halohydrin epoxidase in the present invention was introduced. The site was identified. In other words, the “improved halohydrin epoxidase” in the present invention mainly uses genetic recombination technology to leave an α10 amino acid residue relative to the amino acid sequence I (SEQ ID NO: 15) contained in the wild-type halohydrin epoxidase. The halohydrin epoxidase is composed of an amino acid sequence having a substitution mutation introduced in the group, and has improved stereoselectivity, product accumulation ability, and the like.

  The improved halohydrin epoxidase according to the present invention includes optical properties of the product when epihalohydrin or 4-halo-3-hydroxybutyronitrile is produced from the substrate 1,3-dihalo-2-propanol or epihalohydrin. Those having the attribute that the purity is higher than the optical purity of the product when the product is produced from the substrate by the wild-type halohydrin epoxidase. In other words, those in which the stereoselectivity with respect to the substrate 1,3-dihalo-2-propanol and / or epihalohydrin is improved as compared to the wild-type halohydrin epoxidase. As an essential cause of the improvement in stereoselectivity, any cause may be used as long as it is caused by amino acid substitution mutation.

  In the present invention, “optical activity” means a state of a substance in which one enantiomer is contained more than the other enantiomer, or a state of a substance consisting of only one of the enantiomers. say. In addition, “optical purity” is substantially equal to “enantiomeric excess (% ee)” and is defined by the following formula.

      Optical purity≈enantiomeric excess = 100 × (| [R] − [S] |) / ([R] + [S]) (% ee)

  Here, [R] and [S] indicate the respective concentrations of the enantiomers in the sample. In the present invention, the term “stereoselectivity” refers to a property that halohydrin epoxidase preferentially catalyzes a reaction that one of the enantiomers produces when producing a product from a substrate. say.

  Specifically, the improved halohydrin epoxidase of the present invention is a protein comprising an amino acid sequence in which the following amino acid mutation is introduced into the amino acid sequence of the wild-type halohydrin epoxidase including the amino acid sequence I described above. Is

  (A) An amino acid mutation in which one amino acid residue on the C-terminal side from the starting amino acid residue is substituted with Lys, and α10 residue in amino acid sequence I is substituted with another amino acid.

  In the above (a), the “starting amino acid residue” means an amino acid residue corresponding to the amino acid encoded by the translation initiation codon in the gene (DNA or mRNA) of wild-type halohydrin epoxidase. That is, it means the N-terminal amino acid residue (usually a Met residue) in the amino acid sequence of the wild-type halohydrin epoxidase after translation.

  The amino acid mutation of the protein represented by (a) above is defined when the first amino acid residue (usually a Met residue) located at the N-terminal side in the amino acid sequence of wild-type halohydrin epoxidase is defined as the first. Furthermore, it can be specified by the amino acid mutation in the amino acid residue from the starting amino acid residue toward the C-terminal side. For example, when the wild-type halohydrin epoxidase is HheB (2nd) derived from the Corynebacterium sp. Strain N-1074 consisting of the amino acid sequence shown in SEQ ID NO: 1, The “amino acid residue 1 C-terminal side from the start amino acid residue” and the “α10 residue” correspond to the “second amino acid residue (Ala residue)” and the “128th Ala residue”, respectively. To do. In addition, when the wild-type halohydrin epoxidase is HheB (1st) derived from the Corynebacterium sp. N-1074 strain consisting of the amino acid sequence shown in SEQ ID NO: 2, The “amino acid residue at the C-terminal side from the first amino acid residue” and the “α10 residue” correspond to the “second amino acid residue (Ala residue)” and the “136th Ala residue”, respectively. To do. However, HheB (2nd) is only a protein consisting of an amino acid sequence from which 8 amino acid residues near the N-terminal in the amino acid sequence of HheB (1st) are deleted. Both HheB (1st) and HheB (2nd) The absolute role of each amino acid residue in the amino acid sequence is substantially the same.

  Preferred embodiments of the improved halohydrin epoxidase of the present invention include, for example, those comprising an amino acid sequence into which the following amino acid mutation has been introduced;

  (B) One residue C-terminal amino acid residue from the starting amino acid residue is substituted with Lys, and α10 residue in amino acid sequence I is replaced with Cys, Gln, Ile, Met or Val as another amino acid. Substituted amino acid mutation.

  Here, the amino acid mutation of (b) is a specific example of the amino acid mutation of (a) described above (specifically, a specific example of “another amino acid” is shown).

  Therefore, as in the case of (a) described above, the “amino acid residue 1 residue C-terminal side from the starting amino acid residue” and the “α10 residue” in (b) above are the wild-type halohydrin epoxys, respectively. When the first amino acid residue (usually a Met residue) located in the N-terminal side of a dase amino acid sequence is defined as the first amino acid residue, the number of the amino acid residue from the starting amino acid residue toward the C-terminal side It can also be specified. That is, for example, when the wild-type halohydrin epoxidase is HheB (2nd) derived from Corynebacterium sp. N-1074 strain consisting of the amino acid sequence shown in SEQ ID NO: 1, The aspect of amino acid mutation can be specified as follows.

  (B) The second amino acid residue (Ala residue) is replaced with Lys, and the 128th amino acid residue (Ala residue) is replaced with Cys, Gln, Ile, Met or Val as another amino acid. Amino acid mutations.

  When the wild-type halohydrin epoxidase is HheB (1st) derived from the Corynebacterium sp. N-1074 strain consisting of the amino acid sequence represented by SEQ ID NO: 2, the amino acid of (b) above The mode of mutation can be specified as follows.

  (B) The second amino acid residue (Ala residue) is replaced with Lys, and the 136th amino acid residue (Ala residue) is replaced with Cys, Gln, Ile, Met or Val as another amino acid. Amino acid mutations.

  The improved halohydrin epoxidase in the present invention may be any amino acid sequence having an amino acid mutation selected from the above (a), preferably (b).

  As a more specific and preferred embodiment of the “improved halohydrin epoxidase” in the present invention, the wild-type halohydrin epoxidase has the amino acid sequence shown in SEQ ID NO: 1 (Corynebacterium sp.). In the case of HheB (2nd) derived from the N-1074 strain, an improved halohydrin epoxidase having the amino acid sequence shown in SEQ ID NOs: 16 to 20 can be mentioned. In addition, when the wild-type halohydrin epoxidase is HheB (1st) derived from Corynebacterium sp. N-1074 strain having the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NOs: 21 to 25 And an improved halohydrin epoxidase having the amino acid sequence shown in FIG.

  In the present invention, the type of amino acid may be represented by a three-letter or one-letter alphabet. Furthermore, it may be described by placing a three-letter or one-letter alphabet before or before the number, and in this case, the position of the amino acid residue and the type of amino acid before and after substitution. In other words, unless otherwise specified, as described above, when the amino acid residue (usually methionine) encoded by the translation initiation codon is defined as the first, the number indicates the number of the amino acid residue. It is shown. Also, the three-letter or one-letter alphabet displayed before the number represents the three-letter or one-letter code of the amino acid before the amino acid substitution, and the three-letter or one-letter alphabet displayed after the number causes an amino acid substitution. In this case, the three-letter or one-letter code of the amino acid after substitution is to be expressed. For example, the 128th alanine residue may be expressed as “Ala128” or “A128”, and when the 128th alanine residue is substituted with valine, it may be expressed as “Ala128Val” or “A128V”.

2. Halohydrin Epoxidase Activity In the present invention, “halohydrin epoxidase activity” means an activity of converting 1,3-dihalo-2-propanol to epihalohydrin and an activity of catalyzing the reverse reaction. 1,3-dihalo-2-propanol is a compound represented by the following general formula (1).

（式中、Ｘ_１及びＸ_２は、それぞれ独立して同一のまたは異なるハロゲン原子を表す。）

(Wherein, X ₁ and X ₂ each independently represent the same or different halogen atom)

As the halogen atom, fluorine, chlorine, bromine and iodine are preferable, and chlorine and bromine are particularly preferable. Specifically, 1,3-difluoro-2-propanol, 1,3-dichloro-2-propanol (hereinafter sometimes referred to as “DCP”), 1,3-dibromo-2-propanol, 1,3- Examples include diiodo-2-propanol, and 1,3-dichloro-2-propanol and 1,3-dibromo-2-propanol are preferable.
Epihalohydrin is a compound represented by the following general formula (2).

  As the halogen atom, fluorine, chlorine, bromine and iodine are preferable, and chlorine and bromine are particularly preferable. Specific examples thereof include epifluorohydrin, epichlorohydrin (hereinafter sometimes referred to as “ECH”), epibromohydrin, epiiodohydrin, and the like, and particularly preferably epichlorohydrin, epibromo It is a hydrin.

  In the present invention, the “halohydrin epoxidase activity” can be determined by measuring the amount of epihalohydrin produced or the amount of halide ions produced from 1,3-dihalo-2-propanol per hour. The amount of epihalohydrin produced can be quantified by, for example, liquid chromatography or gas chromatography. In addition, the amount of halide ions generated was, for example, required per hour by adding an alkaline solution continuously or intermittently so as to maintain the pH that decreases with the generation of the halide ions at a certain value. It can be conveniently determined from the amount of alkali. The halohydrin epoxidase activity calculated by this method is sometimes called “dehaloactivity”. The halohydrin epoxidase activity can also be calculated by preparing an antibody against an improved halohydrin epoxidase and using an immunological technique such as Western blot or ELISA. In addition, when it is assumed that the halohydrin epoxidase activity is proportional to the expression level in the transformant, by comparing with a sample having a known halohydrin epoxidase activity, etc., an analytical means such as SDS-PAGE can also be used. It can be obtained indirectly. SDS-PAGE can be performed by those skilled in the art using a known method.

For at least some halohydrin epoxidases, 4-halo-3-hydroxybutyronitrile is obtained by ring-opening cyanation of epihalohydrin in the presence of a cyanide compound in addition to the above-mentioned “halohydrin epoxidase activity”. Has the activity of catalyzing the reaction to produce Cyan compounds in this case include cyanide ions (CN ⁻ ) when added to a reaction solution such as hydrogen cyanide, potassium cyanide (hereinafter sometimes referred to as “KCN”), sodium cyanide, cyanic acid or acetone cyanohydrin. ) Or a compound that generates hydrogen cyanide or a solution thereof. Moreover, 4-halo-3-hydroxybutyronitrile is a compound shown by following General formula (3).

  As the halogen atom, fluorine, chlorine, bromine and iodine are preferable, and chlorine and bromine are particularly preferable. Specifically, 4-fluoro-3-hydroxybutyronitrile, 4-chloro-3-hydroxybutyronitrile (hereinafter sometimes referred to as “CHBN”), 4-bromo-3-hydroxybutyronitrile, 4 -Iodo-3-hydroxybutyronitrile and the like can be mentioned, and 4-chloro-3-hydroxybutyronitrile and 4-bromo-3-hydroxybutyronitrile are preferable.

3. Halohydrin epoxidase gene (1) Wild-type halohydrin epoxidase gene For wild-type halohydrin epoxidase whose gene sequence (base sequence) has been clarified, US Biotechnology Information Center ( NCBI; GenBank database provided by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleides). Registered by;
“Accession No. D90350”: the base sequence of a gene encoding HheB derived from Corynebacterium sp. N-1074 strain,
“Accession No. AY044094”: a base sequence of a gene encoding HheB GP1 derived from Mycobacterium sp. GP1 strain.

  In the present invention, these are referred to as “wild-type halohydrin epoxidase genes”. Here, the term “wild-type” in the wild-type halohydrin epoxidase gene is a term related to “halohydrin epoxidase”, which means that the amino acid sequence of halohydrin epoxidase is wild-type. To do. The base sequence constituting the wild-type halohydrin epoxidase gene in the present invention is not limited as long as it encodes a codon that can be used in the transformant host. The base sequence of the hydrin epoxidase gene is not limited. Note that the wild-type halohydrin epoxidase gene encoding HheB (1st) is referred to as HHEB (1st), and the gene encoding HheB (2nd) is referred to as HHEB (2nd). The base sequence of HHEB (1st) is shown in SEQ ID NO: 4, and the base sequence of HHEB (2nd) is shown in SEQ ID NO: 3.

  As a method for obtaining a wild-type halohydrin epoxidase gene whose base sequence has been clarified, genomic DNA was prepared from the source organism, and the sequence information of the wild-type halohydrin epoxidase gene that was clarified was used. And a method of amplifying a gene encoding halohydrin epoxidase by PCR using the primer. Examples of the derived organism include N-1074 strain, and this strain has the accession number “FERM BP-2643” and is designated by the National Institute of Advanced Industrial Science and Technology as a patent biological deposit center (1-1-1 Higashi Tsukuba, Ibaraki Prefecture). Deposited on the 10th of November 1988 in the center No. 6 (hereinafter the same in this specification).

  It is also possible to chemically synthesize the full length of the wild-type halohydrin epoxidase gene using a PCR method combining synthetic oligo DNA (assembly PCR) based on known gene sequence information. . For example, design a plurality of oligonucleotides that divide the wild-type halohydrin epoxidase gene into several regions (eg, about 50 bases) and overlap with adjacent regions (eg, about 20 bases) at both ends. And synthesize. The wild type halohydrin epoxidase gene can be amplified by annealing the oligonucleotides to each other by PCR.

Moreover, you may change the codon of a wild-type halohydrin epoxidase gene as needed. As means for changing the codon, for example, Molecular Cloning, A Laboratory Manual 2nd ed. , Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), etc., and site-directed mutagenesis using site-directed mutagenesis (site-directed mutagenesis) For example QuickChange ^TM Site-Directed Mutagenesis Kit ^{(Stratagene), GeneTailor TM Site-Directed Mutagenesis} System ( Invitrogen), TaKaRa Site-Directed MutagenesisSystem ( Mutan-K, Mutan-Super Express Km , etc.) (Takara Bio Ltd.)), etc. can be carried out using. In addition, as described above, the codon can be changed by using a primer with a changed codon when performing a PCR method (assembled PCR) in which a synthetic oligo DNA is combined.

(2) Improved halohydrin epoxidase gene The improved halohydrin epoxidase gene in the present invention means a gene encoding the improved halohydrin epoxidase enzyme protein. The improved halohydrin epoxidase gene of the present invention is, for example, the above-mentioned 1. against wild-type halohydrin epoxidase consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2. A gene encoding an improved halohydrin epoxidase having an amino acid sequence introduced with the amino acid mutation described in (2).

  The nucleotide sequence constituting the wild-type halohydrin epoxidase gene and the amino acid residue after introduction of the above-described amino acid mutation may be any sequence as long as it encodes a codon that can be used in the transformant host. It is not limited to the base sequence of the encoded wild type halohydrin epoxidase gene. In addition, HHEB (1st) (sequence number 4) which is the wild-type halohydrin epoxidase gene which codes HheB (1st) described in the wild-type halohydrin epoxidase, and the wild-type halo which codes HheB (2nd) Any HHEB (2nd) (SEQ ID NO: 3), which is a hydrin epoxidase gene, can be used as a wild-type halohydrin epoxidase gene as a base for obtaining an improved halohydrin epoxidase gene. .

  As a more specific and preferred embodiment of the improved halohydrin epoxidase gene of the present invention, HheB (2nd) derived from Corynebacterium sp. N-1074 strain having the amino acid sequence represented by SEQ ID NO: 1 In this case, an improved halohydrin epoxidase gene modified to encode the amino acid sequence after the mutation in the base sequence shown in SEQ ID NO: 3 can be mentioned.

  The improved halohydrin epoxidase gene in the present invention is the above-mentioned 1. Any gene may be used as long as it encodes the amino acid sequence of the improved halohydrin epoxidase introduced with the amino acid mutation described in the item (2). A gene encoding the amino acid sequence of an improved halohydrin epoxidase having the characteristics (a) and (b) described in the section (2) is also included.

  Furthermore, the improved halohydrin epoxidase gene of the present invention encodes the amino acid sequence of the improved halohydrin epoxidase into which any amino acid mutation selected from the above (a) and (b) is introduced. Also included is a DNA that hybridizes with a DNA having a base sequence complementary to the base sequence under a stringent condition and encodes a protein having a halohydrin epoxidase activity.

  Such DNA is, for example, an improved halohydrin epoxidase gene DNA consisting of a base sequence encoding an amino acid sequence selected from the above (a) and (b) or a complementary sequence thereof, or a fragment thereof as a probe. It can be obtained from a cDNA library and a genomic library by known hybridization methods such as colony hybridization, plaque hybridization and Southern blot. A library prepared by a known method can be used, and a commercially available cDNA library and genomic library can also be used.

  “Stringent conditions” are conditions at the time of washing after hybridization, wherein the salt concentration is 300 mM to 2000 mM, the temperature is 40 ° C. to 75 ° C., preferably the salt concentration is 600 mM to 900 mM, and the temperature is 65 ° C. Means. For example, conditions such as 50 ° C. can be given for 2 × SSC. Those skilled in the art will consider the improved halohydrin epoxidase in consideration of other conditions such as probe concentration, probe length, reaction time, etc. in addition to conditions such as salt concentration and temperature of the buffer. Conditions for obtaining a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence encoding the amino acid sequence can be set.

  For details of the hybridization procedure, see Molecular Cloning, A Laboratory Manual 2nd ed. (Cold Spring Harbor Laboratory Press (1989)) and the like can be referred to. The hybridizing DNA has, for example, at least 40%, preferably 60%, more preferably 90% or more identity to the base sequence encoding the amino acid sequence of the improved halohydrin epoxidase. Examples thereof include DNA containing a base sequence or a partial fragment thereof.

  In the present invention, the method for preparing the improved halohydrin epoxidase gene may be any known method for introducing a mutation, and can generally be performed by a known method. For example, using a commercially available kit based on the wild-type halohydrin epoxidase gene, a method for generating site-specific substitution, or selectively cleaving gene DNA and then removing the selected oligonucleotide The method of adding and connecting is mentioned. These site-directed mutagenesis methods are described in “Molecular Cloning, A Laboratory Manual 2nd ed.” (Cold Spring Harbor Press (1989)), “Current Protocols in Molecular Biology” (John 97, S7). . Natl. Acad. Sci. USA, 82: 488-492 (1985), Kramer and Fritz Method. Enzymol. 154: 350-367 (1987), Kunkel, Method. Enzymol. 85: 2763-2766 (1988).

  In addition, when the target mutation introduction site is present in the vicinity of a restriction enzyme site that can be easily digested and linked in the target gene sequence, PCR is performed using a primer (synthetic oligo DNA) into which the target mutation has been introduced, A gene DNA fragment into which the target mutation has been introduced can be easily obtained. Furthermore, it can also be obtained as a synthetic gene by extending by a PCR method (assembling PCR) combining synthetic oligo DNA. In addition, random methods such as a method of contacting and acting a drug that is a mutation source such as hydroxylamine or nitrous acid, a method of inducing a mutation by ultraviolet irradiation, a method of randomly introducing a mutation using PCR (polymerase chain reaction), etc. An improved halohydrin epoxidase gene can be obtained from a wild-type halohydrin epoxidase gene also by a mutation introduction method.

4). Recombinant vector, transformant (1) Recombinant vector In order to express the improved halohydrin epoxidase gene of the present invention obtained by the above method in a host, a transcription promoter is placed upstream of the gene, and a terminator is placed downstream. It can be inserted to construct an expression cassette and this cassette can be inserted into an expression vector. Alternatively, if a transcription promoter and terminator already exist in the expression vector into which the improved halohydrin epoxidase gene is introduced, the promoter and terminator in the vector are used in the meantime without constructing an expression cassette. What is necessary is just to insert a mutated gene. In order to insert the improved halohydrin epoxidase gene into a vector, a method using a restriction enzyme, a method using topoisomerase, or the like can be used. Further, if necessary at the time of insertion, an appropriate linker may be added. In the present invention, such an integration operation can also be performed in combination with an operation for preparing an improved halohydrin epoxidase gene. That is, PCR is performed using a primer having a base sequence substituted with a base sequence encoding another amino acid, a recombinant vector in which a wild-type halohydrin epoxidase gene is cloned as a template, and the obtained amplification product is used as a vector. Can be incorporated into.

  The type of promoter is not particularly limited as long as it allows appropriate expression in the host. Examples of promoters that can be used in E. coli hosts include the tryptophan operon trp promoter, the lactose operon lac promoter, Examples include PL promoters and PR promoters derived from lambda phage, and modified and designed sequences such as tac promoter and trc promoter can also be used. Examples that can be used in a Bacillus subtilis host include gluconate synthase promoter (gnt), alkaline protease promoter (apr), neutral protease promoter (npr), α-amylase promoter (amy), and the like. Examples that can be used in a Rhodococcus genus bacterial host include a promoter related to a nitrilase expression regulatory gene derived from Rhodococcus erythropolis SK92-B1 strain contained in the expression vector pSJ034. pSJ034 is a plasmid that expresses nitrile hydratase in bacteria belonging to the genus Rhodococcus, and can be prepared from pSJ023 by the method described in JP-A-10-337185. In addition, pSJ023 is the transformant ATCC12674 / pSJ023 (Accession number “FERM BP-6232”), which is an independent administrative agency, National Institute of Advanced Industrial Science and Technology (AIST 1-1, Tsukuba City East 1-1-1 Central 6). Deposited on March 4, 1997.

  The terminator is not necessarily required, and the type thereof is not particularly limited, and examples thereof include factor-independent ones such as lipoprotein terminator, trp operon terminator, rrnB terminator and the like.

  Ribosome binding sequences such as an SD sequence and a Kozak sequence are known as base sequences important for translation into amino acids, and these sequences can be inserted upstream of a mutated gene. An SD sequence may be added by a PCR method or the like when a prokaryote is used as a host, and a Kozak sequence may be added when a eukaryotic cell is used as a host. Examples of the SD sequence include sequences derived from Escherichia coli or Bacillus subtilis, but are not particularly limited as long as the sequence functions in a desired host such as Escherichia coli or Bacillus subtilis. For example, a consensus sequence in which a sequence complementary to the 3 'terminal region of 16S ribosomal RNA is continuous for 4 or more bases may be prepared by DNA synthesis and used.

  In general, a vector contains a factor (selection marker) for selecting a target transformant. Examples of selectable markers include drug resistance genes, auxotrophic complementary genes, assimilative genes, and the like, which can be selected according to the purpose and host. For example, drug resistance genes used as selection markers in E. coli include ampicillin resistance gene, kanamycin gene, dihydrofolate reductase gene, neomycin resistance gene and the like.

  The vector used in the present invention is not particularly limited as long as it retains the above mutant gene, and a vector suitable for each host can be used. Examples of the vector include plasmid DNA, bacteriophage DNA, retrotransposon DNA, artificial chromosome DNA and the like. For example, when Escherichia coli is used as a host, pTrc99A having a region capable of autonomous replication in Escherichia coli (Centralalbueau floor Schemes (CBS), Netherlands; http://www.cbs.knaw.nl/), pUC19 (Takara) Bio, Japan), pKK233-2 (Centralalureau voor Schulmelculures (CBS), Netherlands; http://www.cbs.knaw.nl/), pET-12 (Novagen, Germany), pET-26b (Novagen, Germany) ) Etc. can be used. Moreover, what modified these vectors as needed can also be used. An expression vector having high expression efficiency, for example, an expression vector pTrc99A or pKK233-2 having a trc promoter or a lac operator can also be used.

  Recombinant vectors containing the improved halohydrin epoxidase gene are within the scope of the present invention. Specific examples of the recombinant vector containing the improved halohydrin epoxidase gene include pSTTT002, pSTT156, pSTT160, pSTT161, pSTT165, and pSTT167.

(2) Transformant A transformant or a transductant (hereinafter collectively referred to as “transformant”) is produced by transforming or transducing the recombinant vector of the present invention into a host. Such transformants are also included in the scope of the present invention.

  The host used in the present invention is not particularly limited as long as the desired improved halohydrin epoxidase can be expressed after the introduction of the above recombinant vector. Examples of the host include bacteria such as Escherichia coli, Bacillus subtilis, Rhodococcus bacteria, yeast (Pichia, Saccharomyces), mold (Aspergillus), animal cells, insect cells, plant cells, and the like.

  When bacteria are used as hosts, Escherichia coli and Rhodococcus bacteria can be used as preferred hosts in the present invention. Examples of Escherichia coli include Escherichia coli K12 strain and B strain, or JM109 strain, XL1-Blue strain, C600 strain and the like derived from these wild strains. In particular, when the lac promoter of the lactose operon as described above and its derived promoter are used as expression promoters, expression becomes inducible when a host having a lacI repressor gene is used (induced by IPTG or the like), and the lacI repressor gene is present. If a non-host is used, the expression becomes a constitutive type, so that a host as required can be used. These strains are easily available from, for example, American Type Culture Collection (ATCC). Examples of Bacillus subtilis include Bacillus subtilis. Examples of Rhodococcus bacteria include, for example, Rhodococcus rhodochrous ATCC12274, Rhodococcus rhodochrous J1 strain (Accession No. “FERMocro Rhodocus” Is mentioned. Preferred examples include Rhodococcus rhodochrous J1 strain (Accession number “FERM BP-1478”). The ATCC strain is from the American Type Culture Collection, the IFO strain is from the National Institute of Product Evaluation Technology Biotechnology Headquarters, Biogenetic Resources Division (NBRC), and the FERM strain is the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit. Each is available from the center.

  The method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. Examples thereof include a method using calcium ions and an electroporation method. When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris and the like are used. The method for introducing a recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast, and examples thereof include an electroporation method, a spheroplast method, and a lithium acetate method. When animal cells are used as hosts, monkey cells COS-7, Vero, CHO cells, mouse L cells, rat GH3, human FL cells and the like are used. Examples of methods for introducing a recombinant vector into animal cells include an electroporation method, a calcium phosphate method, and a lipofection method. When insect cells are used as hosts, Sf9 cells, Sf21 cells and the like are used. As a method for introducing a recombinant vector into insect cells, for example, calcium phosphate method, lipofection method, electroporation method and the like are used. When a plant cell is used as a host, tobacco BY-2 cells and the like can be mentioned, but are not limited thereto. As a method for introducing a recombinant vector into a plant cell, for example, an Agrobacterium method, a particle gun method, a PEG method, an electroporation method, or the like is used.

  A transformant obtained by the recombinant vector containing the improved halohydrin epoxidase gene is included in the scope of the present invention. Specific examples of the transformant by the recombinant vector containing the improved halohydrin epoxidase gene include, for example, JM109 / pSTT002, JM109 / pSTT156, JM109 / pSTT160, JM109 / pSTT161, JM109 / pSTT165, JM109 / pSTT167, etc. are mentioned.

5. Process for producing improved halohydrin epoxidase In the present invention, the improved halohydrin epoxidase can be produced as a culture itself obtained by culturing the above transformant or by harvesting from the culture. it can. In the present invention, the term “culture” refers to a culture solution, a culture supernatant, cells or cells, cell or cell suspensions, cell or cell disruptions, crude enzyme solutions, and processed products thereof. In addition, it is meant to include both those obtained by culturing a transformant producing an improved halohydrin epoxidase and those resulting therefrom. A culture obtained by culturing the transformant of the present invention is included in the scope of the present invention. The method for culturing the transformant of the present invention can be carried out according to a usual method used for culturing a host. The desired improved halohydrin epoxidase accumulates in any of the cultures.

  The medium for culturing the transformant of the present invention contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the host, and can be a natural medium as long as the transformant can be cultured efficiently. Any of synthetic media may be used. Examples of the carbon source include carbohydrates such as glucose, galactose, fructose, sucrose, raffinose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol. Examples of the nitrogen source include ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, or other nitrogen-containing compounds. In addition, peptone, yeast extract, meat extract, corn steep liquor, various amino acids, and the like may be used. Examples of inorganic substances include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, zinc sulfate, copper sulfate, and calcium carbonate. Moreover, you may add an antifoamer in order to prevent foaming during culture | cultivation as needed. Moreover, you may add a vitamin etc. suitably as needed. During culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary.

  During the culture, the vector and the target gene may be cultured under selective pressure to prevent the loss of the vector and the target gene. That is, a drug corresponding to the case where the selection marker is a drug resistance gene may be added to the medium, and the nutrient factor corresponding to the case where the selection marker is an auxotrophic complementary gene may be removed from the medium. When the selectable marker is an assimilation gene, a corresponding assimilation factor can be added as a sole factor as necessary. For example, when culturing E. coli transformed with a vector containing an ampicillin resistance gene, ampicillin may be added to the medium as needed during the culture.

  When cultivating a transformant transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when a transformant transformed with an expression vector having a promoter inducible with isopropyl-β-D-thiogalactoside (IPTG) is cultured, IPTG or the like can be added to the medium.

  The culture conditions for the transformant are not particularly limited as long as the productivity of the desired improved halohydrin epoxidase and the growth of the host are not hindered. Usually, the culture temperature is 10 ° C to 45 ° C. C., preferably 10 to 40.degree. C., more preferably 15 to 40.degree. C., and even more preferably 20 to 37.degree. C., and the temperature may be changed during the cultivation as necessary. The culture time is 5 hours to 120 hours, preferably 5 hours to 100 hours, more preferably 10 hours to 100 hours, and even more preferably about 15 hours to 80 hours. The pH is adjusted using an inorganic or organic acid, an alkaline solution or the like, and is usually adjusted to 6 to 9 for E. coli. Examples of the culture method include solid culture, stationary culture, shaking culture, and aeration and agitation culture.

  In particular, when culturing E. coli transformants, it is preferable to culture under aerobic conditions by shaking culture or aeration and agitation culture (jar fermenter). In this case, the culture may be performed by a usual solid culture method. However, it is preferable to employ the liquid culture method as much as possible. Examples of the culture medium used for the culture include one or more nitrogen sources such as yeast extract, tryptone, polypeptone, corn steep liquor, and soybean or wheat bran leachate, sodium chloride, monopotassium phosphate, secondary phosphate. One or more inorganic salts such as potassium, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate or manganese sulfate are added, and if necessary, saccharide raw materials, vitamins and the like are appropriately added. The initial pH of the medium is suitably adjusted to 7-9. The culture is performed at 5 ° C to 40 ° C, preferably 10 ° C to 37 ° C for 5 hours to 100 hours. It is preferably carried out by aeration and agitation deep culture, shaking culture, static culture, fed-batch culture or the like. In particular, when producing improved halohydrin epoxidase on an industrial scale, aeration and agitation culture can be used. Furthermore, the operation method of the aeration and agitation culture is not limited and can be selected based on the common general technical knowledge in this field.

Examples of a medium for culturing a transformant obtained using animal cells as a host include generally used RPMI 1640 medium, DMEM medium, or a medium obtained by adding fetal calf serum or the like to these mediums. The culture is usually performed at 37 ° C. for 1 to 30 days in the presence of 5% CO ₂ . During culture, antibiotics such as kanamycin and penicillin may be added to the medium as necessary.

  When the transformed (introduced) body is a plant cell or a plant tissue, the culture can be performed by using a normal plant culture medium, such as an MS basic medium or an LS basic medium. As a culture method, any of a normal solid culture method and a liquid culture method can be employed.

  When cultured under the above culture conditions, the improved halohydrin epoxidase of the present invention is contained in the culture, that is, at least one of the culture solution, the culture supernatant, the cells, the fungus bodies, or the disrupted cells or fungus bodies. Can be accumulated.

  When improved halohydrin epoxidase is produced in cells or cells after culturing, the cells or cells can be used as a catalyst for substance production, or the cells or cells can be disrupted. Thus, the desired improved halohydrin epoxidase can be collected. In either case, if necessary, the medium can be removed and washed by solid-liquid separation operations such as centrifugation and membrane filtration. The method of solid-liquid separation operation is not limited, and can be selected based on the common general technical knowledge in this field.

  After performing the above-mentioned medium removal and washing operations, the bacterial cells or cells can be suspended again in water or, if necessary, in a buffer solution or an isotonic solution to prepare a bacterial cell or cell suspension. The bacterial cell or cell suspension can be used as a catalyst in substance production as it is, or can be used after treatment with a surfactant or the like, if necessary. The bacteria treated with the surfactant may be used after washing with a buffer solution or may be used as it is without washing.

  Moreover, it is also possible to directly perform the treatment with a fungus body or cell surfactant, etc., without performing the above-mentioned medium removal and washing operations.

  In addition, the cells or cells can be immobilized and used. Specific examples include those in which cells or cells after culturing are included in a gel such as acrylamide, or those supported on an inorganic carrier such as alumina, silica, zeolite, or diatomaceous earth.

  On the other hand, the desired improved halohydrin epoxidase can also be collected by disrupting the cells or cells. As a method for disrupting cells or cells, ultrasonic treatment, high-pressure treatment using a French press or homogenizer, grinding treatment using a bead mill, collision treatment using an impact crushing device, enzyme treatment using lysozyme, cellulase, pectinase, etc., freeze-thawing treatment, Examples include hypotonic solution treatment, lysis induction treatment with phage, and the like, and any of these methods can be used alone or in combination as necessary.

  When it is necessary to remove bacterial cells or cell disruption residues from the resulting disrupted solution, for example, centrifugation or filtration (dead-end method or cross-flow method), etc. It can be removed by selection and implementation.

  The supernatant obtained after removing the residue is a cell extract soluble fraction and can be a crude enzyme solution containing an improved halohydrin epoxidase. Thereafter, if necessary, general biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, various chromatographies (eg gel filtration chromatography (eg Sephadex column), ion exchange chromatography (eg DEAE-Toyopearl), By using affinity chromatography, hydrophobic chromatography (for example, butytopearl), anion chromatography (for example, MonoQ column), SDS polyacrylamide gel electrophoresis, etc. alone or in appropriate combination, halohydrin epoxy can be used in the culture. The dase can be isolated and purified.

  The transformant of the present invention is a gene recombinant, and it causes secondary microbial contamination due to leakage of the transformant into the environment in the production process, mixing into the product, or handling after use. In case of fear of possibility, inactivation processing can be performed as necessary. The inactivation method may be any method as long as it does not reduce the improved halohydrin epoxidase activity or the activity recovery rate and can inactivate the transformant. For example, heat treatment, cell disruption treatment, etc. Further, methods such as drug treatment can be used alone or in combination.

  On the other hand, when the improved halohydrin epoxidase is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or filtration as described above. Thereafter, if necessary, the improved halohydrin epoxidase can be purified, for example, by using a general biochemical method used for protein isolation and purification as described above alone or in appropriate combination.

  When the transformant is a plant cell or plant tissue, the cell is destroyed by cell lysis treatment using an enzyme such as cellulase or pectinase, ultrasonic disruption treatment, grinding treatment, or the like. Thereafter, if necessary, for example, as described above, halohydrin epoxidase is isolated and purified from the culture by using a general biochemical method used for protein isolation and purification alone or in combination as appropriate. can do. The isolated halohydrin epoxidase can be retained on a suitable carrier and used as an immobilized enzyme in the same manner as the above-described cells or cells.

  The culture and improved halohydrin epoxidase obtained as described above are included in the scope of the present invention. The production yield of the obtained culture and improved halohydrin epoxidase is determined by measuring the halohydrin epoxidase activity, per culture apparatus, per culture solution, per wet (dry) cell weight (transformant), It can be determined by calculating the activity per unit protein weight in the enzyme solution.

In the present invention, the improved halohydrin epoxidase can also be collected from a recombinant vector containing the improved halohydrin epoxidase gene or the improved halohydrin epoxidase gene. That is, in the present invention, it is possible to produce an improved halohydrin epoxidase by employing a cell-free protein synthesis system without using any living cells. The cell-free protein synthesis system is a system that synthesizes a protein in an artificial container such as a test tube using a cell extract. The cell-free protein synthesis system used in the present invention includes a cell-free transcription system that synthesizes RNA using DNA as a template. In this case, the organism corresponding to the host corresponds to the organism from which the following cell extract is derived. Here, eukaryotic cell-derived or prokaryotic cell-derived extracts such as wheat germ, Escherichia coli and the like can be used as the cell extract. Note that these cell extracts may be concentrated or not concentrated. The cell extract can be obtained by, for example, ultrafiltration, dialysis, polyethylene glycol (PEG) precipitation or the like. Furthermore, in the present invention, cell-free protein synthesis can also be performed using a commercially available kit. Examples of such kits include reagent kits PROTEIOS ^™ (Toyobo), TNT ^™ System (Promega), synthesizer PG-Mate ^™ (Toyobo), RTS (Roche Diagnostics) and the like.

  The improved halohydrin epoxidase obtained by cell-free protein synthesis as described above can be purified, for example, by appropriately selecting chromatography as described above.

6). Process for Producing Epihalohydrin and 4-Halo-3-hydroxybutyronitrile The improved halohydrin epoxidase produced as described above can be used for substance production as an enzyme catalyst. That is, it can use for reaction shown to the following (1)-(3).

(1) Conversion of 1,3-dihalo-2-propanol to epihalohydrin In this conversion reaction, 1,3-dihalo-2-propanol was obtained by the improved halohydrin epoxidase and / or the culture described above. Can be carried out by contacting with the culture to be produced. “Contact” includes the presence of 1,3-dihalo-2-propanol and the improved halohydrin epoxidase and / or culture in the same reaction system or culture system. Specifically, 1,3-dihalo-2-propanol is added to the cell culture vessel, and the improved halohydrin epoxidase and / or the culture is mixed with 1,3-dihalo-2-propanol. Or culturing the cells in the presence of 1,3-dihalo-2-propanol.

  1,3-Dihalo-2-propanol as a substrate is a compound represented by the general formula (1) described above. As the halogen atom, fluorine, chlorine, bromine and iodine are preferable, and chlorine and bromine are particularly preferable. Specific examples include 1,3-difluoro-2-propanol, 1,3-dichloro-2-propanol, 1,3-dibromo-2-propanol, 1,3-diiodo-2-propanol, and preferably 1,3-dichloro-2-propanol and 1,3-dibromo-2-propanol.

  The substrate concentration in the conversion reaction solution is preferably 0.01% to 15 (W / V)%. Within this range, it is preferable from the viewpoint of enzyme stability, and 0.01% to 10% is particularly preferable. The substrate can be added to the reaction solution all at once or in divided portions. It is desirable from the viewpoint of accumulation to make the substrate concentration constant by divided addition.

  As a solvent for the reaction solution, water or a buffer solution having an enzyme activity near the optimum pH of 4 to 10 is preferable. As the buffer solution, for example, a buffer solution composed of a salt such as phosphoric acid, boric acid, citric acid, glutaric acid, malic acid, malonic acid, o-phthalic acid, succinic acid or acetic acid, Tris buffer solution or Good A buffer solution or the like is preferable.

  The reaction temperature is preferably 5 ° C to 50 ° C, and the reaction pH is preferably 4 to 10. The reaction temperature is more preferably 10 ° C to 40 ° C. The reaction pH is more preferably pH 6-9. The reaction time is selected as appropriate depending on the concentration of the substrate, the bacterial cell concentration, other reaction conditions, and the like, but it is preferable to set the conditions so that the reaction is completed in 1 hour to 120 hours. In this reaction, the optical purity can be further improved by removing halide ions generated as the reaction proceeds from the reaction system. This removal of halide ions is preferably carried out by adding silver nitrate or the like.

  Epihalohydrin produced and accumulated in the reaction solution can be collected and purified using a known method. For example, an epihalohydrin syrup can be obtained by performing extraction with a solvent such as ethyl acetate and removing the solvent under reduced pressure. Further, these syrups can be further purified by distillation under reduced pressure.

(2) Conversion of 1,3-dihalo-2-propanol to 4-halo-3-hydroxybutyronitrile In this conversion reaction, 1,3-dihalo-2-propanol was converted into the above-mentioned improved halohydrin epoxy. It can be carried out by contacting with a culture obtained by the above-mentioned culture with a dase and / or.

1,3-Dihalo-2-propanol as a substrate is a compound represented by the above general formula (1), preferably 1,3-dichloro-2-propanol, 1,3-dibromo-2-propanol, etc. It is. Further, as the cyanide compound, a compound that generates cyanide ion (CN ⁻ ) or hydrogen cyanide or a solution thereof when added to a reaction solution such as hydrogen cyanide, potassium cyanide, sodium cyanide, cyanic acid, or acetone cyanohydrin is used. it can. The substrate concentration in the reaction solution is preferably 0.01% to 15 (W / V)%, particularly preferably 0.01% to 10%, from the viewpoint of enzyme stability. The amount of cyanide used is preferably 1 to 3 times (mole) the substrate from the viewpoint of enzyme stability. The reaction conditions are as described in 6. above. It can be performed in the same manner as (1).

  The 4-halo-3-hydroxybutyronitrile produced and accumulated in the reaction solution can be collected and purified using a known method. For example, after removing cells from the reaction solution using a method such as centrifugation, extraction with a solvent such as ethyl acetate is performed, and the solvent is removed under reduced pressure to remove 4-halo-3-hydroxybutyronitrile. You can get syrup. Further, these syrups can be further purified by distillation under reduced pressure.

(3) Conversion of epihalohydrin to 4-halo-3-hydroxybutyronitrile In this conversion reaction, epihalohydrin is contacted with the above-described improved halohydrin epoxidase and / or the culture obtained by the above-mentioned culture. To do.

Epihalohydrin as a substrate is a compound represented by the general formula (2) described above. As the halogen atom, fluorine, chlorine, bromine and iodine are preferable, and chlorine and bromine are particularly preferable. Specific examples include epifluorohydrin, epichlorohydrin, epibromohydrin, epiiodohydrin, and the like, with epichlorohydrin and epibromohydrin being particularly preferred. As the cyanide compound, a compound that generates cyanide ion (CN ⁻ ) or hydrogen cyanide or a solution thereof when added to a reaction solution such as hydrogen cyanide, potassium cyanide, sodium cyanide, cyanic acid or acetone cyanohydrin can be used. Reaction conditions, collection and purification methods are the same as those described in 6. above. It can be performed in the same manner as (2).

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Construction of improved halohydrin epoxidase expression vector by site-directed mutagenesis

  In order to obtain a useful halohydrin epoxidase, a set that expresses a halohydrin epoxidase gene in which a mutation is introduced into the 128th amino acid residue (alanine residue) in SEQ ID NO: 1 by site-directed mutagenesis A replacement vector was prepared.

  In the halohydrin epoxidase HheB (2nd), a gene encoding a protein in which the second amino acid residue (alanine) is replaced with lysine is obtained by PCR using the plasmid pSTT002, which has been cloned on the expression vector pTrc99A, as a template. Site-directed mutagenesis into the halohydrin epoxidase gene was performed as follows. 50 μl of each PCR reaction solution having the composition shown in the following table was prepared.

プライマーとして用いたオリゴヌクレオチドの配列は以下の表の通りである。

The sequences of oligonucleotides used as primers are shown in the following table.

ＰＣＲ反応用チューブに調製した５０μｌのＰＣＲ反応液を、以下の熱サイクル処理に供した。

50 μl of the PCR reaction solution prepared in the PCR reaction tube was subjected to the following thermal cycle treatment.

得られたＰＣＲ反応液を組換ベクター溶液とし、形質転換に供した。

The obtained PCR reaction solution was used as a recombinant vector solution and subjected to transformation.

Confirmation of sequence of improved halohydrin epoxidase gene introduced with site-specific mutation and preparation of transformant Preliminarily prepared by the method described later for each 2 μl of PCR reaction solution subjected to heat cycle treatment in Example 1 200 μl of previously prepared E. coli JM109 strain competent cell was added and left at 0 ° C. for 30 minutes. Subsequently, the competent cell was subjected to a heat shock at 42 ° C. for 45 seconds and cooled at 0 ° C. for 2 minutes. Then, 1 ml of SOC medium (20 mM glucose, 2% bactotryptone, 0.5% bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 1 mM MgSO ₄ , 1 mM MgCl ₂ ) was added and shaken at 37 ° C. for 1 hour. Cultured. After culture, 200 μl of the culture solution was applied to an LB Amp agar medium (LB medium containing ampicillin 100 mg / l, agar 2%) and cultured at 37 ° C. overnight. Colonies that appeared on each agar medium were collected with a sterile toothpick, inoculated into 2 ml of LB Amp liquid medium, and cultured at 37 ° C. overnight. From each of the obtained culture solutions, the plasmid was recovered using PureYield ^™ Miniprep System (Promega). The nucleotide sequence of the halohydrin epoxidase gene in each plasmid was analyzed using a capillary DNA sequencer CEQ2000 (Beckman Coulter) according to the attached manual. Colonies in which mutation introduction was confirmed were used as transformants possessing each improved halohydrin epoxidase. Each plasmid and transformant were named as shown in Table 4 below.

Preparation of competent cells of E. coli JM109 was performed as follows.
E. coli strain JM109 was inoculated into 1 ml of LB medium (1% bactotryptone, 0.5% bacto yeast extract, 1% NaCl) and cultured aerobically at 37 ° C. for 5 hours to obtain a preculture solution. 0.4 ml of the preculture solution is added to 40 ml of SOB medium (2% bactotryptone, 0.5% bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 1 mM MgSO ₄ , 1 mM MgCl ₂ ), and then at 18 ° C. for 20 hours. The main culture solution was obtained by culturing. The main culture was collected by centrifugation (3700 × g, 10 minutes, 4 ° C.), and then a cold TF solution (20 mM PIPES-KOH (pH 6.0), 200 mM KCl, 10 mM CaCl ₂ , 40 mM MnCl ₂ ) was added. 13 ml was added, allowed to stand at 0 ° C. for 10 minutes, centrifuged again (3700 × g, 10 minutes, 4 ° C.) to remove the supernatant, and Escherichia coli cells were obtained. The Escherichia coli cells were suspended in 3.2 ml of a cold TF solution, and 0.22 ml of dimethyl sulfoxide was added to obtain a cell suspension. The bacterial cell suspension was allowed to stand at 0 ° C. for 10 minutes, then frozen using liquid nitrogen, and stored at −80 ° C. to obtain an E. coli JM109 strain competent cell.

(R) -CHBN synthesis reaction with improved halohydrin epoxidase (1) Preparation of improved halohydrin epoxidase crude enzyme solution Five strains (JM109) in which the halohydrin epoxidase gene mutation was confirmed in Example 2 / PSTT156, JM109 / pSTT160, JM109 / pSTT161, JM109 / pSTT165, JM109 / pSTT167) and the control JM109 / pSTT002 total 6 strains in order to examine the optical purity of (R) -CHBN synthesized using each strain First, a crude enzyme solution was prepared from a transformant expressing each improved halohydrin epoxidase. The LB Amp medium was dispensed into each Wasselman test tube in an amount of 1 ml, and 10 secondary selection strains and JM109 / pSTT002 colonies as controls were inoculated into each test tube. After culturing at 37 ° C. and 210 rpm for about 8 hours, 100 μl of each culture solution obtained was inoculated into LB Amp medium (100 ml in a 500 ml Erlenmeyer flask) containing 1 mM IPTG as the final concentration, and at 37 ° C. and 210 rpm. Cultured for 16 hours. Each bacterial cell was collected from 100 ml of each obtained culture solution by centrifugation (3700 × g, 10 minutes, 4 ° C.), washed with 20 mM Tris-sulfate buffer (pH 8.0), and then the bacterial concentration was 12. 5 gD. C. / L was suspended in the same buffer. While cooling 3 ml of the obtained bacterial cell suspension using an ultrasonic crusher VP-15S (Tytec, Japan) under the conditions of output control 4, DUTY CYCLE 40%, PULS, TIMER = B mode 10 s. It was crushed for 10 minutes. The disrupted cell suspension was subjected to centrifugation (12000 rpm, 10 minutes, 4 ° C.), and the supernatant was collected as a crude enzyme solution. Each of the obtained crude enzyme solutions was 6.25 gD. As a bacterial concentration (at the time of cell suspension) before crushing. C. / L was diluted with 20 mM Tris-sulfate buffer (pH 8.0), and halohydrin epoxidase activity (dechlorination activity) was measured by the following method using this crude enzyme solution. 100 ml of a reaction solution for activity measurement (50 mM DCP, 20 mM Tris-sulfate buffer (pH 8.0)) was prepared, and the temperature was adjusted to 20 ° C. The crude enzyme solution was added to the reaction solution to start the reaction. The decrease in pH accompanying the release of chloride ions due to the halohydrin epoxidase activity was continuously adjusted to maintain the pH at 8 by adding 0.01 N aqueous sodium hydroxide solution using an automatic pH controller. From the amount of 0.01N aqueous sodium hydroxide solution added to maintain the pH at 8 during the 10-minute reaction, the amount of chloride ion produced was calculated, and the halohydrin epoxidase activity (dechlorination activity) was calculated. ) (U) was calculated. 1 U was defined as the amount of enzyme desorbing 1 μmol chloride ion per minute from DCP under the above conditions, and the activity of each crude enzyme solution used for activity measurement and the activity was used for activity measurement. The liquid activity of each crude enzyme solution was calculated by dividing by the amount of each crude enzyme solution.

(2) Analytical method DCP, ECH and CHBN concentration analysis and optical purity analysis of the produced CHBN in the reaction solution carried out in the synthesis reaction of (R) -CHBN by the improved halohydrin epoxidase described below are as follows. went.

<Concentration analysis of DCP, ECH and CHBN in reaction solution>
DCP, ECH, and CHBN concentration analysis in the reaction solution was performed by reverse phase HPLC. The reverse phase HPLC analysis conditions are shown in Table 5.

  After 100 μl of the reaction finished solution was diluted and mixed with 5 ml of the mobile phase described in the above table, analysis was performed under the analysis conditions described in the above table. A calibration curve was prepared in advance using DCP, ECH, and CHBN solutions with known concentrations, and the concentrations of DCP, ECH, and CHBN in the reaction solution were determined using the calibration curve.

<Optical purity analysis of produced CHBN>
The optical purity analysis of the produced CHBN was performed by normal phase HPLC after esterification of CHBN. Normal phase HPLC analysis conditions are shown in Table 6.

  Extraction was carried out by adding an equal amount of ethyl acetate to about 400 μl of the reaction end solution. The ethyl acetate phase was separated, and a small amount of anhydrous magnesium sulfate was added and stirred. A portion of the ethyl acetate phase was dispensed into a flask, set on a rotary evaporator (Tokyo Rika), and concentrated at 50 ° C., 250 torr for 60 minutes. 20 μl of dichloromethane and 20 μl of pyridine, and 20 μl of (+)-α-methoxy-α- (trifluoromethyl) phenylacetyl chloride (hereinafter sometimes referred to as (+)-MTPA) were added to the residue. After reacting overnight at room temperature, 400 μl of diisopropyl ether (hereinafter sometimes referred to as IPE) was added, and 1N hydrochloric acid was added for extraction. The IPE phase was separated and extracted by adding 400 μl of a saturated aqueous sodium hydrogen carbonate solution. The IPE phase was fractionated into a 1.5 ml tube, and the IPE phase was volatilized by an aspirator. The residue was suspended in a mixed solution of n-hexane: 2-propanol = 4: 1, and then analyzed under the analysis conditions described in Table 5. Each concentration was calculated from the area area ratio of (R) -CHBN-(+)-MTPA ester and (S) -CHBN-(+)-MTPA ester, and the CHBN optical properties were as described above (described above). Purity was calculated.

(3) Synthesis of (R) -CHBN from DCP by improved halohydrin epoxidase Using each improved halohydrin epoxidase-expressing crude enzyme solution prepared in Example 3 (1), potassium cyanide In the presence, a CHBN synthesis reaction from DCP or ECH was performed. The basic composition of the reaction solution was as shown in the following table, and the reaction scale was 0.5 ml.

  The reaction was carried out at 20 ° C. for 3 hours. After completion of the reaction, the concentrations of DCP, ECH and CHBN in the reaction solution and the optical purity of the produced CHBN were analyzed under the analysis conditions described in Example 3 (2). The results are shown in Table 8.

  The optical purity of (R) -CHBN synthesized using a crude enzyme solution derived from the transformant strain JM109 / pSTT002 as a control is 91.7% e.e. e. Met. On the other hand, it was synthesized using a crude enzyme solution derived from four transformant strains (JM109 / pSTT156, JM109 / pSTT160, JM109 / pSTT161, JM109 / pSTT167) expressing an improved halohydrin epoxidase (R ) -CHBN has an optical purity of 92.8 to 99.9% e.e. e. It was confirmed that the optical purity was improved. Therefore, it can be said that these improved halohydrin epoxidases have improved stereoselectivity for DCP and / or intermediate ECH.

(4) Synthesis of (R) -CHBN from ECH by improved halohydrin epoxidase Using each crude halohydrin epoxidase-expressing transformant-derived crude enzyme solution prepared in Example 3 (1), potassium cyanide In the presence, a CHBN synthesis reaction from ECH was performed. The basic composition of the reaction solution was as shown in the following table, and the reaction scale was 0.5 ml.

  The reaction was carried out at 20 ° C. for 3 hours. After completion of the reaction, the concentrations of DCP, ECH and CHBN in the reaction solution and the optical purity of the produced CHBN were analyzed under the analysis conditions described in Example 3 (2). The results are shown in Table 10.

  The optical purity of (R) -CHBN synthesized using a crude enzyme solution derived from the control transformant strain JM109 / pSTT002 is 78.9% e.e. e. Met. On the other hand, it was synthesized using a crude enzyme solution derived from four kinds of transformant strains (JM109 / pSTT156, JM109 / pSTT160, JM109 / pSTT161, JM109 / pSTT165) expressing an improved halohydrin epoxidase (R ) -CHBN has an optical purity of 79.9-93.6% e.e. e. It was confirmed that the optical purity was improved. Therefore, it can be said that these improved halohydrin epoxidases have improved stereoselectivity for ECH.

  The improved halohydrin epoxidase of the present invention is excellent in stereoselectivity, and an optically active epihalohydrin or 4-halo-3-hydroxybutyronitrile can be efficiently produced by using the enzyme of the present invention. Can do.

  N-1074 strain: under the accession number “FERM BP-2643”, National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (Tsukuba-shi, Ibaraki, 6th, 1-1-1 Chuo 6th November, 1988) Deposited by date.

pSJ023: Transformant ATCC12674 / pSJ023 (Accession number “FERM BP-6232”), which was established in 1997 by the National Institute of Advanced Industrial Science and Technology (National Center for Biological Biology, 1-1-1 Higashi 1-1-1, Tsukuba, Ibaraki) ) Deposited on March 4th of the year.
Rhodococcus rhodochrous J1 strain (Accession number “FERM BP-1478”)
[Explanation of Sequence Listing]
SEQ ID NO: 1: Amino acid sequence of HheB (2nd) derived from Corynebacterium sp. N-1074 strain SEQ ID NO: 2: HheB (1st) derived from Corynebacterium sp. N-1074 strain Amino acid sequence SEQ ID NO: 3: Base sequence of HheB (2nd) derived from Corynebacterium sp. N-1074 strain SEQ ID NO: 4: HheB (1st) derived from Corynebacterium sp. N-1074 strain ) Base sequence SEQ ID NO: 5: primer DH-75
Sequence number 6: Primer DH-76
Sequence number 7: Primer DH-83
Sequence number 8: Primer DH-84
Sequence number 9: Primer DH-85
Sequence number 10: Primer DH-86
Sequence number 11: Primer DH-93
Sequence number 12: Primer DH-94
SEQ ID NO: 13: primer DH-97
Sequence number 14: Primer DH-98
SEQ ID NO: 15: amino acid sequence I
SEQ ID NO: 16: Mutant peptide SEQ ID NO: 17: Mutant peptide SEQ ID NO: 18: Mutant peptide SEQ ID NO: 19: Mutant peptide SEQ ID NO: 20: Mutant peptide SEQ ID NO: 21: Mutant peptide SEQ ID NO: 22: Mutant peptide SEQ ID NO: 23: Mutant peptide sequence number 24: Mutant peptide SEQ ID NO: 25: Mutant peptide

Claims (10)

The following amino acid sequence I:
S-α1-α2-α3-α4-α5-α6-α7-α8-α9-α10-α11-α12-Y-α13-α14-A-R-α15 (SEQ ID NO: 15)
(S represents a Ser residue, Y represents a Tyr residue, A represents an Ala residue, R represents an Arg residue, and α1 to α15 each independently represent an identical or different arbitrary amino acid residue.)
In the amino acid sequence of the wild-type halohydrin epoxidase containing the amino acid residue, one residue C-terminal from the starting amino acid residue was substituted with Lys, and the α10 residue was substituted with another amino acid. An improved halohydrin epoxidase consisting of an amino acid sequence into which an amino acid mutation has been introduced. The improved halohydrin epoxidase according to claim 1, wherein the amino acid mutation at the α10 residue is Cys, Gln, Ile, Met or Val. The improved halohydrin epoxidase according to claim 1 or 2, wherein the wild-type halohydrin epoxidase is classified as type B. The improved halohydrin epoxidase according to any one of claims 1 to 3, wherein the wild type halohydrin epoxidase is derived from a bacterium belonging to the genus Corynebacterium or Mycobacterium. . A gene encoding the improved halohydrin epoxidase according to any one of claims 1 to 4. A recombinant vector comprising the gene according to claim 5. A transformant or a transductant obtained by introducing the recombinant vector according to claim 6 into a host. A culture obtained by culturing the transformant or transductant according to claim 7. An epihalohydrin is produced by contacting the improved halohydrin epoxidase according to any one of claims 1 to 4 and / or the culture according to claim 8 with 1,3-dihalo-2-propanol. A process for producing epihalohydrin, characterized in that The improved halohydrin epoxidase according to any one of claims 1 to 4 and / or the culture according to claim 8 is contacted with 1,3-dihalo-2-propanol or epihalohydrin in the presence of a cyanide compound. To produce 4-halo-3-hydroxybutyronitrile, thereby producing 4-halo-3-hydroxybutyronitrile.

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