KR20060016115A - Novel nitrile hydratase - Google Patents

Abstract

A process for producing an amide compound with the use of a nitrile hydratase of high thermal stability capable of maintaining high activity even in the presence of high-concentration nitrile compound as a substrate and amide compound as a product. A recombinant microbe was produced by searching for a novel microbe capable of expression of the desired nitrile hydratase in nature, obtaining relevant gene from cells of the microbe or treatment product thereof, performing cloning of the gene and introducing the same in a different type of microbe for expression. With the use thereof, nitrile compounds can be efficiently converted to corresponding amide compounds even by the reaction conducted at high temperature and in the presence of high-concentration nitrile compounds and high-concentration amide compounds.

Description

New nitrile hydratase {NOVEL NITRILE HYDRATASE}

The present invention relates to a technique for preparing amide compounds from nitrile compounds by enzymatic catalysis of novel nitrile hydratase derived from microorganisms newly isolated in nature.

In the technique of producing a corresponding amide compound by hydrating the nitrile group of the nitrile compound and converting it into an amide group, a method of using a microorganism enzyme as a catalyst instead of the conventional chemical method using a copper catalyst is becoming the mainstream. Such enzymes are commonly called nitrile hydratase, and since the first reports, many enzymes have been found in various microorganisms. For example, the genus Arthrobacter (Agricultural and Biological Chemistry Vol. 44 p.2251-2252, 1980), the genus Agrobacterium (Japanese Patent Publication No. H05-103681), genus Acinetobacter (Japanese Patent Laid-Open No. 61-282089), Genus Aeromonas (Japanese Patent Laid-Open Publication No. 05-030983), Enterobacter genus (Japanese Patent Laid-Open Publication No. 05-236975), Erwinia genus (Japan Japanese Patent Application Laid-Open No. 05-161496, Genus Xanthobacter (Japanese Patent Application Laid-open No. 05-161495), Klebsiella genus (Japanese Patent Laid-Open Publication No. 05-0030982), Corynebacterium Genus (Japanese Patent Publication No. 54-129190, later identified as Rhodococcus), Pseudomonas genus (Japanese Patent Publication No. 58-86093), Citrobacter genus (Japanese Patent Publication No. 05-030984), Streptomyces genus (Japanese Patent Laid-Open No. 05-236976), Bacillus genus (Japanese Patent Laid-Open) 51-86186, Japanese Patent Application Laid-Open No. 7-255494, Fussumi (Japanese Patent Application Laid-open No. 01-086889), Rhodococcus genus (Japanese Patent Publication No. 63-137688, Japanese Patent Application Laid-Open No. 02-227069, Japan Japanese Patent Laid-Open No. 2002-369697, Japanese Patent Laid-Open No. 2-470), Rhizobium genus (Japanese Patent Laid-Open No. 05-236977), Pseudonocardia genus (Japanese Patent Laid-Open No. Hei 8-56684), etc. Can be mentioned. These enzymes have various physicochemical properties based on the diversity of their amino acid sequences, and research has been conducted for various purposes. Among the physicochemical properties, examples of the elucidation of the stability of heat, amide compounds, and nitrile compounds, etc., are being carried out. 589, 1991. Applied and Microbiology Biotechnology Vol. 40 p. 189-195, 1993.), Pseudonocardia thermothera JCM3095 (Japanese Patent Publication No. Hei 8-187092, Journal of Fermentation and Bioengineering Vol. 83 p.474-477, 1997), Bacillus genus BR449 strain (WO99 / 55719.Applied Biochemistry and Biotechnology Vol.77-79 p.671-679, 1999.), Bacillus Literature on RAPc8 strains (Enzyme and Microbial Technology Vol. 26 p. 368-373, 2000. Extremophiles Vol. 2 p.347-357, 1998), and Bacillus palidus (Dac521 strain) Biochimica et Biophysica Acta Vol. 1431 p. 249-260, 1999) Bacillus smithii SC-J05-1 (Journal of Industrial Microbiology and Biotechnology Vol. 20 220-226, 1998) is mentioned.

On the other hand, when industrially producing an amide compound with a nitrile compound using these unique nitrile hydratases, the production cost of the enzyme, which accounts for the production cost of the amide compound, is an important problem. It is preferable to produce by using. Thus, attempts have been made to clone genes for the purpose of expressing a large amount of nitrile hydratase by a technique of genetic engineering. For example, Pseudomonas genus (Japanese Patent Laid-Open No. Hei 3-251184), Rhodococcus genus (Japanese Laid-Open Patent No. Hei 2-119778, Japanese Patent Laid-Open No. 4-211379, Japanese Patent Laid-Open No. Hei 09-00973, Japanese Patent Laid-Open No. 07-099980) , Japanese Patent Application Laid-Open No. 2001-069978), Rizobium genus (Japanese Patent Laid-Open No. 6-25296), Klebsiella genus (Japanese Patent Laid-Open No. 6-303971), Acromobacter genus (Japanese Patent Laid-open No. , Pseudonocardia genus (Japanese Patent Laid-Open No. Hei 9-275978), Bacillus genus (Japanese Patent Laid-Open No. 09-248188), and the like.

As the physicochemical properties of the enzyme, nitrile hydratase that maintains high activity even in the presence of high concentrations of a nitrile compound, which is a thermal stability or a substrate, and an amide compound, which is a product, is preferred, and its purpose is also reported. Their absolute values may vary depending on the type of nitrile compound, so no enzyme was found. In addition, as a material for improving physicochemical properties using evolutionary engineering in the future, there is no origin so far and development of various enzymes is desired.

It goes without saying that cloning and expression of genes are necessary for improvement of physicochemical properties using evolutionary engineering, and genes using a host whose culture method is established from the cost constraints related to the production of enzymes in the amide compound preparation described above. The production of recombinant bacteria is desired.

That is, an object of the present invention is to isolate nitrile hydratase having high stability against heat and high concentration compounds in nature, and to provide a method for producing the enzyme and a method for producing a corresponding amide compound from the nitrile compound using the enzyme. will be. Also provided is an amino acid sequence and a gene sequence of the enzyme, a recombinant plasmid containing the gene, a transformed strain comprising the recombinant plasmid, the production method of the enzyme using the transformed strain and a nitrile using the transformed strain It is to provide a process for preparing the corresponding amide compound from the compound. It also provides an amino acid sequence and a gene sequence of a protein having a function of activating the nitrile hydratase enzyme of the recombinant.

MEANS TO SOLVE THE PROBLEM As a result of earnestly examining in order to solve the said subject, the present inventor discovered Geobacillus thermoglucosidasius as a microorganism which has nitrile hydratase activity in the soil near the hot spring of Saitama. In addition, it is not known so far that the microorganism of the genus Geobacillus has nitrile hydratase and exhibits nitrile hydratase activity, and the temperature of 65 ° C which is usually used for cultivation of the microorganism has a conventional nitrile hydratase. It is more than normal culture temperature (45-60 degreeC) of bacteria.

In addition, the nitrile hydratase enzyme was purified from the microorganism, and the nitrile hydratase activity was shown to have high stability with respect to heat and high concentration of nitrile compound and amide compound. Moreover, based on the amino acid sequence of the N terminal of each subunit of the purified enzyme, the nitrile hydratase gene was isolated from the chromosomal DNA of the said microorganism, and the amino acid sequence and gene sequence were made clear for the first time, and the known nitrile hydrata was known. The homology with Aze was found to be very low. Furthermore, by simultaneously expressing a gene sequence presumed to be an activating protein present downstream of the gene, the present inventors have also succeeded in constructing a recombinant strain expressing a large amount of the enzyme, thereby achieving the present invention.

That is, this invention provides the following.

(1) A DNA encoding a protein having the following physicochemical properties.

 (a) has nitrile hydratase activity.

 (b) Substrate specificity: It shows activity based on acrylonitrile, adipononitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile and hexanenitrile as a substrate.

 (c) Molecular weight: A protein composed of at least the following two types of subunits, the molecular weight of each subunit being reduced SDS-polyacrylamide electrophoresis as follows.

     Subunit α Molecular weight 25000 ± 2000

     Subunit β Molecular weight 800028000 ± 2000

 (d) Thermal Stability: After heating the enzyme in the sap at a temperature of 70 ° C. for 30 minutes, 35% or more of activity before heating remains.

 (e) Even if 6 wt% of acrylonitrile is used as the substrate, the activity is not decreased as compared with the substrate concentration below that.

 (f) It has the activity which used acrylonitrile as a substrate also in 35 weight% of acrylamide aqueous solution.

(2) The DNA of any one of the following (A) or (B).

 (A) a base sequence containing an α subunit gene encoding an amino acid sequence of SEQ ID NO: 1 of the sequence listing, and a base sequence containing a β subunit gene encoding an amino acid sequence of SEQ ID NO: 2 of the sequence listing; DNA, characterized in that it comprises a combination.

 (B) one or more of any one or both of the α subunit containing the amino acid sequence of SEQ ID NO: 1 of the Sequence Listing and the β subunit containing the amino acid sequence of SEQ ID NO: 2 of the Sequence Listing; The amino acid of dog is substituted, deleted, added, and post-translationally modified, and contains the modified or unmodified α subunit and the modified or unmodified β subunit and encodes a protein having nitrile hydratase activity. DNA.

(3) The DNA of any one of the following (C) or (D).

 (C) a combination of a DNA comprising a sequence at positions 695-1312 of the nucleotide sequence of SEQ ID NO: 3 in the sequence listing and a DNA containing a sequence at positions 1-681 of the nucleotide sequence of SEQ ID NO: 3 in the sequence listing DNA, characterized in that.

 (D) α subunit encoded by DNA containing DNA sequence of positions 695-1312 of the nucleotide sequence of SEQ ID NO: 3 in the sequence listing or DNA hybridized under stringent conditions with respect to the DNA; And a β subunit encoded by any one of DNAs containing the sequences of positions 1-681 of the nucleotide sequence of SEQ ID NO: 3 or DNA hybridized under strict conditions with respect to the DNA, and further including nitrile hydratase. DNA encoding a protein having activity. However, the case where it becomes said (C) is excluded.

(4) The DNA containing the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing or one or more amino acids in the amino acid sequence has been substituted, deleted, added, or post-translationally modified, and further, nitrile hydra The DNA according to any one of (1) to (3), wherein the DNA of any one of the DNAs encoding a protein that is involved in the activation of the ptase is further combined.

(5) Encoding a DNA comprising the sequence No. 1325-1663 of the nucleotide sequence of SEQ ID NO: 3 in the Sequence Listing, or a hybridizing DNA under stringent conditions and participating in nitrile hydratase activation The DNA according to any one of (1) to (3), wherein the DNA of any one of DNAs is further combined.

(6) DNA containing α subunit gene of nitrile hydratase encoding amino acid sequence set forth in SEQ ID NO: 1 in Sequence Listing.

(7) DNA containing β subunit gene of nitrile hydratase encoding amino acid sequence set forth in SEQ ID NO: 2 in Sequence Listing.

(8) DNA containing a gene characterized by being involved in the activation of nitrile hydratase encoding the amino acid sequence set forth in SEQ ID NO: 4 in the Sequence Listing.

(9) The DNA according to any one of (1) to (8), wherein the DNA is derived from the genus Geobacillus.

(10) The DNA according to any one of (1) to (8), wherein the DNA is derived from a Geobacillus thermoglucosidasius species.

(11) The DNA according to any one of (1) to (8), wherein the DNA is derived from Geobacillus thermoglucosidasius Q-6 strain (FERM BP-08658).

(12) The recombinant vector which assembled | assembled the DNA of any one of (1)-(11).

(13) Among microorganisms transformed with DNA according to any one of (1) to (11), or Geobacillus thermoglucosidasius (QM-6 strain) (FERM # BP-08658) and variants thereof Which one microorganism.

(14) A method for producing the above-mentioned protein or cell-containing cell-treated product, wherein the microorganism transformed with the DNA according to any one of (1) to (11) is cultured in a medium.

(15) A method for producing the protein, or a cell-containing treatment product, comprising culturing a microorganism belonging to the genus Geobacilli and capable of producing a protein having the following physicochemical properties in a medium.

 (a) has nitrile hydratase activity.

 (b) Substrate specificity: It shows activity based on acrylonitrile, adipononitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile and hexanenitrile as a substrate.

 (c) Molecular weight: A protein composed of at least the following two types of subunits, the molecular weight of each subunit being reduced SDS-polyacrylamide electrophoresis as follows.

      Subunit α Molecular weight 25000 ± 2000

      Subunit β Molecular weight 28000 ± 2000

(d) Thermal Stability: After heating the enzyme in the sap at a temperature of 70 ° C. for 30 minutes, 35% or more of activity before heating remains.

(e) Even if 6 wt% of acrylonitrile is used as the substrate, the activity is not decreased as compared with the substrate concentration below that.

(f) It has the activity which used acrylonitrile as a substrate also in 35 weight% of acrylamide aqueous solution.

(16) The said protein obtained from the microorganism cultured by the manufacturing method in any one of (14) or (15), or the cell processed material containing the said protein.

(17) A protein having the following physicochemical properties.

(a) has nitrile hydratase activity.

(b) Substrate specificity: It shows activity based on acrylonitrile, adipononitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile and hexanenitrile as a substrate.

(c) Molecular weight: A protein composed of at least the following two types of subunits, the molecular weight of each subunit being reduced SDS-polyacrylamide electrophoresis as follows.

     Subunit α Molecular weight 25000 ± 2000

     Subunit β Molecular weight 28000 ± 2000

(d) Thermal Stability: After heating the enzyme in the sap at a temperature of 70 ° C. for 30 minutes, 35% or more of activity before heating remains.

(e) Even if 6 wt% of acrylonitrile is used as the substrate, the activity is not decreased as compared with the substrate concentration below that.

(f) It has the activity which used acrylonitrile as a substrate also in 35 weight% of acrylamide aqueous solution.

(18) The protein of any one of the following (A) or (B).

(A) A protein comprising α subunit containing an amino acid sequence of SEQ ID NO: 1 of the sequence listing and β subunit containing an amino acid sequence of SEQ ID NO: 2 of the sequence listing.

(B) one or more of any one or both of the α subunit containing the amino acid sequence of SEQ ID NO: 1 of the Sequence Listing and the β subunit containing the amino acid sequence of SEQ ID NO: 2 of the Sequence Listing; A protein having a substitution, deletion, addition and post-translational modification of an amino acid of a dog, the modified or unmodified α subunit, and a modified or unmodified β subunit, and having nitrile hydratase activity.

(19) The protein of any one of the following (C) or (D).

(C) an α subunit encoded by a DNA comprising a sequence at positions 695-1312 of the nucleotide sequence of SEQ ID NO: 3 in the sequence listing, and a sequence at position 1-681 of the nucleotide sequence of SEQ ID NO: 3 in the sequence listing A protein comprising β-subunit encoded by DNA.

 (D) α subunit encoded by DNA containing DNA sequence of positions 695-1312 of the nucleotide sequence of SEQ ID NO: 3 in the sequence listing or DNA hybridized under stringent conditions with respect to the DNA; And a β subunit encoded by any one of DNAs containing the sequences of positions 1-681 of the nucleotide sequence of SEQ ID NO: 3 or DNA hybridized under strict conditions with respect to the DNA, and further including nitrile hydratase. Protein with activity. However, the case where it becomes said (C) is excluded.

(20) a polypeptide containing α subunit which is at least the amino acid sequence of SEQ ID NO: 1 in the sequence listing or a post-translationally modified polypeptide and a polypeptide containing β subunit which is the amino acid sequence of SEQ ID NO: 2 in the sequence listing or post-translation A protein containing any one or both of the modified.

(21) A method for producing an amide compound, characterized by obtaining an amide compound derived from the nitrile compound by acting on a nitrile compound with a protein having the following physicochemical properties or a cell-treated product containing the protein.

(a) has nitrile hydratase activity.

(b) Substrate specificity: It shows activity based on acrylonitrile, adipononitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile and hexanenitrile as a substrate.

(c) Molecular weight: A protein composed of at least the following two types of subunits, the molecular weight of each subunit being reduced SDS-polyacrylamide electrophoresis as follows.

      Subunit α Molecular weight 25000 ± 2000

      Subunit β Molecular weight 28000 ± 2000

(d) Thermal Stability: After heating the enzyme in the sap at a temperature of 70 ° C. for 30 minutes, 35% or more of activity before heating remains.

(e) Even if 6 wt% of acrylonitrile is used as the substrate, the activity is not decreased as compared with the substrate concentration below that.

(f) It has the activity which used acrylonitrile as a substrate also in 35 weight% of acrylamide aqueous solution.

(22) Cultivating any of the microorganisms transformed by the DNA according to any one of (1) to (11), or a microorganism belonging to the genus Geobacillus and capable of producing a protein having the following physicochemical properties: And a method for producing an amide compound, characterized in that it is a protein obtained by treatment or a cell processing product containing the protein.

(a) has nitrile hydratase activity.

(b) Substrate specificity: It shows activity based on acrylonitrile, adipononitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile and hexanenitrile as a substrate.

(c) Molecular weight: A protein composed of at least the following two types of subunits, the molecular weight of each subunit being reduced SDS-polyacrylamide electrophoresis as follows.

     Subunit α Molecular weight 25000 ± 2000

     Subunit β Molecular weight 28000 ± 2000

(d) Thermal Stability: After heating the enzyme in the sap at a temperature of 70 ° C. for 30 minutes, 35% or more of activity before heating remains.

(e) Even if 6 wt% of acrylonitrile is used as the substrate, the activity is not decreased as compared with the substrate concentration below that.

(f) It has the activity which used acrylonitrile as a substrate also in 35 weight% of acrylamide aqueous solution.

BRIEF DESCRIPTION OF THE DRAWINGS The gene composition and restriction enzyme map of the nitrile hydratase (beta) subunit, (alpha) subunit, and downstream gene group of the geobacillus thermoglucosidasius Q-6 (Geobacillus thermoglucosidasius Q-6) strain are shown. Colony

The positions of fragments (Hin 2.3) obtained by hybridization and fragments (βα, βα1, βα12) used for expression in E. coli were shown.

Best Mode for Carrying Out the Invention

First, the nitrile hydratase of this invention is demonstrated. In the present invention, having nitrile hydratase activity is converted to an amide compound by adding water to a nitrile compound such as acetamide in acetonitrile, n-propioamide in n-propionitrile and acrylamide in acrylonitrile. To have activity. The produced compound is fractionated by liquid chromatography, and then classified using gas chromatography / mass spectrometry (GC / MS), infrared absorption spectrum (IR), nuclear magnetic resonance spectrum (NMR), and the like.

In measuring the nitrile hydratase activity in the present invention, for example, 10 µl of a nitrile hydratase enzyme solution is added to 1 ml of a 0.1 wt% nitrile compound solution (0.05 M-phosphate buffer pH7.7), and the reaction temperature is 27. After keeping at 1 to 60 minutes at 0 ° C to 60 ° C, the reaction is stopped by adding 0.1 ml of 1N HCl, and a part of the reaction solution can be analyzed by liquid chromatography to assay the presence or absence of the formation of an amide compound.

In this invention, the nitrile compound used as a substrate is aliphatic nitrile compounds, such as acetonitrile, n-propionitrile, n-butyronitrile, isobutyronitrile, n-valeronitrile, n-hexanenitrile, etc., Nitrile compounds containing halogen atoms, such as 2-chloropropionitrile, hydroxynitriles, such as aliphatic nitrile compounds containing unsaturated bonds, such as acrylonitrile, crotononitrile, and methacrylonitrile, lactonitrile, and mandelonitrile Amino nitrile compounds, such as a compound and 2-phenylglycinonitrile, Aromatic nitrile compounds, such as benzonitrile and a cyano pyridine, Dinitrile compounds, such as malononitrile, succinonitrile, and adiponitrile, and a trinitrile compound are mentioned. have.

The substrate specificity of the nitrile hydratase of the present invention can be judged by measuring various substrates in the above-described measurement conditions to determine whether they have nitrile hydratase activity for each substrate. If the substrate specificity is wide, the kind of the corresponding amide compound that can be produced is also increased and preferable, but the enzyme is at least acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-buty Ronitrile, benzonitrile and hexanenitrile can be used as a substrate.

The nitrile hydratase of the present invention is reduced SDS (sodium dodecyl sulfate) -polyacrylamide electrophoresis, and two subunits of molecular weight 25000 ± 2000 and molecular weight 28000 ± 2000 are stained with Coomassie brilliant blue. The former is called α subunit and the latter is referred to as β subunit.

The nitrile hydratase of the present invention can maintain 35% of its activity before heating even after heat treatment at 70 ° C. for 30 minutes in an aqueous solution containing no stabilizer such as an organic acid prior to activity measurement.

In addition, it has been reported that a high concentration of nitrile substrates chemically deactivates the enzyme, but such a phenomenon is not observed in the nitrile hydratase of the present invention even when 6% by weight of acrylonitrile is used as the substrate.

In addition, it is reported that the amide compound, which is a high concentration of reaction product, inhibits the reaction, which is a big problem when obtaining a high concentration of reaction product. However, the nitrile hydratase of the present invention is a substrate even when 35% by weight of acrylamide is added to the active measurement solution. A decrease in phosphorus acrylonitrile concentration is significantly maintained.

Moreover, as this invention, what was described in said (18) can be illustrated. That is, the nitrile hydratase of the present invention is represented by an α subunit represented by the sequence of 205 amino acids represented by SEQ ID NO: 1 of the sequence listing and a sequence of 226 amino acids represented by SEQ ID NO: 2 of the sequence listing. What is comprised by (beta) subunit is mentioned as a preferable example. In addition to these two subunits, metals, other peptides, and the like can be contained. Many metals especially contain iron and cobalt. It may also be a protein containing either of these subunits. The amino acid sequence of each subunit may have substitution, deletion or insertion of one or more amino acids in the amino acid sequence as long as it forms a complex with another subunit and has nitrile hydratase activity. Depending on the type, it is also expected to receive the formula after translation. In particular, in the α subunit of nitrile hydratase, the cysteine residue is often modified with cysteinesulfinic acid or cysteinesulfenoic acid after translation. Preferred examples include amino acid sequences in which 1 to 30 amino acids are modified, deleted, inserted, or translated after the amino acid sequence. More preferably, 1 to 10, more preferably 1 to 5, Most preferably 1 to 3. In addition, nitrile hydratase enzymes having an amino acid sequence having such substitutions, deletions, or insertions are also known site-directed mutagenesis methods, such as those described in Molecular Cloning 2nd Edition, Cold Spring Harbor Laboratory Press (1989). It can be obtained by introducing into a host microorganism and expressing it using DNA which introduce | transduced substitution, deletion, or insertion in the corresponding site | part of a base sequence as mentioned later, and it improves thermal stability, the improvement of organic solvent tolerance, or the change of substrate specificity Attempts can also be made to produce variant enzymes with added industrially desirable properties. In view of such technical level, the case where they have nitrile hydratase activity shall be included in this invention.

Moreover, although the thing of said (19) can be illustrated as this invention, this is demonstrated in detail in description of the DNA of this invention.

The nitrile hydratase having the above physicochemical properties can be obtained, for example, by culturing microorganisms belonging to the genus Geobacillus. The microorganism used in the present invention belongs to the genus Geobacillus, and any microorganism may be used as long as it is a microorganism having a hydration activity for converting a nitrile compound into an amide compound. The microorganisms belonging to the genus Geobacillus include Geobacillus caldoxylosilyticus, Geobacillus kaustophilus, Geobacillus lituanicus, Geobacillus and stearmophilus. Geobacillus stearothermophilus, Geobacillus subterraneus, Geobacillus thermocatenulatus, Geobacillus thermodenitrificans, Geocobacillus thermosis ), Geobacillus thermoleovorans, Geobacillus toebii, Geobacillus uzenensis. Moreover, it is not specifically limited to the microorganism derived from the genus Geobacilli, and the nitrile hydratase gene derived from another microorganism line is also included. Such microorganisms include the genus Agrobacterium, genus Achromobacter, genus Acinetobacter, genus Aeromonas, genus Enterobacter, genus Erwinia, and genus Erwinia. Genus Xanthobacter, Genus Klebsiella, Genus Corynebacterium, Genus Sinorhizobium, Genus Pseudomonas, Genus Streptomyces, Genocardia Genus Nocardia, Bacillus, Micrococcus, Rhodococcus, Rhodopseudomonas, Rhizobium, Pseudonocardia, etc. have. Specifically, in the present invention, screening was carried out by the following method. First, a small amount of soil collected from various places is taken into a test tube containing water or physiological saline, and shaken in a shaker at 65 ° C. for 2 to 14 days. A part of the culture solution is taken and placed in a liquid medium containing general microbial growth medium, for example, glycerol, polypeptone, yeast extract, and the like, and incubated for 1 to 7 days at a culture temperature of 65 ° C. A microorganism can be isolated by spreading a part of the culture solution obtained on the agar plate medium containing the above-mentioned microorganism growth medium component and culturing again at 65 ° C to form colonies. The microorganism obtained in this way was added to the above-mentioned medium component by using a test tube or a flask in which a liquid medium in which nitrile compounds such as n-valeronitrile or an amide compound such as methacrylamide was added was put in a suitable period, for example, about 12 minutes. For about 7 days, it grows by shaking culture at the culture temperature of 65 degreeC, and selects the target microorganism based on the said conventional method for measuring nitrile hydratase activity. The classification of the representative strains of such microorganisms was performed by 16 SrRNA and the following biochemical properties, and it turned out to be Geobacillus thermoglucosidesius (Geobacillus thermoglucosidasius), and Geobacillus thermoglucosidesius Q-6 (Geobacillus thermoglucosidasius Q-6). In the name of -6), it is deposited as number FERM P-19351 (repair day May 16, 2003) to Industrial Technology General Research Institute (1 Judai 6 Higashi, Tsukuba-shi, Ibaraki, Japan) in the name of independent administrative corporation As BP-08658, it was transferred to the deposit based on the Budapest Treaty (the receipt date March 11, 2004). Although various patents and literature studies have been conducted, there is no description regarding nitrile hydratase activity with respect to microorganisms belonging to geobacilli thermoglucosidesis. In view of this, geobacilli and thermoglucosidesis Q-6 strains are recognized as new strains. The amide compound can also be produced by using variants of the new strain, ie, mutants, cell fusion lines and genetically engineered strains derived from Geobacilli-thermoglucosidesis Q-6 strains. In addition, the properties of Geobacilli and thermoglucosidesis Q-6 strains are as follows.

(a) morphological properties

Culture condition: Nutritional agar (British oxoid) medium 60 degrees Celsius

1. Morphology and size of cells

   Form: Bacteria

   Size: 0.8 × 2.0 to 3.0 μm

2. The presence or absence of cell polymorphism:

3. Mobility or not: +

   The hair growth state of flagella: peritrichous

4. Spore presence:-

Site of spore: single grain

(b) culture properties

Culture condition: Nutritional agar (British oxoid) medium 60 degrees Celsius

1. Color: Cream color

2. Gloss: +

3. Pigment Production:

Culture conditions: Nutritional broth (UK England Oxoid) medium 60 degrees Celsius

1. Whether there is surface development:

2. The presence of medium turbidity: +

Culture condition: Gelatin puncture culture @ 60 degrees Celsius

1. Growth state: +

2. Gelatin liquefaction:

Cultivation conditions: litmus milk 60 ℃

1. Solidification:

2. Liquefaction:

(c) physiological properties

1. Gram dyeing: uneven

2. Reduction of nitrates:

2. Denitrification:

3. MR test:

4. VP test:

5. Generation of indole:

6. Production of Hydrogen Sulfide:

7. Hydrolysis of Starch:-

8. Use of citric acid

              Koser:

  Christensen:

9. Use of Inorganic Nitrogen Sources

     nitrate:-

   Ammonium salt: +

10. Production of Pigment:

11. Urease Activity:

12. Oxidase:

13. Catalase:

14. Scope of growth

    pH: 5.5 to 8.0

    Temperature: 45 degrees Celsius-72 degrees Celsius

15. Attitude to oxygen: breathable anaerobic

16. O-F test:

(d) acid production / gas production from sugars

1.L-arabinose-/-

2. D-xylose +/-

3. D-glucose +/-

4. D-mannose +/-

5. D-fructose +/-

6. D-galactose-/-

7. Maltose +/-

8. Sucrose +/-

9. Lactose-/-

10. Trehalose @ +/-

11.D-sorbitol #-/-

12.D-mannitol +/-

13. Inositol-/-

14. Glycerin-/-

(e) other properties

1. β-galactosidase activity:

2. Arginine Dihydrolase Activity:

3. Lysine decarboxylase activity:

4. Tryptophan deaminase activity:

5. Gelatinase Activity:

The culturing method of the microorganism used in the method of the present invention is carried out in accordance with a general microorganism culturing method, and both solid culture and liquid culture can be performed. Bacillus thermoglucosidesius (Geobacillus thermoglucosidasius), because it is an anaerobic microorganism of the anaerobic, can be cultured under the same culture conditions as conventional anaerobic microorganisms. The culture temperature may be appropriately changed in the range in which the microorganisms grow, but is, for example, in the range of 40 ° C to 75 ° C. The pH of the medium may be 4-9, for example. Especially in case of Bacillus thermoglucosidesis Q-6 (Geobacillus thermoglucosidasius Q-6), medium pH of 5-8 can be mentioned at the culture temperature of 45 to 72 degreeC, Preferably it is 55 to 70 degreeC. have. The incubation time depends on various conditions, but usually about 1 day to about 7 days is preferred. On the other hand, as a medium, various mediums containing an appropriate carbon source, nitrogen source, organic to inorganic salt, etc. used for a general microorganism are used suitably. As the carbon source, glycerol, glucose, sucrose, molasses, organic acids, animal and vegetable oils and the like can be used. Examples of nitrogen sources include yeast extract, peptone, malt extract, meat extract, urea and sodium nitrate. As organic or inorganic salts, sodium chloride, magnesium sulfate, potassium chloride, ferrous sulfate, manganese sulfate, cobalt chloride, zinc sulfate, copper sulfate, sodium acetate, calcium carbonate, monopotassium dihydrogen phosphate, dipotassium hydrogen phosphate and the like can be used. Can be. In the method of the present invention, in order to increase the nitrile hydratase activity of the microorganism used, nitrile compounds such as n-valeronitrile, isovaleronitrile and crotononitrile, and amide compounds such as methacrylamide are added to the medium. desirable. As the addition amount, for example, an appropriate amount in the range of 0.01 g to 10 g can be added to 1 L of the medium. Also preferably Fe or Co ions may be present in the 0.1 μg / ㎖ or more.

In order to obtain amino acid sequence information of the enzyme of the present invention, after purifying the enzyme of the present invention, each subunit is separated by reduced SDS-polyacrylamide electrophoresis, each band is cut out of the gel, and the protein sequence is obtained. A portion of the amino acid sequence can be determined by.

The invention also relates to DNA encoding nitrile hydratase. Specifically, DNA including the 695-1312 position of the nucleotide sequence of SEQ ID NO: 3 of the sequence list, and the DNA containing the 1-681 position of the nucleotide sequence of SEQ ID NO: 3 of the sequence list are exemplified, respectively, The β subunit is encoded, but is not limited thereto, and may be DNA containing this nucleotide sequence. Moreover, even if DNA which can hybridize with DNA containing a nucleotide sequence complementary to these sequences under strict conditions is included in this invention, it is included in this invention as long as it has nitrile hydratase activity. That is, the nitrile hydratase of this invention can be expressed using these DNA. Stringent conditions can be exemplified by the conditions described in the manual (Wash: 42 ° C., primary wash buffer containing 0.5 × SSC) using, for example, an ECL direct nucleic acid labeling and detection system (manufactured by Amarsham Pharmacia Biotech). Examples of DNA that can hybridize under stringent conditions include, for example, DNA including positions 695-1312 of the nucleotide sequence of SEQ ID NO: 3 in the strict sequence described above and 1-681 of the nucleotide sequence of SEQ ID NO: 3 in the sequence list. Any, usually 20 or more, preferably 50 or more, and particularly preferably 100 or more consecutive nucleotide sequences of the complementary nucleotide sequences in the DNA containing the above are used as a detection sample and hybridized thereto. One DNA can be exemplified.

The DNA encoding the nitrile hydratase of the present invention can be obtained by the following method. As long as there is no description in this specification, the genetic recombination technique, the production technique of a recombinant protein, and the analysis method which are known in the art are employ | adopted.

The DNA encoding the nitrile hydratase of the present invention is the nitrile hydratase of the present invention according to sequence information such as the nucleotide sequence disclosed in the present specification or the amino acid sequence, and optionally the amino acid sequence determined by the above-described purification enzyme. Microorganisms containing, for example, thermoglucosidesis Q-6 strains. From the library obtained by using the oligonucleotide synthesize | combined according to the amino acid sequence as a probe, the DNA fragment which digested the chromosomal DNA of the microorganism containing nitrile hydratase by restriction enzyme is introduce | transduced into phage or plasmid, and the host is transformed, DNA encoding the nitrile hydratase of the present invention can also be obtained by plaque hybridization or colony hybridization. Also, primers were prepared according to the amino acid sequence information of the N-terminals of both subunits determined by the above-described purified enzyme without using oligonucleotide as a probe, and a part of the nitrile hydratase gene was amplified by polymerase chain reaction (PCR). One can also be acquired by the same process as a probe. The obtained DNA was inserted into a plasmid vector, for example, pUC118, and cloned, and the nucleotide sequence was determined by a known method such as the dideoxy terminator method (Proceedings of the National Academy of Sciences. USA, 74: 5463-5467, 1977). You can decide. The gene thus produced is confirmed to be a DNA encoding nitrile hydratase by applying the expression product in the E. coli host transformed using the gene to the conventional method for measuring activity described above.

In another aspect, the present invention provides a recombinant vector characterized in that the DNA is linked to the vector.

The recombinant vector according to the present invention is linked downstream of a promoter region suitable for a host microorganism so that the 5 ′ terminal side of the DNA obtained by the above method can function, and a transcription termination sequence is inserted downstream thereof, if necessary, and It can be prepared by assembling into an expression vector.

Suitable expression vectors are not particularly limited as long as they can replicate and propagate in the host microorganism. If the host is capable of gene insertion into a chromosome, it is not necessary to have a region capable of autonomous replication in the host. For example, if E. coli is used as a host, a strong promoter such as a pUC or pGEX system including a promoter of lac, trp, tac, trc, T7, PL or pyruvate oxidase gene (Japanese Patent Publication No. 2579506), etc. and pET, pT7, pBluescript, pKK, pBS, pBC, pCAL, and the like. In addition, the α subunit gene and the β subunit gene may be expressed as independent cystrons from the respective promoters, and may be expressed as polycistrons by a common promoter. Furthermore, for independent cistrons, each subunit gene can be on a different vector.

Moreover, in this invention, when the DNA which expresses the gene downstream of the nitrile hydratase gene of this invention is assembled to said recombinant vector of nitrile hydratase, it discovers that the activity of a nitrile hydratase rises more, This vector is also provided. Specifically, as described above, a gene encoding a protein involved in the activation of nitrile hydratase, an α subunit gene of nitrile hydratase, and β sub using a plasmid vector containing a promoter or transcription termination factor required for expression. Each unit gene may be expressed as an independent cistron, and may be expressed as a polycistron by a common control region. Likewise, each gene can be on a different vector.

Therefore, the present invention provides a DNA encoding a protein involved in the activation of nitrile hydratase. Specifically, positions 1325-1663 of the nucleotide sequence of SEQ ID NO: 3 in the sequence listing can be exemplified, but are not limited to this, and may be DNA containing the nucleotide sequence. Moreover, it is DNA which can hybridize under stringent conditions with respect to DNA containing a base sequence complementary to these sequences, and is included in this invention as long as it is involved in activation of nitrile hydratase. In other words, the nitrile hydratase of the present invention can be further activated using these DNAs. Stringent conditions can be exemplified by the conditions described in the manual (Wash: 42 ° C., primary wash buffer containing 0.5 × SSC) using, for example, an ECL direct nucleic acid labeling and detection system (manufactured by Amarsham Pharmacia Biotech). As DNA which can hybridize under strict conditions, for example, any of the complementary nucleotide sequences in DNA including position 1325-1663 of the nucleotide sequence of SEQ ID NO: 3 in the sequence list under the strict conditions described above, usually 20 As mentioned above, Preferably 50 or more, Especially preferably, 100 or more base sequence is used as a detection sample, The DNA hybridized to this can be illustrated. In addition, the protein involved in the activation of the nitrile hydratase of the present invention is a protein represented by the sequence of 112 amino acids shown in SEQ ID NO: 4 of the sequence list, one amino acid sequence having the ability to participate in the activation of the nitrile hydratase It may have an amino acid sequence having a substitution, deletion, or insertion of one or a plurality of amino acids in, and it is naturally expected to receive a modification after translation, depending on the type of host. Preferred examples include amino acid sequences in which 1 to 25 amino acids are modified, deleted, inserted, or translated after the amino acid sequence, more preferably 1 to 10, more preferably 1 to 5, Most preferably 1 to 3.

In another aspect, the present invention provides a transformant characterized in that the above DNA is introduced into the host cell and transformed.

A transformant can be obtained by transforming a host cell using the expression vector prepared by the above method. Host cells include microorganisms, mammalian cells, plant cells and the like, and microorganisms are preferably used. Examples of the microorganisms include Escherichia coli as in the following examples, but are not particularly limited thereto, and include, but are not limited to, Bacillus genus, Pseudomonas genus, Corynebacterium genus, Brevibacterium genus, Streptococcus genus, Rhodococcus genus, Actinomycetes Yeast and the like.

As a method of introducing a gene into a host microorganism, it can be introduced into a preferred host according to any conventional method well known in the art such as, for example, transformation, transduction, conjugation transfer, or electroshock.

In addition, as the method for producing the nitrile hydratase of the present invention or the cell-treated product containing the same, as described above, a microorganism capable of producing the nitrile hydratase, for example, a microorganism of the genus Geobacillus, particularly preferably geobacillus thermoglucose The enzyme can be obtained by appropriately combining known purification methods from cultures of Sidesius Q-6 strains, but can also be obtained from transformants transformed using the nitrile hydratase gene as described above. .

In acquiring the enzyme, the method of culturing the microorganism of the genus Geobacillus is as described above, but it is preferable that the transformant is usually cultured in a medium containing a nutrient source capable of magnetizing these microorganisms. It can be cultured by a conventional method for producing. The culture can usually be a liquid culture or a solid culture. Carbohydrates such as glucose and sucrose; Alcohols such as sorbitol and glycerol; Organic acids such as citric acid and acetic acid; Carbon sources such as soybean oil or mixtures thereof; Nitrogen-containing inorganic organic nitrogen sources such as yeast extract, meat extract, ammonium sulfate and ammonia; Inorganic nutrients such as phosphate, magnesium, iron, cobalt, manganese and potassium; And a medium in which vitamins such as biotin and thiamine are appropriately mixed. More preferably, at least 0.1 µg / ml of Fe ions or Co ions may be present in such media components. It is preferable to perform culture conditions normally under aerobic conditions. The culture temperature is not particularly limited as long as it is a temperature capable of growing host microorganisms, but it can usually be exemplified by 5 to 80 ° C, preferably 20 to 70 ° C, more preferably 25 to 42 ° C. The pH during the culturing is not particularly limited as long as it is a pH capable of growing host microorganisms, but it can be exemplified that the pH is usually performed at pH 3-9, preferably pH 5-8, more preferably pH 6-7. .

Moreover, in this invention, a nitrile compound can be manufactured using the enzyme of this invention. In the use of the enzyme described above in the present invention, the degree of purification is not particularly limited as long as the action of the enzyme of the present invention is not impaired. The enzyme-containing substance can be used in addition to the purified enzyme of the present invention. The microorganism producing the enzyme or the transformant transformed by introducing the gene of the enzyme can be used. In the case of using microorganisms or transformants, the cells can be used. As the cells, cells which have increased the permeability of the compound by treatment with live cells or by treatment with a solvent such as acetone or toluene or lyophilization can be used. In some cases, it may be an enzyme-containing substance such as cell lysate or cell extract. Illustrating the preparation method of the cell-processing product containing the enzyme, the culture is first separated into a solid solution, and the obtained wet cell is suspended in a buffer solution such as a phosphate buffer or tris hydrochloric acid buffer if necessary, followed by sonication and French press. A cell nitrifying treatment such as a treatment or a grinding treatment using glass beads, or a cell wall lytic enzyme such as lysozyme or protease can be appropriately combined, and the enzyme can be extracted from the cells to obtain a crude nitrile hydratase-containing liquid. . This crude enzyme-containing liquid can be purified again by using isolation and purification means such as known proteins and enzymes, if necessary. For example, a crude enzyme-containing liquid is added to an organic solvent such as acetone or ethanol, and then precipitated separately or salted by adding ammonium sulfate or the like to precipitate and recover a fraction containing nitrile hydratase from an aqueous solution. It can be illustrated. Moreover, it can refine | purify by combining suitably anion exchange, cation exchange, gel filtration, affinity chromatography using an antibody or chelate, etc. Of course, enzymes, cells, and cell-treated products containing enzymes may be packed in a column by a known method, and may be immobilized on a carrier, and in particular, cells are embedded in polymers such as polyacrylamide gels. Can be. The cell or cell treated product is suspended in an aqueous aqueous solution such as a buffer such as water or a phosphate buffer solution, and the reaction is advanced by adding a nitrile compound thereto. The concentration of the cells or cells to be treated is 0.01 to 20% by weight, preferably 0.1 to 10% by weight. Preferably the upper limit of reaction temperature is 90 degreeC, More preferably, it is 85 degreeC, More preferably, 70 degreeC, The lower limit of reaction temperature is 1 degreeC, Preferably it is 4 degreeC, More preferably, it is 10 degreeC. The reaction pH is, for example, 5 to 10, preferably 6 to 8, and the reaction time is, for example, 1 minute to 72 hours. Further, by slowly dropping the nitrile compound, the amide compound can be produced and accumulated at a high concentration. In order to recover the amide compound from the reaction solution, a cell or a cell treated product is removed by filtration, centrifugation or the like, and then removed by a method such as crystallization.

Although an Example is given to the following and this invention is demonstrated in detail, these Examples do not limit the scope of the present invention.

Example 1 Cell Separation

A small amount (about 1 g) of soil collected from the hot spring vicinity of Saitama Prefecture was taken into a test tube containing 5 ml of physiological saline, and cultured in a shaker at 65 ° C. for 3 days. A portion of the culture solution (0.5 ml) was taken and added to a medium (pH 7.0) containing 1.0 wt% glucose, 0.5 wt% polypeptone, and 0.3 wt% yeast extract, followed by reciprocating shaking culture at 65 ° C for 2 days. A part of the culture solution (0.1 ml) thus obtained was spread on agar plate medium containing the above-mentioned media components, incubated at 65 ° C for 2 days to form colonies, and microorganisms were isolated. The isolated microorganism was inoculated into a liquid medium in which 0.1 wt% of n-valeronitrile was added to the medium having the same composition as above, followed by incubation at 65 ° C for 24 hours to obtain a culture solution having a high nitrile magnetization microorganism. 1 ml of this culture solution was added to 9 ml of 1.1% by weight of acrylonitrile solution (0.05 M-phosphate buffer pH7.7), and the reaction was started at a reaction temperature of 27 ° C. After 10 minutes, the reaction was stopped by adding 1 ml of 1N HCl. A part of the reaction solution was analyzed by liquid chromatography (HPLC) to assay for the presence or absence of acrylamide production, thereby screening microorganisms having nitrile hydratase activity. In this way, Geobacilli-thermoglucosidesis Q-6 strain was obtained as a microorganism which has a hydration activity which converts a nitrile compound into an amide compound.

(Liquid chromatography analysis conditions)

 The body: Hitachi (HITACHI) D-7000 (product made by Hitachi)

 Column; Inertsil ODS-3 (made by GL Science Corporation)

 Length; 200mm

 Column temperature; 35 ° C

 Flow rate; 1ml / min

 Sample injection volume; 10 μl

 Solution: 0.1 wt% phosphoric acid aqueous solution

Example 2: Upper growth temperature of cell culture

Geobacillus thermoglucosidesis Q-6 strain obtained in Example 1 was applied to agar plate medium containing the media component used in Example 1, and cultured at a plurality of different temperatures to investigate the growth of the cells. did. The results are shown in Table 1. Geobacilli and thermoglucosidedesus Q-6 strains showed normal growth up to 70 ° C, and growth was possible at 72 ° C.

Evaluation criteria:-did not multiply, + multiplied, + + multiplied well Culture temperature (℃) Proliferation of Geobacilli and Thermoglucosidesis Q-6 20 － 30 － 40 － 50 + 60 + 65 + 70 + 72 + 75 －

Example 3: Determination of nitrile hydratase activity in cells of Geobacilli and thermoglucosidesis Q-6 strains and their temperature dependence

Glycerol 0.2% by weight, 0.2% by weight trisodium citrate dihydrate, 0.1% by weight potassium dihydrogen phosphate, 0.1% by weight hydrogen dipotassium phosphate, 0.1% by weight polypeptone, 0.1% by weight yeast extract, 0.1% by weight sodium chloride, n- 500 ml Erlenmeyer flask with 100 ml of sterile-filled medium (pH7.0) containing 0.1 wt% of valeronitrile, 0.02 wt% of magnesium sulfate heptahydrate, 0.003 wt% of iron (II) sulfate heptahydrate, and 0.0002% of cobalt chloride hexahydrate 1 ml of a culture solution of Geobacilli and thermoglucosidesis Q-6 strains previously cultured in the medium was inoculated. This was subjected to rotation shaking culture at 200 ° C / min at 65 ° C for 1 day to obtain a cell culture solution. The cells were collected by centrifugation (10000 x g, 15 minutes) from 300 ml of the cell culture medium of Giobacilli thermoglucosidicus Q-6 strain, washed with 0.05 M phosphate buffer (pH7.5), and then 50 ml of the same buffer solution. Suspended in The cell suspension thus prepared was reacted for 5 minutes by the method described above to measure the hydration activity of converting the nitrile compound into an amide compound. When the unit (unit) of the enzyme activity is determined to be 1 unit (hereinafter referred to as U) for converting 1 mol of acrylonitrile to acrylamide in 1 minute, nitrile hydrata per wet weight at 27 ° C Aze activity (U / mg) was 9.37 U / mg. Further, a cell suspension containing 0.5% by weight of acrylonitrile was prepared so as to be 5 U / ml at 10 ° C, and the cell suspension was used in the same manner under the conditions of 30 ° C, 40 ° C, 50 ° C, 60 ° C, and 70 ° C. Nitrile hydratase activity was determined and shown in Table 2. As a result, the optimum temperature when the cells were used for the reaction was in the vicinity of 60 ° C., and showed particularly high activity in the high temperature region.

Reaction temperature (℃) Nitrile hydratase activity (U / ml) 10 5.0 30 19.2 40 39.4 50 49.2 60 50.6 70 42.4

Example 4 Thermal Stability of Nitrile Hydratease in Cells of Geobacilli and Thermoglucosidesis Q-6

In order to investigate the thermal stability of the activity of nitrile hydratase in the cells of Geobacillius thermoglucosidesis Q-6 strain, the cells obtained by the culture method of Example 3 were suspended in distilled water at 10 U / ml, and the cells were suspended for 30 minutes. The remaining activity was measured by heat-retaining treatment at temperature. 0.5 mL of 1% by weight acrylonitrile solution (0.05 M potassium phosphate buffer, pH7.5) was added to the cell solution after the heat treatment of 0.5 mL, and the reaction was started while stirring at 27 ° C. After 5 minutes, the reaction was stopped by adding 100 µL of 1 N hydrochloric acid. The activity after the preservation treatment with respect to the activity before the preservation treatment is calculated and shown in Table 3 as a converted value based on the activity before the preservation treatment as a reference (100). From this result, it can be said that the enzyme activity of nitrile hydratase in the cells of Geobacilli and thermoglucosidesis Q-6 strains can be stably maintained even at a high temperature, and can be maintained at 80% or more even at a high temperature of 70 ° C. 30% or more of activity can be maintained even at a high temperature of 80 ° C.

Treatment temperature (℃) Active (U / ml) Remaining activity (%) 30 5 100 40 4.8 96 50 4.5 90 60 4.4 88 70 4.2 84 80 1.6 32

Example 5 Reactions Using Various Nitrile Compounds as Substrates

The nitrile hydratase activity of converting the various nitrile compounds described in Table 4 below into the corresponding amide compounds was investigated. A 1 ml cell suspension was added to 9 ml of a 1.1% nitrile solution (0.05 M potassium phosphate buffer, pH7.5) and the reaction was started at a reaction temperature of 30 deg. After 10 minutes, the reaction was stopped by adding 1 ml of 1N HCl. The analysis conditions by HPLC were the same as Example 1, but the solution was made into distilled water containing 10 wt% acetonitrile. As a result, all of the nitrile compounds listed in Table 4 were used as substrates and had nitrile hydratase activity.

Disclosure nitrile compounds Adiponitrile n-butyronitrile Acetonitrile Hexanenitrile Isobutyronitrile Benzonitrile n-valeronitrile

Amino acid sequences of nitrile hydratase α subunit (ORF2), β subunit (ORF1) and nitrile hydratase activating factor (ORF3) derived from Geobacilli and thermoglucosidesis Q-6 strains described in Example 6 and later The flow of the invention for elucidating the nucleotide sequence is summarized below.

Cells obtained by culturing G. Bacillus thermoglucosidesis Q-6 strain were crushed, and then precipitated with ammonium sulfate, anion exchange column chromatography, DEAE column, hydroxy apatite column, gel filtration chromatography, Dialysis was carried out to purify the nitrile hydratase enzyme.

Determine the amino acid sequence of about 30 residues of the N terminal of the purified nitrile hydratase α subunit and β subunit, and degenerate the oligonucleotide degenerate for gene amplification in consideration of the use of codons of amino acids based on the bacteria. A primer was prepared, and amplified DNA fragments were obtained by degenerate PCR using the chromosomal DNA extracted from the cells as a template. The amplified DNA fragment was cloned and the base sequence of the inserted fragment was determined. The sequence cloned by comparing the amino acid sequence estimated from the nucleotide sequence with the N terminal amino acid sequences of nitrile hydratase α subunit and β subunit purified from Geobacilli and thermoglucosidesis Q-6 strains was nitrile hydratase. You have verified that you are coding.

As a result, it was found that the nitrile hydratase genes were adjacent to each other in the order of β subunit and α subunit in the upstream of the 5 ′ terminal in Geobacilli and thermoglucosidesis Q-6 strains.

Oligonucleotide degenerate primers for gene amplification were prepared using sequences of high homology in the downstream genes of various known nitrile hydratase α subunits, and degenerate PCR using chromosomal DNA extracted from the cells as a template. Amplified DNA fragments were obtained. The amplified DNA fragment of the obtained α subunit portion of the bacterium was cloned to determine the nucleotide sequence.

The nitrile hydratase α subunit and β subunit of the Geobacilli and thermoglucosidesis Q-6 strain obtained from the above were introduced into a suitable expression vector. The appropriate host bacterium was transformed using the constructed expression plasmid. Examples of the host include Rhodococcus, Coryne, E. coli and the like. Preferably a host without amidase is preferred. In addition, acrylamide was produced by contacting the cells obtained by culturing the obtained transformant with acrylonitrile in an aqueous medium, and the production efficiency and the nitrile hydratase activity were compared.

Next, the DNA fragment obtained above was colonized with a probe to clone the peripheral genes including the downstream genes of nitrile hydratase α subunit and β subunit of the Geobacilli and thermoglucosidesis Q-6 strain.

The downstream gene was co-expressed with nitrile hydratase α subunit and β subunit to compare nitrile hydratase activity. As a result, it was found that the downstream gene is a gene involved in activation that significantly increases nitrile hydratase activity.

Example 6: Purification of nitrile hydratase enzyme derived from Geobacillus thermoglucosidesis Q-6 strain

The nitrile hydratase active fraction was purified by culturing Giobacilli and thermoglucosidesis Q-6 strains and providing them to various columns.

The measuring method of the nitrile hydratase active fraction in chromatography was as follows. 1% by weight of acrylonitrile was added to the eluate of each fraction diluted with HEPES buffer (100 mM, pH 7.2) and allowed to react at 27 ° C for 1 minute. The reaction was stopped by adding 1N HCl to 10% by volume of the reaction solution, and the resulting acrylamide concentration was measured by the HPLC analysis method described in Example 1.

In order to purify the nitrile hydratase enzyme derived from geobacillus thermoglucosidesis Q-6 strain, V / F medium containing 0.1 wt% of n-valeronitrile (0.2 wt% glycerol, 0.2 trisodium citrate dihydrate 0.2) % By weight, 0.1% by weight potassium dihydrogen phosphate, 0.1% by weight hydrogen dipotassium phosphate, 0.1% by weight polypeptone, 0.1% by weight yeast extract, 0.1% by weight sodium chloride, 0.1% by weight n-valeronitrile, magnesium sulfate heptahydrate Geobacilli-thermoglucosidesis Q-6 strain was inoculated in 0.02 weight%, 0.003 weight% of iron (II) sulfate hydrates, and 0.0002 weight% of cobalt chloride hexahydrates, and it cultured at 65 degreeC for 24 hours. For culture, 2 ml of a 96 well deep bottom plate (COSTAR) was used. After the completion of the culture, the cells were collected by centrifugation at 8000 g for 10 minutes, and 3 g of the obtained wet cells were resuspended in 20 mL of HEPES buffer (100 mM, pH 7.2). The cells were pulverized with an ultrasonic crusher under cooling, and ammonium sulfate (30% saturation concentration) was added to the cell pulverized solution, gently stirred at 4 ° C. for 30 minutes, and centrifuged at 20000 g for 10 minutes to obtain supernatant. Ammonium sulfate (70% saturation concentration) was added to the centrifugal supernatant, gently stirred at 4 ° C. for 30 minutes, and the precipitate obtained by centrifugation at 20000 g for 10 minutes was reused in 9 ml of HEPES buffer (100 mM, pH 7.2). It decomposed | dissolved, and dialyzed at 4 degreeC in 1 L of the same liquids for 24 hours, and it provided to the anion exchange chromatography (Amarsham Biosciences Co., Ltd .; HiTrap® DEAE®FF (5 mL x 5 column volumes)). The developing solution was eluted by linearly increasing the potassium chloride concentration from 0.0M to 0.5M using HEPES buffer (100 mM, pH 7.2) to obtain a fraction containing nitrile hydratase activity. The fraction was subjected to apatite column chromatography (BIO-RAD, CHT2-I (column volume 2 mL)). A 0.01 M aqueous potassium phosphate solution (pH 7.2) was used as a developing solution, and the fraction was eluted by linearly increasing potassium phosphate from 0.01 M to 0.3 M to obtain a fraction containing nitrile hydratase activity. The fractions were subjected to gel filtration chromatography (Amarsham Biosciences; Superdex: 200 HR 10/30) using 0.05M aqueous sodium phosphate solution (pH7.2) containing 0.15M NaCl as a developing solution. , Nitrile hydratase active fraction was obtained. The following example was performed using the nitrile hydratase active fraction of the gel filtration chromatography thus obtained.

Example 7: Reaction temperature dependence of nitrile hydratase in nitrile hydratase active fraction purified from Geobacilli and thermoglucosidesis Q-6 strains

The nitrile compound was amide at the reaction temperature shown in Table 5 with respect to the nitrile hydratase active fraction solution (3.2 mg / mL, 0.05 M phosphate buffer (pH7.5)) derived from Giobacilli thermoglucosidesis Q-6 strain. Nitrile hydratase activity was converted to compound. A nitrile hydratase active fraction solution was added to 1 mL of 0.5% by weight acrylonitrile solution (0.05 M potassium phosphate buffer, pH7.5), and the reaction was initiated while stirring at each temperature. After 2 minutes, the reaction was stopped by adding 100 µL of 1N hydrochloric acid. The unit (unit) of the enzyme activity is defined as 1 unit (hereinafter referred to as U) for converting 1 mol of acrylonitrile into acrylamide in 1 minute, and the hydration activity (U / mg) per weight of enzyme is given in Table 5 Indicated. From this result, the activity of the nitrile hydratase in the nitrile hydratase active fraction refine | purified from the geobacillus thermoglucosidesis Q-6 strain rises with the rise of reaction temperature to the high temperature of 60 degreeC. The optimum temperature is considered to be around 60 ° C. similarly to the case where the cells are used for the reaction, and shows very high nitrile hydratase activity even at a high temperature of 70 ° C.

Reaction temperature (℃) Active (U / mg) 20 210.4 27 550.7 40 1135.3 50 2228.8 60 2823.3 70 2781.1

Example 8 Thermal Stability of Nitrile Hydratease Purified from Geobacillus Thermoglucosidesis Q-6

Nitrile hydratase active fraction solution (3.2 mg / ml, 0.05 M phosphate buffer (pH7.5)) to investigate the thermal stability of the activity of nitrile hydratase purified from Geobacillus thermoglucosidesis Q-6 strain. Was heat-retained at predetermined temperature for 30 minutes, and residual activity was measured. To 1 mL of 0.5% by weight acrylonitrile solution (0.05 M potassium phosphate buffer, pH7.5) was added 5 µl of the nitrile hydratase solution after thermal treatment, and the reaction was started while stirring at 27 ° C. After 2 minutes, the reaction was stopped by adding 100 μl of 1N HCl. The activity after the preservation treatment (residual activity) with respect to the activity before the preservation treatment is calculated and shown in Table 6 as a converted value based on the activity before the preservation treatment as reference (100). From this result, it can be said that the enzyme activity of nitrile hydratase in the aqueous solution in the nitrile hydratase active fraction purified from the geobacilli thermoglucosidesis Q-6 strain was maintained stably even at high temperature. 60% or more of activity can be maintained even at high temperature, and 35% or more of activity can be maintained even at a high temperature of 70 ° C.

Treatment temperature (℃) Remaining activity (%) 20 89.2 27 85.6 40 82.3 50 78.9 60 66.9 70 38.8

Example 9 Acrylonitrile Concentration Dependency and Concentration Tolerance of Nitrile Hydratase Purified from Geobacilli and Thermoglucosidesis Q-6

Nitrile hydratase purified from Giobacilli thermoglucosidicus Q-6 strain, 4 μL nitrile hydratase active fraction solution (3.2 mg / mL, 0.05M phosphate buffer (pH7.5)) was added to a 5 mL solution (0.05 M potassium phosphate buffer, pH7.5) containing various weight percent acrylonitrile, and the reaction was started with stirring at 27 ° C. After 5 minutes, 10 minutes, 20 minutes, and 40 minutes, 1 ml each was taken out, the reaction was stopped by adding 100 µL of 1N HCl, and the concentration of the resulting acrylamide was quantified by HPLC and shown in Table 7. From these results, it can be said that the enzymatic activity of nitrile hydratase in the aqueous solution in the nitrile hydratase active fraction purified from Geobacilli and thermoglucosidesis Q-6 strains is maintained stably even at high acrylonitrile concentrations. . Even if it was made to react for 40 minutes in the high concentration acrylonitrile solution of 6%, the fall of activity was not observed compared with the case of lower acrylonitrile concentration, On the contrary, activity rose with the increase of substrate concentration.

Acrylonitrile concentration (weight%) at the start of reaction Acrylamide concentration after 5 minutes (% by weight) Acrylamide concentration after 10 minutes (weight%) Acrylamide concentration after 20 minutes (weight%) Acrylamide concentration (weight%) after 40 minutes 0.5% 0.018 0.032 0.052 0.087 2.0% 0.023 0.042 0.070 0.107 4.0% 0.026 0.046 0.077 0.137 6.0% 0.030 0.051 0.082 0.169

Example 10 Acrylamide Concentration Resistance of Nitrile Hydratease Purified from Geobacilli and Thermoglucosidesis Q-6

In regard to nitrile hydratase purified from Giobacilli thermoglucosidesis Q-6 strain, 10 μl of nitrile hydratase active fraction solution (3.2 mg / mL, 0.05M) was investigated to investigate inhibition by acrylamide as a product. Phosphoric acid buffer (pH7.5)) was added to 1 ml of a solution containing 0.5 wt% acrylonitrile and 35 wt% acrylamide (0.05 M potassium phosphate buffer, pH7.5) and reacted for 10 minutes with stirring at 27 ° C. When acrylonitrile concentration in the liquid after reaction was quantified by HPLC, all the acrylonitrile was converted into acrylamide. From these results, the enzyme activity of nitrile hydratase in the aqueous solution in the nitrile hydratase active fraction purified from Geobacilli and thermoglucosidedes Q-6 strain was maintained at high acrylamide concentration of 35%. I can speak.

Example 11: Cloning of genes of nitrile hydratase β subunit and α subunit moiety derived from Geobacilli and thermoglucosidesis Q-6

(1) Identification of nitrile hydratase enzyme from geobacilli and thermoglucosidesis Q-6 strain and N terminal amino acid sequence determination

Nitrile hydratase active fraction eluate in gel filtration chromatography obtained in Example 6 was subjected to reduced SDS-polyacrylamide electrophoresis under reducing conditions. After electrophoresis, destaining by protein staining with Coomassie Brilliant Blue (CBB) identified two major bands with molecular weights of about 25K Daltons and about 28K Daltons. These two major purified proteins are transferred to PVDF membranes (manufactured by MILLIPORE) using a blotting device (Bio-Rad Corporation) and stained with CBB, and the portions of the two bands of interest are adsorbed onto the PVDF membranes. Cut out from Next, the N terminal amino acid sequence of two types of proteins was decoded using the fully automatic protein primary structure analyzer PPSQ-23A (Shimadzu Corporation). As a result, the N-terminal amino acid sequence of the protein having a molecular weight of 25K daltons was the sequence described in SEQ ID NO: 23 in the sequence listing, and the N-terminal amino acid sequence of the protein having a molecular weight of 28K Dalton was the sequence described in SEQ ID NO: 24 in the listing.

As a result of comparison with the amino acid sequence of the known nitrile hydratase enzyme, the 25K dalton polypeptide chain showed low homology with the nitrile hydratase α subunit and the 28 K dalton polypeptide chain with the nitrile hydratase β subunit. It has been suggested to encode the protein.

(2) Synthesis of Oligonucleotide Primer Corresponding to N-Term Amino Acid Sequence

From the N-terminal amino acid sequence of the two types of proteins decoded above, the following 12 types of oligonucleotide primers for degenerate PCR were synthesized based on the use of the above codon. Primer 1 (αF1) described in SEQ ID NO: 5 of the Sequence Listing, Primer 2 (αF2) described in SEQ ID NO: 6 of the Sequence Listing, Primer 3 (αF3) described in SEQ ID NO: 7 of the Sequence Listing, and SEQ ID NO: 8 of the Sequence Listing Primer 4 (αR1), Primer 5 (αR2) as shown in SEQ ID NO: 9, Primer 6 (αR3) as shown in SEQ ID NO: 10 in Sequence Listing, Primer 7 (βF1) as shown in SEQ ID NO: 11 in Sequence Listing, Sequence Listing Primer 8 as described in SEQ ID NO: 12 (βF2), primer 9 as described in SEQ ID NO: 13 (βF3), primer 10 as described in SEQ ID NO: 14 (βR1) as listed in SEQ ID NO: 15 (βR2), Primer 12 (βR3) described in SEQ ID NO: 16 of the Sequence Listing. And y represents c or t, r represents a or g, m represents a or c, k represents g or t, s represents c or g, w represents a or t, d Represents a, g or t, and n represents a, c, g or t. In addition, primers were prepared in consideration of the positions on the chromosomes of the genes encoding the α subunit and the β subunit.

(3) Extraction and Degenerate PCR of Chromosome DNA from Geobacilli and Thermoglucosidesis Q-6

Q6 strain of Geobacilli and thermoglucosidedesius were cultured and recovered in the same manner as in Example 6, using a QIAGEN Genomic-Tip System (500 / G) kit. The chromosomal DNA was extracted from the cells. Degenerate PCR was carried out using 0.1 µg of chromosomal DNA of Geobacilli and thermoglucosidesis Q-6 strains dissolved in TE solution as a template. Degenerate PCR performed 36 combinations of primers 1 to 6 described in SEQ ID NOs: 5 to 10 in the sequence listing and primers 7 to 12 described in SEQ ID NOs: 11 to 16 in the Sequence listing. Two 100 mmol primers were subjected to degenerate PCR using 100 µl of a reaction solution containing 5 U of Ex Taq® DNA polymerase from Takara Co., Ltd. and a buffer solution, and amplification of DNA fragments was attempted. Reaction conditions are as follows. After 96 DEG C and 3 minutes of heat denaturation, 96 DEG C, 30 seconds of heat denaturation, 42 DEG C, 30 seconds annealing, 72 DEG C, 1 minute 30 seconds extension reaction was performed 35 cycles, followed by extension reaction of 72 DEG C and 5 minutes. Cooled at ℃. Each PCR product was subjected to 1% by weight agarose electrophoresis, and the amplification of the DNA was confirmed. As a result, primer 5 (αR2) described in SEQ ID NO: 9 in the Sequence Listing and primer 7 described in SEQ ID NO: 11 in the Sequence Listing. Amplification of the DNA fragment of about 700 bp was confirmed only by PCR performed with a combination of (βF1) and primer 5 (αR2) as set out in SEQ ID NO: 9 in the Sequence Listing and primer 8 (βF2) as set out in SEQ ID NO: 12 in the Sequence Listing. It became.

(4) Cloning of Degenerate PCR Products and Nucleotide Sequence Translation of Amplified DNA Fragments

The amplified DNA fragments were cut from the gel, extracted using QIAquick Gel® Extraction Kit (QIAGEN), and extracted with T4DNA ligase (p4) from the pGEM-T® vector (Promega). I ligated using Takara). As a result of PCR by Ex Taq, the property of adding one base to the 3 ′ terminal was used. After the ligation reaction, E. coli JM109 strains were transformed, and LB agar medium (50 μg / ml ampicillin, 0.5 wt% bacterium yeast extract, 1 wt% bactotriptone, 0.5 wt% NaCl, 2.0 wt% bacterium agar (pH7. 5)) was incubated overnight at 37 ℃ to select a transformant with ampicillin. Plasmid DNA was extracted by a conventional method from a transformant cultured in LB medium containing ampicillin, and the base sequence was deciphered using an insert sequence of about 700 bp and the sequences of the SP6 and T7 promoters on the vector as primers.

As a result, an open reading frame of 681 bp in the amplified DNA fragment (hereinafter referred to as ORF1) was confirmed. It was 13bp between the translation stop codon of ORF1 and the translation start codon ATG of the next open reading frame (henceforth called ORF2). The 25 amino acid sequences of the N-terminal side estimated from the nucleotide sequence of ORF1 and the 25-amino acid sequence of the N-terminal side of the 28K Dalton polypeptide chain refine | purified above completely correspond, and the amino acid sequence of SEQ ID NO: 2 of a sequence list It corresponds to the first to 25th sequences. The amino acid sequence of ORF1 shows low homology with the amino acid sequence of the β subunit of known nitrile hydratase, and it has been suggested to encode the protein.

The nitrile hydratase β subunit of Giobacilli thermoglucosidesis Q-6 strain encodes 226 amino acids, and the degree of concordance of the amino acid sequence with the homologous protein in the existing database is cleve in order from the higher one. Nitrile hydratase β subunit of Siela genus MC12609 strain 43%, nitrile hydratase β subunit of Agrobacterium 42%, and 40% with nitrile hydratase β subunit of Rhodoshudomonas CGA009 strain. In addition, the degree of amino acid correspondence with a protein derived from Bacillus genus, which is closely related to the genus Bacillus genus, 35.0% of nitrile hydratase β subunit of thermophilic Bacillus BR449 strain, and nitrile hydra of thermophilic bacillus Smisch SC-J05-1 strain It was extremely low (34.5%) with the acetylase subunit. On the other hand, the nitrile hydratase β subunit of thermophilic Bacillus BR449 strain and the nitrile hydratase β subunit of thermophilic Bacillus smice SC-J05-1 strain have high concordance of 85.6%. Moreover, the amino acid sequence of the N terminal estimated from the nucleotide sequence of ORF2 and the amino acid sequence of the N terminal of the 25K Dalton polypeptide chain refine | purified above were exactly identical.

From the above, in the Q-6 strain of Geobacilli and thermoglucosidicus, genes encoding a 28K dalton nitrile hydratase β subunit and a gene encoding a 25K dalton nitrile hydratase α subunit It turns out that it exists adjacent in this order.

(5) Cloning of gene of geobacillus thermoglucosidesis Q-6 nitrile hydratase α subunit moiety

The degenerate PCR was performed to clone the genes of the genes of the nitrile hydratase β subunit moiety and the gene of the α subunit moiety obtained from the above-described geobacilli thermoglucosidesis Q-6 strain. The following two kinds of oligonucleotide primers for degenerate PCR were prepared with reference to the gene located downstream of the known nitrile hydratase α subunit. Primer 13 (pR1) set forth in SEQ ID NO: 17 of the Sequence Listing, primer 14 (pR2) set forth in SEQ ID NO: 18 of the Sequence Listing.

In addition, the following two kinds of oligonucleotide primers for PCR amplification were prepared in the nitrile hydratase β subunit of the geobacilli and thermoglucosidesis Q-6 strains obtained by deciphering the nucleotide sequence. Primer 15 (Q6AposF) described in SEQ ID NO: 19 of the Sequence Listing, primer 16 (Q6abF1) described in SEQ ID NO: 20 of the Sequence Listing. Degenerate PCR was carried out using 0.1 μg of chromosomal DNA of Geobacilli and thermoglucosidesis Q-6 strains as a template. The degenerate PCR performed four combinations of primers 13 and 14 described in SEQ ID NOs: 17 and 18 in the sequence listing and primers 15 and 16 described in SEQ ID NOs: 18 and 19 in the annealing temperature at 50 ° C. As a result, the presence of the amplified DNA product of about 0.8 kb was confirmed by PCR performed by the combination of primer 13 (pR1) described in SEQ ID NO: 17 of the Sequence Listing and primer 15 (Q6AposF) described in SEQ ID NO: 18 of the Sequence Listing. In addition, the presence of 1.5k amplified DNA product was confirmed by PCR performed by the combination of primer 13 (pR1) described in SEQ ID NO: 16 of the Sequence Listing and primer 16 (Q6abF1) described in SEQ ID NO: 19 of the Sequence Listing. In the case of PCR performed in other combinations, the presence of the amplified DNA product could not be confirmed.

A 0.8 kb DNA fragment amplified by degenerate PCR by a combination of Primer 13 (pR1) as set out in SEQ ID NO: 16 in Sequence Listing and Primer 15 (Q6AposF) as set out in SEQ ID NO: 19 in Sequence Listing was cut out from an agarose gel. DNA was extracted by a conventional method, assembled into a pGEM-T vector (Promega) to transform E. coli JM109 strain, and recombinants were selected by 50 μg / ml ampicillin. The transformant was cultured in LB medium containing ampicillin, plasmid DNA was extracted by a conventional method, and the base sequence of the insert portion of about 0.8 kb was read. As a result, an open reading frame of 618 bp (hereinafter referred to as ORF2) was confirmed. The 29 amino acid sequences of the N-terminal side estimated from the nucleotide sequence of ORF2 and the 29 amino acid sequences of the N-terminal side of the 25K Dalton polypeptide chain refine | purified above completely correspond, and the amino acid sequence of sequence number 1 of a sequence list is described. It corresponds to the 1st to 29th sequence of. The amino acid sequence of ORF2 shows low homology with the amino acid sequence of a known nitrile hydratase α subunit, and it has been suggested to encode the protein.

The nitrile hydratase α subunit of the geobacilli thermoglucosidesis Q-6 strain encodes 205 amino acids, and the degree of concordance of the amino acid sequence with the homologous protein in the existing database is higher than that of thermophilic bacteria. It is 66.3% with nitrile hydratase β subunit of Bacillus BR449 strain and 63.9% with nitrile hydratase β subunit of Bacillus bacilli S. SC-J05-1 strain. On the other hand, the nitrile hydratase β subunit of thermophilic Bacillus BR449 strain and the nitrile hydratase β subunit of thermophilic Bacillus smice SC-J05-1 strain have 88.8% and have high homology.

Example 12 Assembly of nitrile hydratase α-subunit and β-subunit moieties of geobacilli-thermoglucosidesis Q-6 strains into expression vectors and expression in E. coli

(1) Assembly of nitrile hydratase α subunit and β subunit moiety of geobacillus thermoglucosidesis Q-6 strain into expression vector

Based on the base sequence read out above, two types of oligonucleotide primers for amplifying nitrile hydratase α subunit and β subunit moiety by PCR were prepared. Primer 17 (Q6ab-F1-T) described in SEQ ID NO: 21 of the Sequence Listing, and primer 18 (Q6ABall-R1-BglII-T) described in SEQ ID NO: 22 of the Sequence Listing. Primer 17 described in SEQ ID NO: 21 of the Sequence Listing designed a translation initiation codon of nitrile hydratase β subunit of the geobacilli-thermoglucosidesis Q-6 strain in the restriction enzyme site NdeI. In primer 18 described in SEQ ID NO: 22 of the Sequence Listing, a restriction enzyme BglII region was introduced just below the translation end codon of the nitrile hydratase α subunit of the geobacilli-thermoglucosidesis Q-6 strain. PCR was carried out using chromosomal DNA of the Geobacillus thermoglucosidesis Q-6 strain as a template using 100 mmol of primers 17 and 18 described in SEQ ID NOs: 21 and 22, respectively. PCR was performed under conditions of 96 ° C. and 3 minutes of thermal denaturation at 96 ° C. and 3 minutes using Ex Taq DNA polymerase, followed by elongation reaction at 96 ° C. for 30 seconds, heat denaturation at 60 ° C., 30 seconds for annealing, and 72 ° C. for 1 minute and 30 seconds. After cycling, it extended | stretched for 5 minutes at 72 degreeC, and then cooled to 4 degreeC. The solution after PCR was subjected to 1.5% by weight agarose electrophoresis to confirm DNA fragment amplification of about 1.3 kb. This amplified DNA product was extracted from the agarose gel by a conventional method and ligated to pGEM-T easy vector from Promega to transform E. coli JM109 strain. Plasmid DNA was extracted from the transformant to decode the nucleotide sequence of the insert portion, and it was confirmed that there was no error in amplification by PCR.

Next, this plasmid was digested with NdeI and EcoRI restriction enzymes, subjected to 1.5% by weight agarose electrophoresis, and about 1.3 Kb of insert DNA was cut out of the agarose gel and extracted by a conventional method. As an expression vector, the pET-26b (+) 'vector and the pET-28a (+)' vector by Novagene were used. These two types of vector DNA were digested with NdeI and EcoRI restriction enzymes and subjected to 1% by weight agarose electrophoresis, and about 5.3 Kb of DNA fragments were extracted by a conventional method. These inserts and vectors were ligated according to a conventional method to transform E. coli JM109 strain, plasmid DNA was extracted from a transformant selected for kanamycin resistance, and an insert-inserted plasmid was selected. From the above, the expression plasmid into which nitrile hydratase α subunit and β subunit moiety of geobacilli thermoglucosidesis Q-6 strain were introduced as an insert was obtained. These completed plasmids are referred to as pET-26b (+)-βα and pET-28a (+)-βα below.

(2) Nitrile hydratase activity of Geobacilli and thermoglucosidesis Q-6 strains expressed in Escherichia coli

The expression plasmids pET-26b (+)-βα and pET-28a (+)-βα were used to transform Novagen's Escherichia coli BL21 (DE3) LysE strain, and nitrile hydra of Geobacilli-thermoglucosidesis Q-6 strain. Proteins of the kinase β subunit and α subunit were coexpressed as polycistrons by the T7 promoter.

Each expression plasmid was transformed into Novazen Co. BL21 (DE3) LysE strain competing cells, and the LB agar medium containing 0.5 µg / ml kanamycin (0.5 wt% bacterium yeast extract). , 1% by weight bactotryptone, 0.5% by weight NaCl, 2.0% by weight agar; This transformant was inoculated into 2 ml of LB medium containing 30 µg / ml kanamycin, and the culture was shaken at 200 ° C. overnight at 200 rpm. The whole culture was inoculated with 2% by weight in 10 ml of LB medium containing 20 µg / ml of cobalt chloride hexahydrate (CoCl ₂ · 6H ₂ O) and 30 µg / ml of kanamycin, and at 30 ° C for about 3 hours at 200 rpm. Shake culture was performed until OD600 = 0.5, 0.1 mM IPTG was added, and expression from the T7 promoter was induced, followed by shaking culture at 200 rpm for 4 hours to recover the cells.

Nitrile hydratase hydration activity which converts a nitrile compound into an amide compound at 27 degreeC was measured using this cell.

E. coli cells expressing nitrile hydratase derived from Geobacilli thermoglucosidesis Q-6 strain were re-dissolved in TBS buffer (pH7.5) containing 20 mM Tris-HCl and 15 mM NaCl. It diluted so that it might become = 0.2, and made the reaction liquid of the final concentration 0.2weight% acrylonitrile solution. The reaction was stirred while stirring at 27 ° C., and 30 minutes later, 100 µL of 1 N hydrochloric acid was added to terminate the reaction. The unit (unit) of enzymatic activity is defined as 1 unit (hereinafter referred to as U) for converting 1 mol of acrylonitrile into acrylamide in 1 minute, and the hydration activity (U / mg) per weight of the wet cell is shown. 8 is shown.

Expression plasmid Activity (U / mg) pET-26b (+) vector 0 pET-26b (+)-βα 0.200 pET-28a (+) vector 0 pET-28a (+)-βα 0.212

From these results, when nitrile hydratase α-subunit and β-subunit of Geobacilli-thermoglucosidesis Q-6 strain were expressed in Escherichia coli, those having nitrile hydratase activity for converting acrylonitrile to acrylamide were identified. It turned out.

Example 13: Acquisition of nitrile hydratase surrounding genes derived from Geobacilli and thermoglucosidesis Q-6 strains by colony hybridization

(1) Preparation of Fluorescent Labeled DIG Probe

Fluorescent labeling probes were prepared by Roche's DIG-DNA labeling kit using DNA of the nitrile hydratase α subunit moiety of the geobacilli-thermoglucosidesis Q-6 strain described in SEQ ID NO: 25 in the sequence listing as a template. . How to write is according to Roche's DIG manual.

(2) chromosome Southern hybridization

Chromosomal DNA of Geobacilli and thermoglucosidesis Q-6 strains prepared in Example 11 were digested with various restriction enzymes and subjected to 1% by weight agarose gel electrophoresis. The DNA in the agarose gel was transferred to a nylon membrane Hybond-N + (Amarsham Co., Ltd.), and then chromosomal hybridization was performed using a fluorescently labeled DIG probe prepared above. Transferring and immobilizing the DNA-immobilized membrane into 10 ml of hybridization buffer (5 × SSC containing 1 wt% skim milk, 0.1 wt% N-lauroyl zarcosine, 0.02 wt% SDS, 50 wt% formamide) Dip and prehybridized at 42 degreeC for 2 hours. 100 ng of the fluorescent labeling probe prepared as described above was thermally denatured by boiling and quenching at 95 ° C. for 10 minutes, added to a prehybridization buffer, and hybridized at 42 ° C. overnight. The membrane after hybridization was washed twice at room temperature with 2 x SSC containing 150 ml of 0.1 wt% SDS. Next, washing for 5 minutes was performed twice in 1 * SSC containing 150 ml of 0.1 weight% SDS heated at 65 degreeC. Subsequently, after washing for 5 minutes with 100 ml maleic acid buffer (0.1 M maleic acid, 0.15 M NaCl, NaOH and pH 7.5), 50 ml of blocking solution (0.3 wt% Tween) Blocking was carried out for 30 minutes at room temperature in 0.1 M maleic acid buffer (pH 7.5) containing 20, 0.15 M NaCl and 1 wt% skim milk. The antidigoxigenin AP was diluted with 20 ml of blocking solution to 75 mU / ml, followed by antibody reaction at room temperature for 30 minutes, followed by 100 ml of washing buffer (0.3 M by weight Tween 20, 0.15 M NaCl). Unbound antibody was washed by washing the membrane five times in maleic acid buffer; pH 7.5). After equilibrating in 20 ml of detection buffer (0.1 M (Tris-HCl, 0.1 M NaCl, pH9.5) for 5 minutes, 10 mg of detection buffer was added 100 mg / ml of NBT (nitrosoblue tetrazolium chloride) solution. A 34 μl, 50 mg / mL BCIP solution was prepared with NBT / BCIP (5-bromo-4-chloro-3-indolylphosphate) diluted in 35 μl, and poured and shaded so that the membrane was completely submerged. Incubate for minutes to 16 hours. During incubation, the color was checked without moving or shaking the disk. As a result, it was found that the gene downstream of the nitrile hydratase α subunit portion was contained in the about 2.3 kb gene fragment digested by the restriction enzyme HindIII.

(3) Acquisition of target clone by colony hybridization

① Preparation of plasmid library for colony hybridization

Next, colony hybridization was performed using the same fluorescently labeled DIG probe. 10 μg of chromosomal DNA of Giobacilli-thermoglucosidesis Q-6 strain was subjected to 1% by weight agarose gel electrophoresis, and the portion containing the DNA fragment of about 2.0 kb to 2.6 kb was cut out from the agarose gel. DNA fragments were extracted and purified in the same manner as above. The resulting DNA fragment was introduced into the HindIII restriction enzyme site in the multicloning region of the pUC118 plasmid vector (manufactured by Takara Corporation) using a DNA ligation kit (manufactured by Takara Corporation). The pUC118 plasmid vector DNA used for ligation was digested with the restriction enzyme HindIII, purified by phenol / chloroform treatment and ethanol precipitation, followed by dephosphorylation treatment at the 5 'terminal using alkaline phosphatase (manufactured by Takara). Phenol / chloroform treatment and ethanol precipitation were carried out again and used for agarose electrophoresis, and the thing refine | purified by extraction from an agarose gel was used.

E. coli JM109 strain was transformed using a solution obtained by ligating the chromosomal DNA of Geobacilli-thermoglucosidesis Q-6 strain fragmented at about 2.0 kb to 2.6 kb at the pUC118 plasmid vector and HindIII restriction enzyme site, and 50 µg / Ml of ampicillin, 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) and 2% by weight X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galacto It was sprinkled on LB agar medium (0.5% by weight Bacterial Yeast Extract, 1% by weight Bacterium tryptone, 0.5% by weight NaCl, 2.0% by weight Agar; pH 7.5) containing pyranoside) and incubated overnight at 37 ° C. As a result, a large number of Petri dishes in which 50 to 500 white colonies appeared per sheet could be obtained.

The plasmid libraries of these chromosomal DNAs were colonized using the fluorescently labeled DIG probes prepared above to screen clones containing the downstream genes of the desired nitrile hydratase α subunit.

② Acquisition of target clone by colony hybridization

First, about 1000 clones of the emerged white colonies were streaked again on fresh LB agar medium using sterile toothpicks. At this time, streaking was similarly carried out to the LB agar medium for hybridizing the membrane and the LB agar medium for preservation and incubated overnight at 30 ° C.

Next, a nylon membrane high bond-N + made by Amarsham Co., Ltd. was carefully placed on a petri dish with colonies, and then slowly removed using tweezers from the end after 1 minute. The peeled membrane was immersed in a denatured solution (0.5M aqueous NaOH solution containing 1.5 M NaCl) for 7 minutes with the cell surface attached upwards, followed by a neutralization solution (containing 1.5 M NaCl and 1 mM EDTA.2Na). Immersed in 0.5 M Tris hydrochloric acid aqueous solution; Next, the film was washed once with a 2 × SSC solution (containing 8.76 g of NaCl and 4.41 g of sodium citrate in 1 liter of 1 × SSC), and the membrane was air dried on a dry filter paper. In addition, DNA was fixed on the membrane by UV irradiation of 120mJ / cm ² .

③ Detection by DIG antibody and isolation of target clone

5 x containing 10 ml of hybridization buffer (1 wt% skim milk, 0.1 wt% N-lauroyl zarcosine, 0.02 wt% SDS, 50 wt% formamide) per sheet of the DNA-fixed membrane treated above SSC), and prehybridized at 42 ° C. for 2 hours. 100 ng of the fluorescent labeling probe prepared as described above was thermally denatured by boiling and quenching at 95 ° C. for 10 minutes, and added to the prehybridization buffer, and hybridized at 42 ° C. overnight. The membrane after hybridization was washed twice at room temperature with 2 x SSC containing 150 ml of 0.1 wt% SDS. Next, washing for 5 minutes was performed twice in 1 * SSC containing 150 ml of 0.1 weight% SDS heated at 65 degreeC. Subsequently washed with 100 ml maleic acid buffer (0.1 M maleic acid, 0.15 M NaCl, adjusted to pH7.5 using NaOH) for 5 minutes, followed by 50 ml of blocking solution (0.3 wt% Tween 20, 0.15 M). Blocking was performed for 30 minutes at room temperature in 0.1 M maleic acid buffer (pH 7.5) containing NaCl and 1 wt% skim milk. The antidigoxigenin AP was diluted with 20 ml of blocking solution to 75 mU / ml, followed by antibody reaction at room temperature for 30 minutes, followed by 100 ml of washing buffer (0.3 M by weight Tween 20, 0.15 M NaCl). Unbound antibody was washed by washing the membrane five times in maleic acid buffer; pH 7.5). After equilibrating for 5 minutes in 20 ml of detection buffer (0.1 M－Tris-HCl, 0.1 M NaCl, pH9.5), 34 µl of 100 mg / ml NBT solution was added to 10 ml of detection buffer and 50 mg / ml BCIP. The solution was prepared with a color substrate solution NBT / BCIP diluted 35 μl, poured to completely submerge the membrane, and incubated for 1 minute to 16 hours. During incubation, the color was checked without moving or shaking the disk. As a result, four positive signals were found among the 1000 clones on the membrane, and the positive clones overlapping with the positions were confirmed on the original Petri dishes.

(4) Analysis of positive clones containing downstream genes of nitrile hydratase α subunit moiety of geobacilli and thermoglucosidesis Q-6 strains

The identified positive clones were inoculated from a Petri dish into LB liquid medium containing ampicillin, shaken overnight at 37 ° C and 250 rpm to recover the cells by centrifugation, and plasmid DNA was extracted by a conventional method. After digesting the plasmid DNA with the restriction enzyme HindIII, it was subjected to 1.5% by weight agarose electrophoresis and confirmed the size of the inserted fragment, which was about 2.3 kb in size. In addition, it was confirmed by digestion patterns by PCR and restriction enzymes of several patterns that the inserted fragment contained the nitrile hydratase α subunit moiety of the Geobacilli and thermoglucosidesis Q-6 strain.

This plasmid obtained from the above was named pUC118-Q6Hin 2.3, and the entire nucleotide sequence of the inserted fragment was determined. The restriction map and gene configuration of nitrile hydratase and downstream genogroups of Geobacilli and thermoglucosidedes Q-6 strains are shown in FIG. 1.

As a result, it was confirmed that an open reading frame (hereinafter referred to as ORF3) containing a 339 bp base sequence in the insertion fragment was present in the same direction downstream of the 5 ′ terminal side of the nitrile hydratase α subunit (ORF2). . The translation end codon of ORF2 and the translation start codon of ORF3 were 12bp, and the translation start codon of ORF3 and the translation start codon of ORF located further downstream was 145bp. ORF3 encodes 112 amino acids and had very low homology with the following proteins in existing databases. In the existing database, the proportion of amino acids with highly homologous sequences was 31% with P12K of the Bacillus BR449 strain, NhhG with 31% of the Rhodococcus-Rhodochross J1, and NhlE with 21% of the Rhodococcus-Rhodochross J1. % And P16 and 23% of pseudonocardia thermophila JCM3095 strain.

Example 14 Construction of nitrile hydratase α subunit and β subunit of geobacillus thermoglucosidesis Q-6 strain and expression plasmid of downstream gene ORF3

The pET-26b (+)-βα and pET-28a (+)-βα plasmids were digested with the restriction enzyme HindIII, dephosphorylated and subjected to dephosphorylation by extraction with phenol chloroform. This digested product was subjected to 1% by weight agarose electrophoresis, and a DNA fragment of about 6.1 Kb was extracted by a conventional method. This DNA fragment contains the pET vector and the nitrile hydratase β subunit of the geobacilli-thermoglucosidesis Q-6 strain and the HindIII restriction enzyme site located at the 60th amino acid of the α subunit.

The pUC118-Q6Hin 2.3 plasmid was then digested with restriction enzyme HindIII, subjected to 1% by weight agarose electrophoresis, and the insert DNA of about 2.3 kb was extracted. This insert was ligated with the previously extracted fragment to transform E. coli JM109 strain, and a plasmid containing insert DNA was selected from a transformant selected for kanamycin resistance. The nitrile hydratase α-subunit and β-subunit and downstream gene ORF3 of the geobacilli-thermoglucosidesis Q-6 strain in the pET-26b (+) and pET-28a (+) vectors were confirmed by PCR. And a plasmid containing the downstream region was obtained. The plasmid thus completed is referred to hereinafter as pET-26b (+)-βα12 and pET-28a (+)-βα12.

Next, the expression plasmid which introduce | transduces nitrile hydratase (alpha) subunit, (beta) subunit, and the translation end codon of the downstream gene ORF3 of the geobacilli thermoglucosidesis Q-6 strain is introduced, and three types of proteins are co-expressed. Built. The previously constructed plasmid pET-26b (+)-βα12 was digested with restriction enzymes NdeI and BglII to provide 1.5% by weight agarose gel electrophoresis to translation end codons of α subunit, β subunit and downstream gene ORF3. 1.7kb gene fragment containing was extracted by the conventional method. At the same time, the expression vectors pET-26b (+) and pET-28a (+) were digested with NdeI and BamHI to be subjected to 1% by weight agarose gel electrophoresis, and the gene fragment of the 5.3 kb vector portion was conventionally obtained. Extracted. These vectors were subjected to the ligation reaction according to a conventional method by inserting a 1.7kb gene fragment including the α subunit, β subunit and the translation end codon of the downstream gene ORF3, which were extracted before. In this reaction, a terminal digested with BglII restriction enzyme and a terminal digested with BamHI restriction enzyme are attached. E. coli JM109 strain was transformed using the solution after the ligation reaction, plasmid DNA was extracted from the transformant selected for kanamycin resistance, and the plasmid to which the insert was introduced was selected. The nitrile hydratase α subunit, the β subunit moiety, and the downstream gene ORF3 of the geobacillus thermoglucosidesis Q-6 strain were introduced as inserts to obtain an expression plasmid in which three kinds of proteins were co-expressed. These target plasmids are referred to below as pET-26b (+)-βα1 and pET-28a (+)-βα1.

Example 15 Nitrile hydratase activity of geobacilli and thermoglucosidesis Q-6 strains co-expressing downstream genes in E. coli

The expression plasmids pET-26b (+)-βα and pET-28a (+)-βα were used to transform Novagen's Escherichia coli BL21 (DE3) LysE strain, and nitrile hydra of Geobacilli-thermoglucosidesis Q-6 strain. Proteins of the kinase β subunit and α subunit were coexpressed by the T7 promoter. Similarly, Novagen's Escherichia coli BL21 (DE3) LysE strain was transformed using pET-26b (+)-βα1 and pET-28a (+)-βα1, and nitrile hydrata of Geobacilli and thermoglucosidesis Q-6 strains. Proteins of the aze β subunit, α subunit and downstream gene ORF3 were co-expressed by the T7 promoter. As a control, a transformant transformed from Novagen's Escherichia coli BL21 (DE3) LysE strain using expression vectors pET-26b (+) and pET-28a (+) was used.

Each expression plasmid was transformed into Novagen E. coli BL21 (DE3) LysE strain reactive cells, LB agar medium containing 0.5 µg / ml kanamycin (0.5% by weight Bacteria yeast extract, 1% by weight). % Bactotryptone, 0.5 wt% NaCl, 2.0 wt% agar; pH 7.5) After the transformation process, the bacterial solution was sprinkled and incubated overnight at 30 ° C. for selection by kanamycin resistance. This transformant was inoculated into 2 ml of LB medium containing 30 µg / ml kanamycin, and the culture was shaken at 200 ° C. overnight at 200 rpm. The whole culture was inoculated with 2% by weight in 10 ml of LB medium containing 20 µg / ml of cobalt chloride hexahydrate (CoCl ₂ · 6H ₂ O) and 30 µg / ml of kanamycin, and at 30 ° C for about 3 hours at 200 rpm. Shake culture was performed until OD600 = 0.5, 0.1 mM IPTG was added to induce expression from the T7 promoter, and shake culture was performed at 200 rpm for about 4 hours to recover the cells.

E. coli cells expressing nitrile hydratase derived from Geobacilli and thermoglucosidesis Q-6 strain thus obtained are reproduced in TBS buffer (pH7.5) containing 20 mM Tris-HCl and 15 mM NaCl. It was turbid and diluted so that OD = 0.2, and the reaction liquid of the final concentration of 0.2 weight% acrylonitrile solution was prepared. The reaction was stirred while stirring at 27 ° C, and after 10 minutes, the reaction was stopped by adding 100 µL of 1 N hydrochloric acid after 30 minutes. The unit (unit) of enzymatic activity is defined as 1 unit (hereinafter referred to as U) for converting 1 mol of acrylonitrile into acrylamide in 1 minute, and the hydration activity (U / mg) per weight of the wet cell is shown. 9 is shown.

Expression plasmid Activity (U / mg) pET-26b (+) vector 0 pET-26b (+)-βα 0.20 pET-26b (+)-βα1 2.74 pET-28a (+) vector 0 pET-28a (+)-βα 0.21 pET-28a (+)-βα1 4.22

From these results, the nitrile hydratase α subunit and β subunit of the geobacilli thermoglucosidicus Q-6 strain were expressed compared with the case where the downstream gene ORF3 was coexpressed. It can be seen that the hydratase activity is significantly increased. It has been found that ORF3 has a function of remarkably elevating the enzymatic activity of nitrile hydratase of geobacilli-thermoglucosidesis Q-6 strain.

Example 16: Thermal Stability of Nitrile Hydratease from Geobacilli and Thermoglucosides Q-6

E. coli solution (20 mM Tris-HCl and 15 mM NaCl) containing an expression vector pET26b (+)-? TBS buffer (pH7.5) containing) was insulated at 30, 65 and 70 ° C for 30 minutes. After the heat treatment, the mixture was cooled on ice and kept at 27 ° C to keep the temperature constant, and then nitrile hydratase activity was measured at the reaction temperature of 27 ° C. E. coli, which expresses nitrile hydratase of Geobacilli and thermoglucosidesis Q-6 strains, was suspended with a turbidity of OD = 0.2, and a reaction solution of 0.5% by weight acrylonitrile solution in final concentration was prepared and stirred at 27 ° C. The reaction was started while the reaction was stopped after 30 minutes by adding 10% by volume of 1N hydrochloric acid. It shows in Table 10 as conversion value which made the activity (at 100%) the activity at the time of heat-retaining processing at 30 degreeC.

Treatment temperature (℃) Remaining activity (%) 30 100.0 65 102.5 70 83.9

From these results, the enzymatic activity of nitrile hydratase in E. coli expressing nitrile hydratase of Geobacilli thermoglucosidesis Q-6 strain can maintain about 80% of its activity even at a high temperature of 70 ° C. Even when expressed in E. coli, it has high heat resistance and can be said to be an extremely useful enzyme industrially.

The composition of this invention shows high stability with respect to heat, a high concentration of nitrile, and an amide compound, and has the effect of efficiently converting a nitrile compound into a corresponding amide compound. The composition of the present invention can be suitably used in the field of converting a nitrile compound to a corresponding amide compound even at a high temperature and in reaction under a high nitrile compound concentration or a high amide compound concentration.

SEQUENCE LISTING <110> Asahi Kasei Corporation <120> New nitrile hydratase from Geobacillus thermoglucosidasius <130> A41354A <160> 25 <210> 1 <211> 205 <212> PRT <213> Geobacillus thermoglucosidasius Nitrile hydratase alpha-subunit <400> 1 Met Ser Val Gln Lys Val His His Asn Val Leu Pro Glu Lys Pro Ala   1 5 10 15 Gln Thr Arg Thr Lys Ala Leu Glu Ser Leu Leu Ile Glu Ser Gly Leu              20 25 30 Val Ser Thr Asp Ala Leu Asp Ala Ile Ile Glu Ala Tyr Glu Asn Asp          35 40 45 Ile Gly Pro Met Asn Gly Ala Lys Val Val Ala Lys Ala Trp Val Asp      50 55 60 Pro Asp Tyr Lys Glu Arg Leu Leu Arg Asp Gly Thr Ser Ala Ile Ala  65 70 75 80 Glu Leu Gly Phe Leu Gly Leu Gln Gly Glu His Met Val Val Val Glu                  85 90 95 Asn Thr Pro Lys Val His Asn Val Val Val Cys Thr Leu Cys Ser Cys             100 105 110 Tyr Pro Trp Pro Val Leu Gly Leu Pro Pro Ser Trp Tyr Lys Ser Ala         115 120 125 Ser Tyr Arg Ala Arg Ile Val Ser Glu Pro Arg Thr Val Leu Lys Glu     130 135 140 Phe Gly Leu Glu Leu Asp Asp Asp Val Glu Ile Arg Val Trp Asp Ser 145 150 155 160 Ser Ala Glu Ile Arg Tyr Leu Val Leu Pro Glu Arg Pro Ala Gly Thr                 165 170 175 Glu Gly Trp Ser Glu Glu Glu Leu Ala Lys Leu Val Thr Arg Asp Ser             180 185 190 Met Ile Gly Val Ala Lys Ile Lys Ser Pro Val Lys Lys         195 200 205 <210> 2 <211> 226 <212> PRT <213> Geobacillus thermoglucosidasius Nitrile hydratase beta-subunit <400> 2 Met Asn Gly Pro His Asp Leu Gly Gly Lys Arg Asp Phe Gly Pro   1 5 10 15 Ile Ile Lys His Asp Gln Glu Pro Leu Phe His Glu Glu Trp Glu                  20 25 30 Ala Lys Val Leu Ala Met His Phe Ala Leu Leu Gly Gln Gly Val                  35 40 45 Ile Asn Trp Asp Glu Phe Arg His Gly Ile Glu Arg Met Gly Tyr                  50 55 60 Val Tyr Tyr Leu Thr Ser Ser Tyr Tyr Glu His Trp Leu Ala Ser                  65 70 75 Leu Glu Thr Val Leu Ala Glu Lys Asn Ile Ile Asn Ser Glu Gln                  80 85 90 Tyr Arg Lys Arg Ile Arg Glu Ile Glu Tyr Gly Met Ser Val Pro                  95 100 105 Val Ser Glu Lys Pro Glu Leu Lys Glu Ser Leu Leu Ser Glu Val                 110 115 120 Ile Tyr Gly Thr Lys Ile Ser Ser Glu Arg Arg Glu Ser Thr Val                 125 130 135 Ser Pro Arg Phe Arg Pro Gly Asp Arg Val Arg Val Lys His Phe                 140 145 150 Tyr Thr Asn Lys His Thr Arg Cys Pro Gln Tyr Val Met Gly Lys                 155 160 165 Val Gly Val Val Glu Leu Leu His Gly Asn His Val Phe Pro Asp                 170 175 180 Ser Asn Ala His Gly Asp Gly Glu Ala Pro Gln Pro Leu Tyr Asn                 185 190 195 Val Arg Phe Glu Ala Arg Glu Leu Trp Gly Gly Glu Ala His Glu                 200 205 210 Lys Asp Ser Leu Asn Leu Asp Leu Trp Asp Ser Tyr Leu Thr His                 215 220 220 Ala 226 <210> 3 <211> 1663 <212> DNA <213> Geobacillus thermoglucosidasius <223> Nitrile hydratase beta -subunit, alpha-subunit, orf3 <221> CDS <222> 1..681 Nitrile hydratase beta-subunit <221> CDS <222> 695..1312 Nitrile hydratase alpha-subunit <221> CDS <222> 1325..1663 <223> orf3 <400> 3 atg aac ggc ccg cac gat tta ggt gga aaa cgt gat ttt ggc cca 45 Met Asn Gly Pro His Asp Leu Gly Gly Lys Arg Asp Phe Gly Pro   1 5 10 15 atc att aaa cat gat caa gaa cct ctt ttt cat gaa gaa tgg gaa 90 Ile Ile Lys His Asp Gln Glu Pro Leu Phe His Glu Glu Trp Glu                  20 25 30 gca aaa gta ctg gcg atg cat ttt gct tta ctt gga caa gga gta 135 Ala Lys Val Leu Ala Met His Phe Ala Leu Leu Gly Gln Gly Val                  35 40 45 atc aac tgg gat gaa ttt agg cat ggt ata gaa cgg atg gga tat 180 Ile Asn Trp Asp Glu Phe Arg His Gly Ile Glu Arg Met Gly Tyr                  50 55 60 gtt tat tac ctt act tca agc tat tat gaa cat tgg ctt gct tca 225 Val Tyr Tyr Leu Thr Ser Ser Tyr Tyr Glu His Trp Leu Ala Ser                  65 70 75 cta gaa acc gta ttg gcc gag aaa aat atc att aac agt gaa cag 270 Leu Glu Thr Val Leu Ala Glu Lys Asn Ile Ile Asn Ser Glu Gln                  80 85 90 tat aga aag aga att agg gaa ata gaa tat ggc atg agt gta cct 315 Tyr Arg Lys Arg Ile Arg Glu Ile Glu Tyr Gly Met Ser Val Pro                  95 100 105 gtc agc gaa aag cct gag tta aaa gag tct ttg tta tcc gaa gtg 360 Val Ser Glu Lys Pro Glu Leu Lys Glu Ser Leu Leu Ser Glu Val                 110 115 120 atc tat ggc acg aaa ata tca tcc gaa cgg aga gaa agc act gta 405 Ile Tyr Gly Thr Lys Ile Ser Ser Glu Arg Arg Glu Ser Thr Val                 125 130 135 tct ccg cgg ttt cgt cct gga gat aga gtg agg gta aaa cac ttt 450 Ser Pro Arg Phe Arg Pro Gly Asp Arg Val Arg Val Lys His Phe                 140 145 150 tat aca aac aag cat act aga tgt cct caa tat gtc atg ggg aaa 495 Tyr Thr Asn Lys His Thr Arg Cys Pro Gln Tyr Val Met Gly Lys                 155 160 165 gta gga gtt gta gaa ctt ctt cat ggg aat cat gtt ttc cca gac 540 Val Gly Val Val Glu Leu Leu His Gly Asn His Val Phe Pro Asp                 170 175 180 tct aac gct cat ggt gat ggc gag gct ccg caa ccg ctt tac aat 595 Ser Asn Ala His Gly Asp Gly Glu Ala Pro Gln Pro Leu Tyr Asn                 185 190 195 gtg cgc ttt gaa gca aga gaa ctg tgg gga ggc gag gct cac gaa 630 Val Arg Phe Glu Ala Arg Glu Leu Trp Gly Gly Glu Ala His Glu                 200 205 210 aaa gat agt cta aat ctc gac tta tgg gat agc tat cta act cac 675 Lys Asp Ser Leu Asn Leu Asp Leu Trp Asp Ser Tyr Leu Thr His                 215 220 225 gcg taa aggaggaaaa atc 694 Ala 226 atg agt gta caa aaa gtt cat cac aac gtt ctg cct gaa aag cct 739 Met Ser Val Gln Lys Val His His Asn Val Leu Pro Glu Lys Pro   1 5 10 15 gct caa act cgg aca aag gct ttg gaa tcg ctg ttg atc gaa tct 784 Ala Gln Thr Arg Thr Lys Ala Leu Glu Ser Leu Leu Ile Glu Ser                  20 25 30 gga ttg gtc tcc act gat gcc ctt gat gcg att att gaa gcc tat 829 Gly Leu Val Ser Thr Asp Ala Leu Asp Ala Ile Ile Glu Ala Tyr                  35 40 45 gaa aat gat att ggg cct atg aat ggg gca aaa gtt gtt gca aaa 874 Glu Asn Asp Ile Gly Pro Met Asn Gly Ala Lys Val Val Ala Lys                  50 55 60 gct tgg gtt gat cct gat tac aaa gaa aga ttg ctt cgg gat ggg 910 Ala Trp Val Asp Pro Asp Tyr Lys Glu Arg Leu Leu Arg Asp Gly                  65 70 75 act tcg gct att gca gag ctt ggc ttt tta ggg ttg cag ggg gag 964 Thr Ser Ala Ile Ala Glu Leu Gly Phe Leu Gly Leu Gln Gly Glu                  80 85 90 cac atg gtt gtt gtc gaa aat acg cct aaa gtt cat aat gta gta 1009 His Met Val Val Val Glu Asn Thr Pro Lys Val His Asn Val Val                  95 100 105 gtt tgt acg cta tgt tcc tgc tat ccg tgg cct gtc cta ggc ttg 1054 Val Cys Thr Leu Cys Ser Cys Tyr Pro Trp Pro Val Leu Gly Leu                 110 115 120 cct cct tca tgg tat aaa agt gct tca tac agg gct cga att gtt 1099 Pro Pro Ser Trp Tyr Lys Ser Ala Ser Tyr Arg Ala Arg Ile Val                 125 130 135 tca gag cca aga act gta ctt aaa gag ttt ggg ctt gaa ctg gat 1144 Ser Glu Pro Arg Thr Val Leu Lys Glu Phe Gly Leu Glu Leu Asp                 140 145 150 gat gat gtt gaa att agg gtt tgg gac agc agt gct gaa att cga 1189 Asp Asp Val Glu Ile Arg Val Trp Asp Ser Ser Ala Glu Ile Arg                 155 160 165 tat tta gtt ctt cca gaa aga cct gca ggt act gaa ggg tgg tcg 1234 Tyr Leu Val Leu Pro Glu Arg Pro Ala Gly Thr Glu Gly Trp Ser                 170 175 180 gaa gag gaa ctg gct aaa ctt gta acg cgt gac tct atg atc ggt 1279 Glu Glu Glu Leu Ala Lys Leu Val Thr Arg Asp Ser Met Ile Gly                 185 190 195 gtg gcc aag ata aag tcg cct gtt aaa aaa taa ggggggacaa aa 1324 Val Ala Lys Ile Lys Ser Pro Val Lys Lys                 200 205 atg gtt caa tca aat ctt caa ata aaa ccg gat gag att cta cct 1369 Met Val Gln Ser Asn Leu Gln Ile Lys Pro Asp Glu Ile Leu Pro   1 5 10 15 gaa cct agg aga aca gaa aat gag ccg gtt ttt aat tcc ccg tgg 1414 Glu Pro Arg Arg Thr Glu Asn Glu Pro Val Phe Asn Ser Pro Trp                  20 25 30 gaa gcc cgg att ttt gct atg aca atc aat ttg tat gac aaa aaa 1459 Glu Ala Arg Ile Phe Ala Met Thr Ile Asn Leu Tyr Asp Lys Lys                  35 40 45 ttc ttt gat tgg gag gac ttt cga caa gga tta ata gct gaa att 1504 Phe Phe Asp Trp Glu Asp Phe Arg Gln Gly Leu Ile Ala Glu Ile                  50 55 60 gca gtt gcg gac agc ctt cct gag aat gaa cga cca acc tac tac 1549 Ala Val Ala Asp Ser Leu Pro Glu Asn Glu Arg Pro Thr Tyr Tyr                  65 70 75 gaa agt tgg ctg gcc gct ttg gaa aag ttg tta atc aag gat ggt 1594 Glu Ser Trp Leu Ala Ala Leu Glu Lys Leu Leu Ile Lys Asp Gly                  80 85 90 ata tta aca aaa gaa caa ata gat gaa cgc act aaa gaa ttg aaa 1639 Ile Leu Thr Lys Glu Gln Ile Asp Glu Arg Thr Lys Glu Leu Lys                  95 100 105 gaa ggt ata aga aaa agt tgc tag 1663 Glu Gly Ile Arg Lys Ser Cys                 110 <210> 4 <211> 112 <212> PRT <213> Geobacillus thermoglucosidasius <223> ORF3 <400> 4 Met Val Gln Ser Asn Leu Gln Ile Lys Pro Asp Glu Ile Leu Pro Glu   1 5 10 15 Pro Arg Arg Thr Glu Asn Glu Pro Val Phe Asn Ser Pro Trp Glu Ala              20 25 30 Arg Ile Phe Ala Met Thr Ile Asn Leu Tyr Asp Lys Lys Phe Phe Asp          35 40 45 Trp Glu Asp Phe Arg Gln Gly Leu Ile Ala Glu Ile Ala Val Ala Asp      50 55 60 Ser Leu Pro Glu Asn Glu Arg Pro Thr Tyr Tyr Glu Ser Trp Leu Ala  65 70 75 80 Ala Leu Glu Lys Leu Leu Ile Lys Asp Gly Ile Leu Thr Lys Glu Gln                  85 90 95 Ile Asp Glu Arg Thr Lys Glu Leu Lys Glu Gly Ile Arg Lys Ser Cys             100 105 110 <210> 5 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Degenerate PCR primer for amplification of partial DNA fragment of Geobacillus thermoglucosidasius Q6 <400> 5 gtncaraarg tncaycayaa yg 22 <210> 6 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Degenerate PCR primer for amplification of partial DNA fragment of Geobacillus thermoglucosidasius Q6 <400> 6 ccncaraarc cngcncarac 20 <210> 7 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Degenerate PCR primer for amplification of partial DNA fragment of Geobacillus thermoglucosidasius Q6 <400> 7 caycancanc ayytncc 17 <210> 8 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Degenerate PCR primer for amplification of partial DNA fragment of Geobacillus thermoglucosidasius Q6 <400> 8 acrttrtgrt gnacyttytg nac 23 <210> 9 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Degenerate PCR primer for amplification of partial DNA fragment of Geobacillus thermoglucosidasius Q6 <400> 9 gtytgngcng gyttytgngg 20 <210> 10 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Degenerate PCR primer for amplification of partial DNA fragment of Geobacillus thermoglucosidasius Q6 <400> 10 ggnarrtgnt gntgrtg 17 <210> 11 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Degenerate PCR primer for amplification of partial DNA fragment of Geobacillus thermoglucosidasius Q6 <400> 11 atgaayggnc cncayga 17 <210> 12 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Degenerate PCR primer for amplification of partial DNA fragment of Geobacillus thermoglucosidasius Q6 <400> 12 aarmgngayt tyggncc 17 <210> 13 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Degenerate PCR primer for amplification of partial DNA fragment of Geobacillus thermoglucosidasius Q6 <400> 13 athathaarc aygaycarga rcc 23 <210> 14 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Degenerate PCR primer for amplification of partial DNA fragment of Geobacillus thermoglucosidasius Q6 <400> 14 arrtcrtgng gnccrttcat 20 <210> 15 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Degenerate PCR primer for amplification of partial DNA fragment of Geobacillus thermoglucosidasius Q6 <400> 15 thrtcrtgyt tdatdatngg 20 <210> 16 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Degenerate PCR primer for amplification of partial DNA fragment of Geobacillus thermoglucosidasius Q6 <400> 16 tcytcytcra araanarngg 20 <210> 17 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Degenerate PCR primer for amplification of partial DNA fragment of Geobacillus thermoglucosidasius Q6 <400> 17 gcgraartcy yccca 15 <210> 18 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Degenerate PCR primer for amplification of partial DNA fragment of Geobacillus thermoglucosidasius Q6 <400> 18 ccartgytcr tarttcc 17 <210> 19 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Degenerate PCR primer for amplification of partial DNA fragment of Geobacillus thermoglucosidasius Q6 <400> 19 agatagtcta aatctcgact tatgg 25 <210> 20 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Degenerate PCR primer for amplification of partial DNA fragment of Geobacillus thermoglucosidasius Q6 <400> 20 atgaacggcc cgcacgattt aggtgg 26 <210> 21 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Degenerate PCR primer for amplification of partial DNA fragment of Geobacillus thermoglucosidasius Q6 <400> 21 catatgaacg gcccgcacga tttaggtgg 29 <210> 22 <211> 37 <212> DNA <213> Artificial sequence <220> <223> Degenerate PCR primer for amplification of partial DNA fragment of Geobacillus thermoglucosidasius Q6 <400> 22 cagatcttat tttttaacag gcgactttat cttggcg 37 <210> 23 <211> 29 <212> PRT <213> Geobacillus thermoglucosidasius N-terminal amino acid sequence of nitrile hydratase alpha-subunit <400> 23 Met Ser Val Gln Lys Val His His Asn Val Leu Pro Glu Lys Pro   1 5 10 15 Ala Gln Thr Arg Thr Lys Ala Leu Glu Ser Leu Leu Ile Glu                  20 25 <210> 24 <211> 25 <212> PRT <213> Geobacillus thermoglucosidasius N-terminal amino acid sequence of nitrile hydratase beta-subunit <400> 24 Met Asn Gly Pro His Asp Leu Gly Gly Lys Arg Asp Phe Gly Pro   1 5 10 15 Ile Ile Lys His Asp Gln Glu Pro Leu Phe                  20 25 <210> 25 <211> 618 <212> DNA <213> Geobacillus thermoglucosidasius <223> Probe for colony hybridization (nitrile hydratase alpha-subunit) <400> 25 atgagtgtac aaaaagttca tcacaacgtt ctgcctgaaa agcctgctca aactcggaca 60 aaggctttgg aatcgctgtt gatcgaatct ggattggtct ccactgatgc ccttgatgcg 120 attattgaag cctatgaaaa tgatattggg cctatgaatg gggcaaaagt tgttgcaaaa 180 gcttgggttg atcctgatta caaagaaaga ttgcttcggg atgggacttc ggctattgca 240 gagcttggct ttttagggtt gcagggggag cacatggttg ttgtcgaaaa tacgcctaaa 300 gttcataatg tagtagtttg tacgctatgt tcctgctatc cgtggcctgt cctaggcttg 360 cctccttcat ggtataaaag tgcttcatac agggctcgaa ttgtttcaga gccaagaact 420 gtacttaaag agtttgggct tgaactggat gatgatgttg aaattagggt ttgggacagc 480 agtgctgaaa ttcgatattt agttcttcca gaaagacctg caggtactga agggtggtcg 540 gaagaggaac tggctaaact tgtaacgcgt gactctatga tcggtgtggc caagataaag 600 tcgcctgtta aaaaataa 618  1/16

Claims (22)

(a) has nitrile hydratase activity, (b) Substrate specificity: It shows activity based on acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile and hexanenitrile as a substrate, (c) Molecular weight: A protein composed of at least the following two types of subunits, the molecular weight of each subunit being reduced SDS-polyacrylamide electrophoresis is as follows,      Subunit α Molecular weight 25000 ± 2000      Subunit β Molecular weight 800028000 ± 2000 (d) Thermal Stability: After heating the enzyme in the sap at a temperature of 70 ° C. for 30 minutes, at least 35% of the activity before heating remains, (e) 6% by weight of acrylonitrile as a substrate does not decrease the activity as compared to the substrate concentration below that, (f) has an acrylonitrile-based activity in 35% by weight aqueous acrylamide solution DNA encoding a protein characterized by having physicochemical properties. (A) a base sequence containing an α subunit gene encoding an amino acid sequence of SEQ ID NO: 1 of the sequence listing, and a base sequence containing a β subunit gene encoding an amino acid sequence of SEQ ID NO: 2 of the sequence listing; DNA comprising a combination; or (B) one or more of any one or both of the α subunit containing the amino acid sequence of SEQ ID NO: 1 of the Sequence Listing and the β subunit containing the amino acid sequence of SEQ ID NO: 2 of the Sequence Listing; A dog amino acid has a substitution, deletion, addition, and post-translational modification, and contains a modified or unmodified α subunit and a modified or unmodified β subunit and encodes a protein having nitrile hydratase activity. DNA Any one of DNA. (C) a combination of a DNA comprising a sequence at positions 695-1312 of the nucleotide sequence of SEQ ID NO: 3 in the sequence listing and a DNA containing a sequence at positions 1-681 of the nucleotide sequence of SEQ ID NO: 3 in the sequence listing DNA, characterized in that; or (D) α subunit encoded by DNA containing DNA sequence of positions 695-1312 of the nucleotide sequence of SEQ ID NO: 3 in the sequence listing or DNA hybridized under stringent conditions with respect to the DNA; Contains a β subunit encoded by any one of DNAs containing the sequences 1-681 of the nucleotide sequence of SEQ ID NO: 3, or DNA hybridized under strict conditions with respect to the DNA, and contains nitrile hydratase activity. DNA encoding a protein having a protein, except for the case of (C) above Any one of DNA. The substitution, deletion, addition to DNA according to any one of claims 1 to 3, wherein the DNA comprises a nucleotide sequence encoding an amino acid sequence as set forth in SEQ ID NO: 4 in the sequence listing, or one or a plurality of amino acids thereof. DNA, characterized in that the post-translational modification is further combined with any one of the DNA encoding the protein, characterized in that involved in the activation of nitrile hydratase. The nitrile hydratase activation according to any one of claims 1 to 3, wherein the DNA comprises a sequence of positions 1325-1663 of the nucleotide sequence of SEQ ID NO: 3 in the sequence listing, or hybridizes under stringent conditions to nitrile hydratase activation. The DNA of any one of the DNA which codes the protein characterized by being involved in further combining. DNA comprising the α subunit gene of nitrile hydratase encoding the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing. DNA containing the β subunit gene of nitrile hydratase encoding the amino acid sequence set forth in SEQ ID NO: 2 of the Sequence Listing. A DNA comprising a gene characterized by being involved in the activation of a nitrile hydratase encoding an amino acid sequence as set forth in SEQ ID NO: 4 in the Sequence Listing. The DNA according to any one of claims 1 to 8, wherein the DNA is derived from the genus Geobacillus. The DNA according to any one of claims 1 to 8, wherein the DNA is derived from a Geobacillus thermoglucosidasius species. The DNA according to any one of claims 1 to 8, wherein said DNA is derived from Geobacillus thermoglucosidasius (QM) -6 strain (FERM # BP-08658). The recombinant vector which assembled the DNA of any one of Claims 1-11. The microorganism transformed by the DNA of any one of Claims 1-11, or Geobacillus thermoglucosidasius Q-6 strain (FERM # BP-08658) and its variants is either microbe. A method for producing the protein, or a cell processing product containing the protein, wherein the microorganism transformed with the DNA according to any one of claims 1 to 11 is cultured in a medium. Belongs to the genus Geobacilli, (a) has nitrile hydratase activity, (b) Substrate specificity: It shows activity based on acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile and hexanenitrile as a substrate, (c) Molecular weight: A protein composed of at least the following two types of subunits, the molecular weight of each subunit being reduced SDS-polyacrylamide electrophoresis is as follows,       Subunit α Molecular weight 25000 ± 2000       Subunit β Molecular weight 28000 ± 2000 (d) Thermal Stability: After heating the enzyme in the sap at a temperature of 70 ° C. for 30 minutes, at least 35% of the activity before heating remains. (e) 6% by weight of acrylonitrile as a substrate does not decrease the activity as compared to the substrate concentration below that, (f) has an acrylonitrile-based activity in 35% by weight aqueous acrylamide solution A method for producing the protein, or a cell-containing treatment product, comprising culturing a microorganism capable of producing a protein having physicochemical properties in a medium. The cell-processing product containing the said protein obtained from the microorganism cultured by the manufacturing method of any one of Claims 14 or 15, or the said protein. (a) has nitrile hydratase activity, (b) Substrate specificity: It shows activity based on acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile and hexanenitrile as a substrate, (c) Molecular weight: A protein composed of at least the following two types of subunits, the molecular weight of each subunit being reduced SDS-polyacrylamide electrophoresis is as follows,    Subunit α Molecular weight 25000 ± 2000    Subunit β Molecular weight 28000 ± 2000 (d) Thermal Stability: After heating the enzyme in the sap at a temperature of 70 ° C. for 30 minutes, at least 35% of the activity before heating remains, (e) 6% by weight of acrylonitrile as a substrate does not decrease the activity as compared to the substrate concentration below that, (f) has an acrylonitrile-based activity in 35% by weight aqueous acrylamide solution Protein characterized by having physicochemical properties. (A) a protein comprising an α subunit containing an amino acid sequence of SEQ ID NO: 1 in the sequence listing and a β subunit containing an amino acid sequence of SEQ ID NO: 2 in the sequence listing; or (B) one or more of any one or both of the α subunit containing the amino acid sequence of SEQ ID NO: 1 of the Sequence Listing and the β subunit containing the amino acid sequence of SEQ ID NO: 2 of the Sequence Listing; The amino acid substitution, deletion, addition, post-translational modification of the dog, containing a modified or unmodified α subunit and a modified or unmodified β subunit, characterized in that it has nitrile hydratase activity protein Either protein. (C) an α subunit encoded by a DNA comprising a sequence at positions 695-1312 of the nucleotide sequence of SEQ ID NO: 3 in the sequence listing, and a sequence at position 1-681 of the nucleotide sequence of SEQ ID NO: 3 in the sequence listing A protein comprising β subunit encoded by DNA; or (D) α subunit encoded by DNA containing DNA sequence of positions 695-1312 of the nucleotide sequence of SEQ ID NO: 3 in the sequence listing or DNA hybridized under stringent conditions with respect to the DNA; Contains a β subunit encoded by any one of DNAs containing the sequences 1-681 of the nucleotide sequence of SEQ ID NO: 3, or DNA hybridized under strict conditions with respect to the DNA, and contains nitrile hydratase activity. Protein with the exception of (except for the case of (C)) Either protein. Polypeptides containing at least a subunit of the amino acid sequence of SEQ ID NO: 1 in the sequence listing or post-translationally modified, Polypeptides containing β subunits at the amino acid sequence of SEQ ID NO: 2 of the sequence listing or modified after translation A protein containing any one or both of them. (a) has nitrile hydratase activity, (b) Substrate specificity: It shows activity based on acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile and hexanenitrile as a substrate, (c) Molecular weight: A protein composed of at least the following two types of subunits, the molecular weight of each subunit being reduced SDS-polyacrylamide electrophoresis is as follows,    Subunit α Molecular weight 25000 ± 2000    Subunit β Molecular weight 28000 ± 2000 (d) Thermal Stability: After heating the enzyme in the sap at a temperature of 70 ° C. for 30 minutes, at least 35% of the activity before heating remains, (e) 6% by weight of acrylonitrile as a substrate does not decrease the activity as compared to the substrate concentration below that, (f) has an acrylonitrile-based activity in 35% by weight aqueous acrylamide solution A method for producing an amide compound, characterized by obtaining a amide compound derived from the nitrile compound by acting on a nitrile compound with a protein having a physicochemical property or a cell processing product containing the protein. Belongs to the microorganism transformed by the DNA according to any one of claims 1 to 11, or genus Geobacillus, (a) has nitrile hydratase activity, (b) Substrate specificity: It shows activity based on acrylonitrile, adiponitrile, acetonitrile, isobutyronitrile, n-valeronitrile, n-butyronitrile, benzonitrile and hexanenitrile as a substrate, (c) Molecular weight: A protein composed of at least the following two types of subunits, the molecular weight of each subunit being reduced SDS-polyacrylamide electrophoresis is as follows,    Subunit α Molecular weight 25000 ± 2000    Subunit β Molecular weight 28000 ± 2000 (d) Thermal Stability: After heating the enzyme in the sap at a temperature of 70 ° C. for 30 minutes, at least 35% of the activity before heating remains, (e) 6% by weight of acrylonitrile as a substrate does not decrease the activity as compared to the substrate concentration below that, (f) has an acrylonitrile-based activity in 35% by weight aqueous acrylamide solution A nitrile compound derived from the nitrile compound is obtained by acting a nitrile compound on a protein obtained by culturing any of the microorganisms capable of producing a protein having physicochemical properties in a medium or a cell processing product containing the protein. The manufacturing method of the amide compound made into.

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