JP3026367B2 - Method for producing 3-hydroxynitrile compound by a microorganism transformed with a recombinant plasmid having halohydrin epoxidase gene - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to 1,3-dihalo-2-
A transformant obtained by introducing into a host microorganism a recombinant plasmid obtained by linking a gene plasmid to an enzyme having the activity of converting propanol to epihalohydrin and the activity of catalyzing the reverse reaction (hereinafter abbreviated as halohydrin epoxidase) into a vector plasmid. , 3-hydroxynitrile compounds. A 3-hydroxynitrile compound is known as a useful substance as a raw material for synthesizing various pharmaceuticals and physiologically active substances.

[0002]

BACKGROUND OF THE INVENTION A method for producing 4-halo-3-hydroxybutyronitrile from epihalohydrin by the action of a dehalogenase has been previously found by some of the present inventors (Japanese Patent Application No. Hei 10-284). 1-118592).
However, these microorganisms having a dehalogenase have a low catalytic ability and are not satisfactory for industrial use.

[0003]

In the conversion reaction of epihalohydrin into 4-halo-3-hydroxybutyronitrile by a dehalogenase gene cloned by a gene recombination method, a large number of genes are introduced into the cells. Since it can be present, it is expected that the catalytic ability of the microorganism will be dramatically increased as compared with the conventional method. Under these circumstances, some of the present inventors have described epihalohydrin as 4-
An enzyme that converts to halo-3-hydroxybutyronitrile was found to be halohydrin epoxidase, and further, a recombinant plasmid was obtained by inserting a halohydrin epoxidase enzyme gene DNA derived from a microorganism into a vector plasmid, Halohydrin epoxidase was successfully expressed by the transformant into which the recombinant plasmid was introduced.

[0004]

According to the present invention, the enzyme reaction of halohydrin epoxidase by the transformant can be applied to 1,2-epoxy compounds other than epihalohydrin, and is useful for producing 3-hydroxynitrile compounds. We have found what we can do and completed the present invention. That is, the present invention provides a method for transforming a culture solution, cells or treated cells of a transformed microorganism transformed with a recombinant plasmid obtained by linking a microorganism-derived halohydrin epoxidase gene DNA to a vector plasmid. In the presence of an alkali, the compound is allowed to act on a 1,2-epoxy compound represented by the following general formula (I) to convert it to a 3-hydroxynitrile compound represented by the following general formula (II): A method for producing a hydroxynitrile compound.

[0005]

[R represents an alkyl group having 1 to 4 carbon atoms]

The transformed microorganism in the present invention is a microorganism transformed by a recombinant plasmid in which halohydrin epoxidase gene DNA is ligated to a vector plasmid. A DNA sequence encoding a polypeptide having an amino acid sequence represented by 1 or a partial sequence thereof and having halohydrin epoxidase activity; (2) an amino acid sequence represented by SEQ ID NO: 2 or a partial sequence thereof; And (3) a DNA sequence encoding a polypeptide having halohydrin epoxidase activity described in (1) above, which is represented by SEQ ID NO: 3. (4) The halohydr of the above item (2) A DNA sequence encoding a polypeptide having a phosphoepoxidase activity includes at least one of the DNA sequence represented by SEQ ID NO: 4 or a partial sequence thereof.

Hereinafter, the present invention will be described specifically. Examples of the DNA donor microorganism in the transformed microorganism that can be used in the present invention include Corynebacterium sp. N-1074
(Microtechnical Laboratory No. 2643), Microbacterium s
p. N-4701 (Pierce of Engineering, No. 2644) and the bacteriological properties thereof are described in JP-A-2-29128, respectively.
No. 0 publication. As the vector, a plasmid vector (for example, pUC18, pUC19, pU
C118, pUC119, etc.) and phage vectors (eg, λgt11 etc.). Examples of the host microorganism used for transformation include Escherichia coli (E. coli) strain JM105 or JM109, but are not particularly limited thereto, and other host organisms can be used. . As an example,
Corynebacterium sp. The halohydrin epoxidase gene of E.N. Cloning into the E. coli JM109 strain is shown below.

(1) Corynebacterium sp. N-10
Preparation of chromosome 74 DNA and construction of DNA library: Corynebacterium sp. Saito from N-1074
and Miura's method [Biochim. Bio
phys. Acta 72, 619 (1963)]
Chromosomal DNA is separated by a restriction enzyme (Bam
HI or BglII) and inserted into the vector plasmid pUC18 to prepare a recombinant DNA library.

(2) Preparation of Transformant and Recombinant D
Selection of NA: A transformant using the recombinant library prepared in step (1) was used as a host organism for E. coli. coli JM109 strain using the calcium chloride method [J. Mol. Biol. 5
3, 154 (1970)], from which those exhibiting halohydrin epoxidase activity were selected. Sorting was performed as follows. A transformant prepared on an LB agar medium (1% bactotryptone, 0.5% bactoeast extract, 0.5% NaCl, 1.5% agar) containing ampicillin (100 μg / ml) and IPTG (1 ml) Colonies were allowed to form. 10 mM
The colonies were transferred to a piece of paper impregnated with Tris-HCl buffer (pH 7.5), 0.02% bromocresol purple, and 1% 1,3-dichloro-2-propanol, and left at room temperature for several hours. Colonies with halohydrin epoxidase activity release hydrochloric acid and decrease the pH near the colonies, and bromocresol purple, a pH indicator, changes from blue-violet to yellow, so it has a halohydrin epoxidase gene by visual observation Strains can be selected.

Whether these transformants actually have halohydrin epoxidase activity can be examined as follows. These strains are cultured overnight at 37 ° C. in an LB medium (1% bactotryptone, 0.5% bactoeast extract, 0.5% NaCl) containing ampicillin (50 μg / ml) and IPTG (1 mM).
The cells were washed twice with 50 mM Tris-sulfate buffer (pH 8), suspended in 1 M Tris-sulfate buffer (pH 8) containing 1% 1,3-dichloropropanol, and incubated at 20 ° C. After a certain time, the formed epichlorohydrin was quantified by gas chromatography. Plasmid DNA was again taken out from the thus obtained transformant, and two kinds of target plasmids selected were obtained. These plasmids were designated as pST001 and pST005, and the transformants in which these plasmids had been introduced were designated as JT.
M109 / pST001 and JM109 / pST00
No. 5.

(3) Preparation of restriction enzyme map and determination of position of halohydrin epoxidase gene: A restriction enzyme map was prepared for the plasmid obtained in step (2). Thereafter, a plasmid having a smaller DNA fragment was prepared. The location containing the target gene was determined by the presence or absence of halohydrin epoxidase activity of the strain transformed with these plasmids in the same manner as in step (2). In this process, Ba of pST001
pST015 (p) containing the mHI-Bgl 1.3 Kb fragment
UC118 vector) and BamHI of pST005.
-PST111 containing the PstI1.1Kb fragment (pUC
118 vector) plasmid (FIG. 1). Transformants into which these plasmids have been introduced are referred to as JM109.
/ PST015 and JM109 / pST111.

(4) Determination of base sequence: Plasmids pST015 and pST15 obtained in step (3)
The nucleotide sequence of the DNA related to the halohydrin epoxidase gene of ST111 was determined (SEQ ID NO: 5 and SEQ ID NO: 6). The transformants JM109 / pST001 and JM109 / pST00 obtained here were used.
5, JM109 / pST015 and JM109 / pS
T111 was sent to the Microbial Industry Research Institute (MIC) of the National Institute of Advanced Industrial Science and Technology (NIKEN), No. 11961, No. 11962, No. 12064, and No. 12064. Deposit No. 12065.

The culture of the transformed microorganism of the present invention is usually carried out by liquid culture, but can also be carried out by solid culture. As the medium, for example, an LB medium is used.
The cultivation is performed at a temperature of 10 to 50 ° C and a pH range of 2 to 11. Aeration and agitation may be performed to promote the growth of microorganisms.

The transformed microorganism obtained by culturing may be obtained by adding a substrate to a culture solution or a suspension of cells obtained by centrifugation, etc., or by treating the cells (eg, crushed cells, crude enzyme / purification). A method in which a substrate is added to a suspension of bacterial cells or processed cells, etc. immobilized by a conventional method, or a substrate extract added to a culture solution during the culture of microorganisms. By reacting the 1,2-epoxy compound represented by the general formula [I] in the presence of an alkali cyanide by a method of conducting a reaction, the compound is reacted with the general formula [II]
Can be converted to a 3-hydroxynitrile compound represented by the formula:

The 1,2-epoxy compound represented by the general formula [I] is, for example, 1,2-epoxypropane, 1,2
-Epoxybutane, 1,2-epoxyhexane and the like. The alkali cyanide is potassium cyanide, sodium cyanide, or the like. Although the substrate concentration in the reaction solution is not particularly limited, it is preferably 0.1 to 10 (W / V)%, and the amount of alkali cyanide used is usually 1 to 3 times (mol) the substrate. It is. The substrate can be added to the reaction solution all at once or in portions. The reaction is preferably performed at a reaction temperature of 5 to 50 ° C and a reaction pH of 4 to 10. The reaction time varies depending on the concentration of the substrate and the like, the concentration of the bacterial cells, and other reaction conditions.
It is preferable to set the condition so that the operation ends in 0 hours.

[0016] Thus, the 3-hydroxyl formed and accumulated in the reaction solution.
The hydroxynitrile compound can be collected and purified using a known method. For example, after removing cells from the reaction solution by 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 obtain a syrup of the 3-hydroxynitrile compound. it can. Further, these syrups can be further purified by distillation under reduced pressure.

[0017]

According to the present invention, the use of a transformed microorganism in which a large number of halohydrin epoxidase genes cloned by a gene recombination method are present in the cells enables the production of 1,1 in the presence of alkali cyanide. It is possible to efficiently produce a 3-hydroxynitrile compound from a 2-epoxy compound.

[0018]

EXAMPLES Example 1 JM109 / pST001 was inoculated into 18 l of an LB medium containing ampicillin (50 μg / ml) and 1 mM IPTG, and cultured with shaking at 37 ° C. for 16 hours. The cells were collected from the obtained culture by centrifugation, washed with 50 ml of 100 mM Tris-sulfate buffer (pH 8.0), and suspended in the same buffer to prepare a cell suspension. After sonication of the cells, the precipitate was removed by centrifugation, and the supernatant was used as a cell extract. Ammonium sulfate fractionation was performed by a conventional method, and DEAE-Sephacel, Phenyl-Sepharose, and Octyl-
The enzyme was purified by column chromatography using Sepharose (Pharmacia). Potassium cyanide was dissolved in a 400 mM Tris-sulfate buffer (pH 8.0) to a concentration of 200 mM, and the pH was adjusted with 1N sulfuric acid.
Was adjusted to 8.0, and 0.1 ml of purified enzyme solution (protein concentration: 30 mg / ml) and 400 ml were added to 25 ml of this solution.
25 ml of a 1,2-epoxybutane solution of mM
The reaction was performed at 0 ° C. for 2 hours. When the reaction solution was analyzed by gas chromatography, 100 mM 3-hydroxyvaleronitrile was found to have been formed. The reaction product was isolated from the reaction solution by extraction with ethyl acetate, and analyzed by NMR, infrared absorption spectrum, and mass spectrum to confirm that the product was 3-hydroxyvaleronitrile.

Example 2 The same reaction as in Example 1 was carried out using 1,2-epoxypropane instead of 1,2-epoxybutane, and 71 mM of 3-hydroxybutyronitrile was produced. This reaction product was identified in the same manner as in Example 1.

Example 3 In a medium (100 ml) prepared in the same manner as in Example 1,
JM109 / pST015 and JM109 /
pST111 was inoculated and cultured at 37 ° C. for 16 hours. Each of these cultures was centrifuged to collect the cells, and 50 mM 100 mM Tris-sulfate buffer (pH 8.0) was added.
After washing with the same buffer, the cells were suspended in 25 ml of the same buffer to prepare a cell suspension. 400 mM Tris-sulfate buffer (pH 8.
In 0), potassium cyanide was dissolved to a concentration of 200 mM, and the solution was adjusted to pH 8.0 with 1N sulfuric acid.
ml, and the above cell suspension and 200 mM were added to this solution.
25 ml of a 1,2-epoxybutane solution was added and reacted at 20 ° C. for 30 minutes (JM109 / pST015) and at 20 ° C. for 15 minutes (JM109 / pST111). When the reaction liquid was analyzed by gas chromatography, it was 11.1 mM (JM109 / pST01).
5) and 7.2 mM (JM109 / pST111) of 3-hydroxyvaleronitrile were produced.

Example 4 The same reaction as in Example 3 was carried out using 1,2-epoxypropane instead of 1,2-epoxybutane.
3.6 mM (JM109 / pST015) and 6.2 mM (JM109 / pST111) of 3-hydroxybutyronitrile were produced, respectively.

[0022]

[Sequence list]

[0023]

[0024]

[0025]

[0026]

[0027]

[Brief description of the drawings]

FIG. 1. Recombinant plasmids pST001, pST00
5 shows a restriction map of pST015 and pST111.

[Sequence list]

SEQ ID NO: 1 Sequence length: 244 Sequence type: amino acid Topology: linear Sequence type: peptide Origin Organism name: Corynebacterium Strain name: N-1074 Sequence: Met Lys Ile Ala Leu Val Thr His Ala Arg His Phe Ala Gly Pro Ala 1 5 10 15 Ala Val Glu Ala Leu Thr Arg Asp Gly Tyr Thr Val Val Cys His Asp 20 25 30 Ala Thr Phe Ala Asp Ala Ala Glu Arg Gln Arg Phe Glu Ser Glu Asn 35 40 45 Pro Gly Thr Val Ala Leu Ala Glu Gln Lys Pro Glu Arg Leu Val Asp 50 55 60 Ala Thr Leu Gln His Gly Glu Ala Ile Asp Thr Ile Val Ser Asn Asp 65 70 75 80 Tyr Ile Pro Arg Pro Met Asn Arg Leu Pro Ile Glu Gly Thr Ser Glu 85 90 95 Ala Asp Ile Arg Gln Val Phe Glu Ala Leu Ser Ile Phe Pro Ile Leu 100 105 110 Leu Leu Gln Ser Ala Ile Ala Pro Leu Arg Ala Ala Gly Gly Ala Ser 115 120 125 Val Ile Phe Ile Thr Ser Ser Val Gly Lys Lys Pro Leu Ala Tyr Asn 130 135 140 Pro Leu Tyr Gly Pro Ala Arg Ala Ala Thr Val Ala Leu Val Glu Ser 145 150 155 160 Ala Ala Lys T hr Leu Ser Arg Asp Gly Ile Leu Leu Tyr Ala Ile Gly 165 170 175 Pro Asn Phe Phe Asn Asn Pro Thr Tyr Phe Pro Thr Ser Asp Trp Glu 180 185 190 Asn Asn Pro Glu Leu Arg Glu Arg Val Glu Arg Asp Val Pro Leu Gly 195 200 205 Arg Leu Gly Arg Pro Asp Glu Met Gly Ala Leu Ile Thr Phe Leu Ala 210 215 220 Ser Arg Arg Ala Ala Pro Ile Val Gly Gln Phe Phe Ala Phe Thr Gly 225 230 235 240 Gly Tyr Leu Pro

SEQ ID NO: 2 Sequence length: 235 Sequence type: amino acid Topology: linear Sequence type: peptide Origin Organism: Corynebacterium Strain: N-1074 Sequence: Met Ala Asn Gly Arg Lys Arg Glu Met Ala Asn Gly Arg Leu Ala Gly 1 5 10 15 Lys Arg Val Leu Leu Thr Asn Ala Asp Ala Tyr Met Gly Glu Ala Thr 20 25 30 Val Gln Val Phe Glu Glu Glu Gly Ala Glu Val Ile Ala Asp His Thr 35 40 45 Asp Leu Thr Lys Val Gly Ala Ala Glu Glu Val Val Glu Arg Ala Gly 50 55 60 His Ile Asp Val Leu Val Ala Asn Phe Ala Val Asp Ala His Phe Gly 65 70 75 80 Val Thr Val Leu Glu Thr Asp Glu Glu Leu Trp Gln Thr Ala Tyr Glu 85 90 95 Thr Ile Val His Pro Leu His Arg Ile Cys Arg Ala Val Leu Pro Gln 100 105 110 Phe Tyr Glu Arg Asn Lys Gly Lys Ile Val Val Tyr Gly Ser Ala Ala 115 120 125 Ala Met Arg Tyr Gln Glu Gly Ala Leu Ala Tyr Ser Thr Ala Arg Phe 130 135 140 Ala Gln Arg Gly Tyr Val Thr Ala Leu Gly Pro Glu Ala Ala Arg His 145 150 155 160 Asn Val Asn V al Asn Phe Ile Ala Gln His Trp Thr Gln Asn Lys Glu 165 170 175 Tyr Phe Trp Pro Glu Arg Ile Ala Thr Asp Glu Phe Lys Glu Asp Met 180 185 190 Ala Arg Arg Val Pro Leu Gly Arg Leu Ala Thr Ala Arg Glu Asp Ala 195 200 205 Leu Leu Ala Leu Phe Leu Ala Ser Asp Glu Ser Asp Phe Ile Val Gly 210 215 220 Lys Ser Ile Glu Phe Asp Gly Gly Trp Ala Thr 225 230 235

SEQ ID NO: 3 Sequence length: 732 Sequence type: number of nucleic acid strands: single-stranded Topology-: linear Sequence type: Genomic DNA Origin Organism: Corynebacterium Strain name: N-1074 Sequence: ATG AAG ATC GCC CTC GTG ACT CAT GCA CGG CAT TTT GCA GGC CCC GCC 48 GCC GTC GAG GCG CTT ACG CGG GAT GGC TAT ACC GTG GTT TGC CAC GAC 96 GCG ACG TTC GCT GAT GCA GCT GAA CGA CAG CGT TTC GAG TCG GAG AAC 144 CCG GGC ACC GTC GCG CTC GCC GAG CAG AAG CCC GAG CGT CTG GTC GAC 192 GCC ACG CTG CAG CAC GGG GAA GCG ATC GAC ACG ATC GTC TCG AAC GAT 240 TAC ATT CCG CGC CCG ATG AAT CGG CTC CC ATC GAG GGA ACG AGC GAG 288 GCC GAC ATC CGA CAG GTG TTC GAG GCG CTC AGC ATC TTC CCG ATC CTG 336 CTC CTG CAG TCG GCC ATC GCG CCG CTA CGG GCT GCA GGC GGC GCC TCC 384 GTT ATC TTC ATC GTC TGC GCA AAG AAG CCG CTC GCC TAC AAC 432 CCT CTC TAT GGG CCC GCG CGC GCC GCT ACC GTC GCG CTT GTC GAA TCG 480 GCA GCG AAG ACG CTG TCC CGT GAC GGA ATC TTG CTC TAC GCG ATC GGT 528 CCG AAC TTC TTC AAC AAC CCG ACG TAC TTC CCG ACG TCG GAT TGG GAG 576 AAC AAC CCC GAG CTC CGG GAG CGT GTC GAG CGG GAC GTG CCG CTC GGT 624 CGC CTC GGC CGT CCG GAC GAG ATG GGT GCG CTG ATC ACC TTC CCT GTC TCG CGT CGT GCA GCG CCC ATC GTG GGG CAG TTC TTC GCT TTC ACC GGT 720 GGC TAT CTG CCC 732

SEQ ID NO: 4 Sequence length: 705 Sequence type: number of nucleic acid strands: single-stranded Topology-: linear Sequence type: Genomic DNA Origin Organism: Corynebacterium Strain name: N-1074 Sequence: ATG GCT AAC GGA AGG AAA AGG GAA ATG GCT AAC GGA AGA CTG GCA GGC 48 AAG CGG GTC CTA CTC ACG AAC GCC GAT GCC TAC ATG GGT GAG GCC ACG 96 GTC CAG GTG TTC GAG GAG GAG GGC GCA GAG GTC ATC GCT GAC CAC ACC 144 GAC TTG ACG AAG GTC GGC GCG GCG GAG GAG GTC GTC GAG AGG GCT GGG 192 CAC ATC GAT GTC CTG GTG GCC AAC TTC GCG GTC GAC GCC CAC TTC GGG 240 GTG ACC GTG CTG GAG ACC GAC GAG CAG TGG CAG ACG GCC TAC GAG 288 ACC ATC GTG CAC CCG CTG CAT CGG ATC TGC CGT GCG GTG CTC CCG CAG 336 TTC TAC GAG CGG AAC AAG GGC AAG ATC GTT GTC TAC GGA AGT GCC GCA 384 GCG ATG CGG TAC CAG GATG GGT GCC TAC AGC ACG GCG CGT TTC 432 GCT CAG CGC GGG TAC GTC ACC GCC CTC GGT CCC GAG GCA GCG AGG CAC 480 AAC GTC AAC GTG AAC TTC ATC GCC CAG CAC TGG ACC CAA AAC AAG GAG 528 TAC TTC TGG CCC GAG CGC ATC GCC ACC GAC GAG TTC AAG GAG GAT ATG 576 GCG CGC CGA GTT CCC CTG GGT CGG CTC GCG ACT GCC CGA GAG GAC GCG 624 CTG CTC GCG TTG TTC CTG GCC TCG GAC GAG AGT GAC TTC ATC GTC GTC AAG TCG ATC GAG TTC GAC GGC GGC TGG GCC ACC 705

SEQ ID NO: 5 Sequence length: 829 Sequence type: nucleic acid Number of strands: single-stranded Topology-: linear Sequence type: Genomic DNA Origin Organism: Corynebacterium Strain name: N-1074 Sequence: GAATTCCAGA ACCAATTGAG AGGAAATGAA CA ATG AAG ATC GCC CTC GTG ACT 53 Met Lys Ile Ala Leu Val Thr 15 CAT GCA CGG CAT TTT GCA GGC CCC GCC GCC GTC GAG GCG CTT ACG CGG 101 His Ala Arg His Phe Ala Gly Pro Ala Ala Val Glu Ala Leu Thr Arg 10 15 20 GAT GGC TAT ACC GTG GTT TGC CAC GAC GCG ACG TTC GCT GAT GCA GCT 149 Asp Gly Tyr Thr Val Val Cys His Asp Ala Thr Phe Ala Asp Ala Ala 25 30 35 GAA CGA CAG CGT TTC GAG TCG GAG AAC CCG GGC ACC GTC GCG CTC GCC 197 Glu Arg Gln Arg Phe Glu Ser Glu Asn Pro Gly Thr Val Ala Leu Ala 40 45 50 55 GAG CAG AAG CCC GAG CGT CTG GTC GAC GCC ACG CTG CAG CAC GGG GAA 245 Glu Gln Lys Pro Glu Arg Leu Val Asp Ala Thr Leu Gln His Gly Glu 60 65 70 GCG ATC GAC ACG ATC GTC TCG AAC GAT TAC ATT CCG CGC CCG ATG AAT 293 Ala Ile Asp Thr Ile Val Ser Asn Asp Tyr Ile Pro Arg Pro Met Asn 75 80 85 CGG CTC CCG ATC GAG GGA ACG AGC GAG GCC GAC ATC CGA CAG GTG TTC 341 Arg Leu Pro Ile Glu Gly Thr Ser Glu Ala Asp Ile Arg Gln Val Phe 90 95 100 GAG GCG CTC AGC ATC TTC CCG ATC CTG CTC CTG CAG TCG GCC ATC GCG 389 Glu Ala Leu Ser Ile Phe Pro Ile Leu Leu Leu Gln Ser Ala Ile Ala 105 110 115 CCG CTA CGG GCT GCA GGC GGC GCC TCC GTT ATC TTC ATC ACG TCC TCA 437 Pro Leu Arg Ala Ala Gly Gly Ala Ser Val Ile Phe Ile Thr Ser Ser 120 125 130 135 GTT GGC AAG AAG CCG CTC GCC TAC AAC CCT CTC TAT GGG CCC GCG CGC 485 Val Gly Lys Lys Pro Leu Ala Tyr Asn Pro Leu Tyr Gly Pro Ala Arg 140 145 150 GCC GCT ACC GTC GCG CTT GTC GAA TCG GCA GCG AAG ACG CTG TCC CGT 533 Ala Ala Thr Val Ala Leu Val Glu Ser Ala Ala Lys Thr Leu Ser Arg 155 160 165 GAC GGA ATC TTG CTC TAC GCG ATC GGT CCG AAC TTC TTC AAC AAC CCG 581 Asp Gly Ile Leu Leu Tyr Ala Ile Gly Pro Asn Phe Phe Asn Asn Pro 170 175 180 ACG TAC TTC CCG ACG TCG GAT TGG GAG AAC AAC CCC GAG CTC CGG GAG 629 Thr Tyr Phe Pro Thr Ser Asp Trp Glu Asn Asn Pro Glu Leu Arg Glu 185 190 195 CGT GTC GAG CGG GAC GTG CCG CTC GGT CGC CTC GGC CGT CCG GAC GAG 677 Arg Val Glu Arg Asp Val Pro Leu Gly Arg Leu Gly Arg Pro Asp Glu 200 205 210 215 ATG GGT GCG CTG ATC ACC TTC CTC GCT TCG CGT CGT GCA GCG CCC ATC 725 Met Gly Ala Leu Ile Thr Phe Leu Ala Ser Arg Arg Ala Ala Pro Ile 220 225 230 GTG GGG CAG TTC TTC GCT TTC ACC GGT GGC TAT CTG CCC TAACCCGCGC 774 Val Gly Gln Phe Phe Ala Phe Thr Gly Gly Tyr Leu Pro 235 240 CGGTACGGCA ACAGGAAGGA CTGTCTGACA CGGTTCGTCC TCCCAACGCG CCGGC 829

SEQ ID NO: 6 Sequence length: 843 Sequence type: nucleic acid Number of strands: single-stranded Topology-: linear Sequence type: Genomic DNA Origin Organism: Corynebacterium Strain name: N-1074 Sequence: GTCGACTAGA GAAGGTATTC CGACTGCTGC GGTGCCTGGC ACCGCAGCAA AAGATTCAAG 60 GATTCTCGAA GAAAGGAAAA GGGAA ATG GCT AAC GGA AGG AAA AGG GAA ATG 112 Met Ala Asn Gly Arg Lys Arg Glu Met 1 5 GCT AAC GGA CGA GGA CGA GGA CGA GGA CGA CGA GCC GAT 160 Ala Asn Gly Arg Leu Ala Gly Lys Arg Val Leu Leu Thr Asn Ala Asp 10 15 20 25 GCC TAC ATG GGT GAG GCC ACG GTC CAG GTG TTC GAG GAG GAG GGC GCA 208 Ala Tyr Met Gly Glu Ala Thr Val Gln Val Phe Glu Glu Glu Gly Ala 30 35 40 GAG GTC ATC GCT GAC CAC ACC GAC TTG ACG AAG GTC GGC GCG GCG GAG 256 Glu Val Ile Ala Asp His Thr Asp Leu Thr Lys Val Gly Ala Ala Glu 45 50 55 GAG GTC GTC GAG AGG GCT GGG CAC ATC GAT GTC CTG GTG GCC AAC TTC 304 Glu Val Val Glu Arg Ala Gly His Ile Asp Val Leu Val Ala Asn Phe 60 65 70 GCG G TC GAC GCC CAC TTC GGG GTG ACC GTG CTG GAG ACC GAC GAG GAG 352 Ala Val Asp Ala His Phe Gly Val Thr Val Leu Glu Thr Asp Glu Glu 75 80 85 CTG TGG CAG ACG GCC TAC GAG ACC ATC GTG CAC CCG CTG CAT CGG ATC 400 Leu Trp Gln Thr Ala Tyr Glu Thr Ile Val His Pro Leu His Arg Ile 90 95 100 105 TGC CGT GCG GTG CTC CCG CAG TTC TAC GAG CGG AAC AAG GGC AAG ATC 448 Cys Arg Ala Val Leu Pro Gln Phe Tyr Glu Arg Asn Lys Gly Lys Ile 110 115 120 GTT GTC TAC GGA AGT GCC GCA GCG ATG CGG TAC CAG GAA GGT GCG CTG 496 Val Val Tyr Gly Ser Ala Ala Ala Met Arg Tyr Gln Glu Gly Ala Leu 125 130 135 GCC TAC AGC ACG GCG CGT TTC GCT CAG CGC GGG TAC GTC ACC GCC CTC 544 Ala Tyr Ser Thr Ala Arg Phe Ala Gln Arg Gly Tyr Val Thr Ala Leu 140 145 150 GGT CCC GAG GCA GCG AGG CAC AAC GTC AAC GTG AAC TTC ATC GCC CAG 592 Gly Pro Glu Ala Ala Arg His Asn Val Asn Val Asn Phe Ile Ala Gln 155 160 165 CAC TGG ACC CAA AAC AAG GAG TAC TTC TGG CCC GAG CGC ATC GCC ACC 640 His Trp Thr Gln Asn Lys Glu Tyr Phe Trp Pro Glu Arg Ile Ala Thr 170 175 1 80 185 GAC GAG TTC AAG GAG GAT ATG GCG CGC CGA GTT CCC CTG GGT CGG CTC 688 Asp Glu Phe Lys Glu Asp Met Ala Arg Arg Val Pro Leu Gly Arg Leu 190 195 200 GCG ACT GCC CGA GAG GAC GCG CTG CTC GCG TTG TTC CTG GCC TCG GAC 736 Ala Thr Ala Arg Glu Asp Ala Leu Leu Ala Leu Phe Leu Ala Ser Asp 205 210 215 GAG AGT GAC TTC ATC GTC GGC AAG TCG ATC GAG TTC GAC GGC GGC TGG 784 Glu Ser Asp Phe Ile Val Gly Lys Ser Ile Glu Phe Asp Gly Gly Trp 220 225 230 GCC ACC TGAGAGACGT CACAGCCCCC TCGGGCAGGC GCTCGTCGTC GTTGTAGCTG CAG 843 Ala Thr 235

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12R 1:19) (C12N 9/00 C12R 1:19) (C12N 15/09 ZNA C12R 1:15) (C12P 13/00 C12R 1:19) (72) Wataru Minazushi 10-1 Ogurocho, Tsurumi-ku, Yokohama-shi, Kanagawa Prefecture Nitto Chemical Industry Co., Ltd. Central Research Laboratory (72) Inventor Fujio Yu 10-1 Ogurocho, Tsurumi-ku, Yokohama-shi, Kanagawa No. Nitto Chemical Industry Co., Ltd. Central Research Laboratory Examiner Koichi Niimi (58) Fields investigated (Int. Cl. ⁷ , DB name) C12P 13/00 C12N 9/00 C12N 15/09 GenBank / EMBL / DDBJ

Claims (5)

(57) [Claims] 1. A culture solution, cells or treated cells of a transformed microorganism transformed with a recombinant plasmid obtained by linking a microorganism-derived halohydrin epoxidase gene DNA to a vector plasmid, In the presence of a compound represented by the following general formula [I] to convert it into a 3-hydroxynitrile compound represented by the following general formula [II]. A method for producing a hydroxynitrile compound. Embedded image [R represents an alkyl group having 1 to 4 carbon atoms] 2. A DNA sequence wherein the halohydrin epoxidase gene DNA has the amino acid sequence represented by SEQ ID NO: 1 or a partial sequence thereof and encodes a polypeptide having halohydrin epoxidase activity. The method for producing a 3-hydroxynitrile compound according to claim 1, comprising: 3. A DNA sequence wherein the halohydrin epoxidase enzyme gene DNA has the amino acid sequence represented by SEQ ID NO: 2 or a partial sequence thereof and encodes a polypeptide having halohydrin epoxidase activity. The method for producing a 3-hydroxynitrile compound according to claim 1, comprising: 4. The DNA sequence encoding a polypeptide having halohydrin epoxidase activity has the sequence of SEQ ID NO:
3. The method for producing a 3-hydroxynitrile compound according to claim 2, comprising the DNA sequence represented by 3 or a partial sequence thereof. 5. A DNA sequence encoding a polypeptide having halohydrin epoxidase activity, comprising the sequence:
4. The method for producing a 3-hydroxynitrile compound according to claim 3, comprising a DNA sequence represented by 4 or a partial sequence thereof.