JP2010022334A - Nitrile hydratase gene having stereoselectivity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a transformant, etc., expressing a number of nitrile hydratase proteins having high activity and stereoselectivity, and exhibiting increased catalytic power. <P>SOLUTION: A transformant having remarkably increased catalytic power compared with conventional microorganisms is provided by including a number of nitrile hydratase genes comprising a gene encoding a protein composed of a specific amino acid sequence having stereoselectivity in a microbial cell. An optically active S-(+)-mandelamide or its derivative can be produced in high efficiency from racemic mandelonitrile and its derivative derived e.g. from a mixture of benzaldehyde and its derivative and hydrocyanic acid by using the transformant or its cultured product. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a nitrile hydratase having the ability to stereospecifically hydrate the nitrile group of racemic mandelonitrile and its derivatives to produce optically active S-(+)-mandelamide or a derivative thereof, and It relates to a gene encoding a protein.

  The optically active amide is a compound useful as a raw material for synthesizing pharmaceuticals and agricultural chemicals and as a raw material for producing optically active carboxylic acids.

  As a method for producing an optically active amide, a method using an asymmetric catalyst or a method using a stereoselective enzyme such as a nitrile hydratase having stereoselectivity is known. .

  As microorganisms producing nitrile hydratase having stereoselectivity, Rhodococcus erythropolis NCIMB 11540 (Non-patent Document 1) Rhodococcus sp. AJ270 (Non-patent Document 2), Pseudomonas putida NRRL-18668 (Patent Document 1) From the racemic mandelonitrile and its derivatives produced from a mixture of racemic mandelonitrile and its derivatives, for example, benzaldehyde and its derivatives and hydrocyanic acid, S- A Rhodococcus sp. HT40-6 strain that produces (+)-mandelamide and its derivatives has been found (Patent Document 2).

11-513255 JP-A-4-22591 Biotechol.j.2006,1,596-573 Tetrahedron: Asymmetry 11 (2000) 1123-1135

When producing optically active amides industrially, microorganisms with high catalytic ability are required from the viewpoint of cost. However, conventional microorganisms are not sufficient in terms of catalytic activity.
In view of the above-described circumstances, the present invention provides a nitrile hydratase having high activity and stereoselectivity, a transformant that expresses a large number of the enzyme and has increased catalytic ability, and a method for producing an amide using the same. The purpose is to provide.

  As a result of intensive studies in view of the above problems, the present inventors have identified a nitrile hydratase gene of Rhodococcus sp. HT40-6 that produces nitrile hydratase having stereoselectivity, and a number of such genes in the bacterial body. The present invention has been completed by finding the knowledge that the presence of the present invention can provide a transformant with dramatically increased catalytic ability.

That is, the present invention is as follows.
(1) A nitrile hydratase protein comprising the following protein (a) and protein (b):
(a) A protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
(b) A protein comprising the amino acid sequence set forth in SEQ ID NO: 4.
(2) A protein having the function of a nitrile hydratase α subunit consisting of the amino acid sequence set forth in SEQ ID NO: 2.
(3) A gene encoding the protein described in (2) above.
(4) A protein having the function of a nitrile hydratase β subunit consisting of the amino acid sequence set forth in SEQ ID NO: 4.
(5) A gene encoding the protein described in (4) above.
(6) A recombinant vector containing the gene according to (3) above and / or the gene according to (5) above.
(7) A transformant having the recombinant vector according to (6) above.
(8) A method for producing a nitrile hydratase protein, comprising culturing the transformant according to (7) above and recovering the nitrile hydratase protein from the culture.
(9) A method for producing an amide compound, wherein the culture of the transformant according to (7) or a treated product thereof is contacted with a nitrile compound to recover the amide compound.

  According to a preferred embodiment of the present invention, a large number of nitrile hydratase genes having stereoselectivity cloned by a gene recombination method are present in the microbial cells, so that they can be catalyzed compared to conventional microorganisms. Transformants with increased capacity are provided. By using this transformant or a culture thereof, it is possible to efficiently optically extract racemic mandelonitrile and its derivatives, for example, racemic mandelonitrile and its derivatives resulting from a mixture of benzaldehyde and its derivatives and hydrocyanic acid. Active S-(+)-mandelamide or a derivative thereof can be prepared.

Hereinafter, the present invention will be described in detail.
It should be noted that all publications cited in the present specification, for example, prior art documents, publications, patent publications and other patent documents are incorporated herein by reference.

1. Nitrile hydratase is an enzyme that catalyzes a hydration reaction that acts on a nitrile group of a nitrile compound and converts it into a corresponding amide compound, as shown in the following reaction formula.

(Where R is a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted saturated or unsaturated group) Means a heterocyclic group.)

  The “nitrile hydratase activity” means an activity that acts on a nitrile group of a nitrile compound to convert it into a corresponding amide compound as described above. Examples of the method for measuring nitrile hydratase activity include a method using mandelonitrile, which is one of nitrile compounds. In this method, for example, nitrile hydratase activity is measured by contacting the transformant of the present invention or a protein purified from its culture with mandelonitrile and quantifying the increase in the amount of mandelamide produced. can do. Alternatively, the nitrile hydratase activity can also be measured by quantifying the consumption of mandelonitrile.

  The nitrile hydratase of the present invention is a complex comprising an α subunit protein (hereinafter referred to as “nitrile hydratase α subunit protein”) and a β subunit protein (hereinafter referred to as “nitrile hydratase β subunit protein”). (Hereinafter sometimes referred to as “nitrile hydratase protein”).

The nitrile hydratase protein of the present invention is a nitrile hydratase protein comprising the following protein (a) and protein (b).
(a) A protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
(b) A protein comprising the amino acid sequence set forth in SEQ ID NO: 4.

The present invention also provides the following protein (a).
(a) A protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
Furthermore, the present invention provides the following protein (b).
(b) A protein comprising the amino acid sequence set forth in SEQ ID NO: 4.

  The nitrile hydratase protein according to a preferred embodiment of the present invention includes a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (α subunit) and a protein consisting of the amino acid sequence represented by SEQ ID NO: 4 (β subunit). It is a protein. According to a preferred embodiment of the present invention, the nitrile hydratase is efficiently optically active from racemic mandelonitrile and its derivatives, such as racemic mandelonitrile and its derivatives resulting from a mixture of benzaldehyde and its derivatives and hydrocyanic acid. S-(+)-mandelamide or a derivative thereof can be stereoselectively hydrated. Due to such nitrile hydratase activity, the nitrile hydratase protein according to a preferred embodiment of the present invention is optically active by acting on racemic mandelonitrile and its derivatives, or a mixture of benzaldehyde and its derivatives and hydrocyanic acid. S-(+)-mandelamide or a derivative thereof can be produced.

  Here, examples of the mandelonitrile or derivatives thereof, benzaldehyde and derivatives thereof, and mandelamide or derivatives thereof include compounds represented by the following formulas (1) to (3), respectively.

(Here, X represents a hydrogen atom, a methyl group, a methoxy group, a hydroxyl group or a halogen.)

  Examples of the halogen include a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

  Further, whether or not the nitrile hydratase protein expressed in the transformant of the present invention has stereoselective nitrile hydratase activity (stereoselective nitrile hydratase activity) is determined by, for example, mandelonitrile. It can be confirmed by the method of measuring the activity. In this method, in order to racemize the substrate, mandelonitrile or a derivative thereof, the reaction system is preferably kept near neutral to basic, for example, pH 4 to 11, preferably pH 6 to 10. The concentration of the substrate cannot be generally specified due to the sensitivity of the enzyme to benzaldehyde or hydrocyanic acid. Usually, mandelonitrile and its derivatives in the reaction solution are, for example, 0.1 to 10% by weight, preferably 0.2 to 5.0% by weight. Benzaldehyde or a derivative thereof is, for example, 0.1 to 10% by weight, preferably 0.2 to 5.0% by weight, and hydrocyanic acid is, for example, 0.1 to 1.0% by weight, preferably 0.1 to 0.5% by weight. The amount of microorganisms used relative to the raw material substrate is, for example, 0.01 to 5.0% by weight as dry cells, the reaction temperature is 0 to 50 ° C., preferably 10 to 30 ° C., and the reaction time is about 0.1 to 24 hours, for example.

  The present inventors have identified a gene encoding nitrile hydratase from the microorganism Rhodococcus sp. HT40-6 strain (hereinafter sometimes simply referred to as HT40-6 strain). As a result, the base sequence of the gene encoding the α subunit was the base sequence shown in SEQ ID NO: 1. On the other hand, the base sequence of the gene encoding the β subunit was the base sequence shown in SEQ ID NO: 3. Further, the amino acid sequences of the α subunit and the β subunit were the amino acid sequences shown in SEQ ID NOs: 2 and 4, respectively.

  The HT40-6 strain is deposited under the deposit number FERM BP-5231 at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (Chuo 6-1-1, Tsukuba City, Ibaraki Prefecture).

2. Gene The gene encoding the nitrile hydratase α subunit protein according to the present invention is a gene (polynucleotide) encoding the following protein (a).
(a) A protein comprising the amino acid sequence set forth in SEQ ID NO: 2.

  The gene encoding the protein described in (a) above is a gene encoding a nitrile hydratase α subunit protein consisting of the amino acid sequence described in SEQ ID NO: 2 (hereinafter referred to as “nitrile hydratase α subunit gene”). is there.

  The nitrile hydratase α subunit gene according to the present invention can be isolated from the aforementioned HT40-6 strain. For example, using a chromosomal DNA derived from HT40-6 as a template, a primer designed based on an amino acid sequence corresponding to a highly homologous region in a known nitrile hydratase α subunit gene derived from other microorganisms was used. The nitrile hydratase α subunit gene according to the present invention can be isolated by PCR. Alternatively, when the obtained PCR product is a part of the nitrile hydratase α subunit gene according to the present invention, a recombinant plasmid incorporating a chromosomal DNA fragment using chromosomal DNA derived from HT40-6 strain A recombinant DNA library such as (recombinant vector) is prepared. A recombinant DNA is introduced into Escherichia coli or the like to produce a transformant. Next, a transformant containing the nitrile hydratase α subunit gene according to the present invention is selected by colony hybridization using the above PCR product as a probe for the transformant. Then, the nitrile hydratase α subunit gene according to the present invention is isolated from the selected transformant.

  The base sequence of the nitrile hydratase α subunit gene according to the present invention is not limited in terms of the degeneracy of the genetic code, but includes, for example, the base sequence set forth in SEQ ID NO: 1.

  Table 1 shows the results of homology analysis at the amino acid level between the nitrile hydratase α subunit gene consisting of the base sequence represented by SEQ ID NO: 1 and the nitrile hydratase α subunit gene derived from other microorganisms. The homology analysis was performed using Genetyx ver6 (Genetics Co., Ltd.).

  Nitrogen hydratase α derived from Rhodococcus rhodochrous J1 (L-type), Rhodococcus sp., Arthrobacter aurecense TC1, Pseudocardia thermophila, Comamonas testosteroni 5-MGAM-4D, and Mesolysobiium sp. F28 The nucleotide sequence of the subunit gene and the corresponding amino acid sequence are registered under the registration numbers shown in Table 2 below.

  As shown in Table 1, the nitrile hydratase α subunit gene consisting of the base sequence represented by SEQ ID NO: 1 is compared with the known nitrile hydratase α subunit gene derived from Rhodococcus rhodochrous J1 (L type) at the amino acid sequence level. It has 90% or more homology.

  On the other hand, nitrile hydratase is already known to have a non-heme iron atom or a non-choline nuclear cobalt atom as a prosthetic molecule at the active center. A nitrile hydratase having a non-heme iron atom is called an iron-type nitrile hydratase, and a nitrile hydratase having a non-choline nuclear cobalt atom is called a cobalt-type nitrile hydratase. Cobalt-type nitrile hydratase has an active site called a cysteine cluster (Cys Thr Leu Cys Ser Cys) (Eur. J. Biochem. 271,429-438 (2004)). In SEQ ID NO: 2 showing the amino acid sequence of the nitrile hydratase α subunit protein according to the present invention, the cysteine cluster is found at positions 109-114.

  From the above, the nitrile hydratase α subunit gene consisting of the base sequence represented by SEQ ID NO: 1 can be identified as a novel nitrile hydratase α subunit gene.

On the other hand, the gene encoding the nitrile hydratase β subunit protein according to the present invention is a gene encoding the following protein (b).
(b) A protein comprising the amino acid sequence set forth in SEQ ID NO: 4.

  The gene encoding the protein described in (b) above is a nitrile hydratase β subunit gene encoding a nitrile hydratase β subunit protein consisting of the amino acid sequence set forth in SEQ ID NO: 4.

  The nitrile hydratase β subunit gene according to the present invention can be isolated from, for example, the microorganism HT40-6 strain in the same manner as the nitrile hydratase α subunit gene according to the present invention. In the isolation method, the nitrile hydratase α described above is used except that a primer designed based on an amino acid sequence corresponding to a region having high homology in a known nitrile hydratase β subunit gene derived from another microorganism or the like is used. This is the same as the method for isolating the subunit gene.

  The nucleotide sequence of the nitrile hydratase β subunit gene according to the present invention is not limited in terms of the degeneracy of the genetic code, but includes, for example, the nucleotide sequence set forth in SEQ ID NO: 3.

  Table 3 shows the results of homology analysis at the amino acid level between the nitrile hydratase β subunit gene consisting of the base sequence represented by SEQ ID NO: 3 and the nitrile hydratase β subunit gene derived from another microorganism. The homology analysis was performed using Genetyx ver6 (Genetics Co., Ltd.).

  Nitrogen hydratase β from Rhodococcus rhodochrous J1 (type L), Mesozobium sp. F28, Pseudocardia thermophila, Arthrobacter aurescens TC1, Rhodococcus sp., And Comamonas testosteroni 5-MGAM-4D The amino acid sequences of the subunits are registered with the registration numbers shown in Table 4, respectively.

  As shown in Table 3, the nitrile hydratase β subunit gene consisting of the nucleotide sequence represented by SEQ ID NO: 3 is compared with the known nitrile hydratase β subunit gene derived from Rhodococcus rhodochrous J1 (L type) at the amino acid sequence level. It has 90% or more homology.

  Hereinafter, the nitrile hydratase α subunit gene and the nitrile hydratase β subunit gene according to the present invention may be collectively referred to as “the gene according to the present invention”.

  Once the base sequence of the gene according to the preferred embodiment of the present invention has been determined, thereafter, for example, chemical synthesis, PCR using a cloned clone as a template, or a DNA fragment having the base sequence is probed. As a result, the gene according to the present invention can be obtained.

3. The recombinant plasmid (recombinant vector) according to the present invention is a recombinant plasmid (recombinant vector) containing the nitrile hydratase α subunit gene according to the present invention and / or the nitrile hydratase β subunit gene according to the present invention. ). A recombinant plasmid (recombinant vector) according to the present invention can be obtained by inserting the gene according to the present invention into an appropriate vector. The vector for inserting the gene according to the present invention is not particularly limited as long as it can be introduced into the host. For example, plasmid DNA, shuttle vector, helper plasmid, bacteriophage DNA, retrotransposon DNA, artificial chromosome DNA, etc. Is mentioned. If the vector itself does not have replication ability, it may be a DNA fragment that can be replicated by inserting it into the host chromosome.

As plasmid DNA, plasmids derived from E. coli (for example, pET systems such as pET30b, pBR systems such as pBR322 and pBR325, pUC systems such as pUC118, pUC119, pUC18 and pUC19, pBluescript, etc.), plasmids derived from Bacillus subtilis (for example, pUB110, pTP5, etc.), yeast-derived plasmids (eg, YEp systems such as YEp13, YCp systems such as YCp50), and plasmids derived from bacteria belonging to the genus Rhodococcus (pRC001, pRC002, pRC003, pRC004, etc.). Plasmids pRC004 are derived from Rhodococus rhodochrous ATCC 4276, ATCC 14349, ATCC 14348, and IFO 3338 strains, respectively, and disclosed in JP-A-2-84198, JP-A-4-48685, and JP-A-5-48685. No. 64589 and JP-A-5-68566.
Examples of the phage DNA include λ phage (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.). Furthermore, animal viruses such as retrovirus or vaccinia virus, plant viruses such as cauliflower mosaic virus, or insect virus vectors such as baculovirus can be used.

  When the host into which the recombinant plasmid (recombinant vector) according to the present invention is introduced is E. coli, a vector having high expression efficiency in E. coli, for example, an expression vector pKK233-2 (Centraalbureau voor Schimmelcultures (CBS) having a trc promoter. , Netherlands; http://www.cbs.knaw.nl/Amplicon Express) or pTrc99A (Centraalbureau voor Schimmelcultures (CBS), Netherlands; http://www.cbs.knaw.nl/Amplicon Express) It is preferable.

  In addition, when the host is a Rhodococcus bacterium, a recombinant vector containing a DNA region capable of replicating and growing in the Rhodococcus bacterium, pSJ020, pSJ034, and the like can be mentioned. A preferred vector is pSJ034.

  Note that pSJ034 is a plasmid that expresses nitrile hydratase in bacteria belonging to the genus Rhodococcus, and can be prepared from pSJ023 by the method described in JP-A-10-337185. In addition, pSJ023 was transformed into ATCC12674 / pSJ023 (FERM BP-6232) at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st East, 1-chome, Tsukuba City, Ibaraki Prefecture), March 4, 1997. Deposited on the date.

  pSJ034 is a vector that expresses a recombinant gene using a nitrilase expression regulatory gene derived from Rhodococus erythropolys SK92-B1. When an enzyme gene is introduced into the multiple cloning site, several amino acids are placed at the N-terminus. It is expressed as a bound fusion protein.

  To insert the gene according to the present invention into a vector, first cleave the cDNA of the gene according to the present invention with an appropriate restriction enzyme, and then insert it into the restriction enzyme site or multicloning site of the appropriate vector DNA and link it to the vector Is used. In addition, a method of linking both the vector and the cDNA of the gene according to the present invention by linking the both by an in vitro method using PCR or an in vivo method using yeast, etc. Also good.

  In addition to the gene according to the present invention, the recombinant plasmid (recombinant vector) according to the present invention controls gene control such as a promoter, terminator, enhancer, splicing signal, poly A addition signal, and ribosome binding sequence (SD sequence). It may contain sequences as well as selectable markers. Examples of the selection marker include, but are not limited to, a dihydrofolate reductase gene, an ampicillin resistance gene, and a kanamycin resistance gene. The method for inserting the gene regulatory sequence and the selection marker may be the same as the method for introducing the gene according to the present invention into the vector, and is linked to the vector so that the gene regulatory sequence and the selection marker can function in the host.

4). Transformant and method for producing nitrile hydratase protein The transformant according to the present invention comprises a recombinant plasmid (recombinant vector) containing the nitrile hydratase α subunit gene according to the present invention and the nitrile hydra according to the present invention. It can be obtained by introducing a recombinant plasmid (recombinant vector) containing a tase β subunit gene into the same host. Further, it can be obtained by introducing a recombinant plasmid (recombinant vector) containing the nitrile hydratase α subunit gene and the nitrile hydratase β subunit gene according to the present invention into a host. Here, the host is not particularly limited as long as it can express the gene according to the present invention, and examples thereof include bacteria. Examples of the bacterium include, for example, Escherichia genus such as Escherichia coli, Bacillus genus such as Bacillus subtilis, and Rhodococcus rhodochrous specified as accession numbers ATCC12674, ATCC17895 and ATCC19140. Examples include bacteria belonging to the genus Rhodococcus such as strains.

  The method for introducing the recombinant plasmid (recombinant vector) according to the present invention into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. Examples thereof include a method using calcium ions and an electroporation method.

  Not only the recombinant plasmid (recombinant vector) according to the present invention is introduced into the above bacterial host, but also yeast such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, COS cells and Chinese hamsters. Transformants can also be obtained by introduction into animal cells such as ovary cells (CHO cells), insect cells such as Sf9, or plants belonging to the Brassicaceae family.

  The method for introducing the recombinant plasmid (recombinant vector) according to the present invention into yeast is not particularly limited as long as it is a method for introducing DNA into yeast. For example, electroporation (electroporation), spheroplast method And the lithium acetate method. Alternatively, a YIp-type vector or a substitution / insertion type yeast transformation method into a chromosome using a DNA sequence homologous to an arbitrary region in a chromosome may be used.

  When animal cells are used as hosts, monkey cells COS-7, Vero, CHO cells, mouse L cells and the like are used. Examples of the method for introducing the recombinant plasmid (recombinant vector) according to the present invention into animal cells include the electroporation method, the calcium phosphate method, and the lipofection method.

  When insect cells are used as hosts, Sf9 cells and the like are used. Examples of the method for introducing the recombinant plasmid (recombinant vector) according to the present invention into insect cells include the calcium phosphate method, lipofection method, electroporation method and the like.

  When using a plant as a host, the whole plant body, plant organ (for example, leaf, petal, stem, root, seed, etc.), plant tissue (for example, epidermis, phloem, soft tissue, xylem, vascular bundle, etc.) or plant culture Cells are used. Examples of the method for introducing the recombinant plasmid (recombinant vector) according to the present invention into plants include an electroporation method, an Agrobacterium method, a particle gun method, and a PEG method.

  The method for introducing the recombinant vector of the present invention into yeast is not particularly limited as long as it can introduce DNA into yeast, and examples thereof include electroporation, spheroplast method, lithium acetate method and the like. Is mentioned.

  Whether or not the gene according to the present invention has been incorporated into the host can be confirmed by PCR, Southern hybridization, Northern hybridization, or the like. For example, DNA is prepared from the transformant, and PCR is performed by designing a primer specific for the gene according to the present invention. Thereafter, the PCR product was transformed by performing agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., staining with ethidium bromide, SYBR Green solution, etc., and detecting the PCR product. Confirm. PCR products can also be detected by performing PCR using primers previously labeled with a fluorescent dye or the like. Furthermore, a method of binding the PCR product to a solid phase such as a microplate and confirming the PCR product by fluorescence or enzyme reaction may be employed.

  On the other hand, in the method for producing the nitrile hydratase protein according to the present invention, the microorganism or the transformant according to the present invention is cultured, and the nitrile hydratase protein is recovered from the culture, whereby the nitrile hydratase according to the present invention is recovered. Protein can be obtained. Here, the “culture” means any of culture supernatant, cultured cells or cultured cells, or disrupted cells or cells.

  In the method for producing a nitrile hydratase protein according to the present invention, the method for culturing the microorganism according to the present invention is the same as the method described above. On the other hand, the method for culturing the transformant according to the present invention can be appropriately selected according to the type of host into which the recombinant plasmid (recombinant vector) according to the present invention has been introduced.

  In the transformant according to the present invention obtained using a microorganism such as bacteria or yeast as a host, the medium used for the culture is a medium that can efficiently culture the transformant according to the present invention. Either a natural medium or a synthetic medium may be used. The medium can contain a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the transformant according to the present invention. Examples of the carbon source include carbohydrates such as glucose, fructose, sucrose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol. Examples of nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic acids such as ammonium phosphate or other nitrogen-containing compounds, and peptone, meat extract and corn steep liquor. Can be mentioned. Furthermore, examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

  Further, an antibiotic such as ampicillin or tetracycline may be added to the medium according to the selection marker possessed by the recombinant plasmid (recombinant vector) according to the present invention.

  When culturing a host transformed with a recombinant plasmid (recombinant vector) according to the present invention having an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when cultivating a microorganism transformed with a recombinant plasmid (recombinant vector) having a promoter inducible with isopropyl-β-D-thiogalactoside (IPTG), IPTG or the like is added to the medium. can do. Further, when culturing a microorganism transformed with a recombinant plasmid (recombinant vector) according to the present invention having a trp promoter inducible with indoleacetic acid (IAA), IAA or the like can be added to the medium.

  Furthermore, the amount of nitrile hydratase produced by the transformant according to the present invention can be increased by adding a nitrile hydratase inducer to the medium. Examples of the nitrile hydratase inducer include nitrile compounds such as acetonitrile, propionitrile, butyronitrile, valeronitrile, and benzonitrile, and amide compounds such as urea, acetamide, and propionamide.

  Culture of the transformant according to the present invention obtained using a microorganism such as bacteria or yeast as a host is usually 20 ° C. to 40 ° C., preferably 30 ° C. to 30 ° C. under aerobic conditions such as shaking culture or aeration stirring culture. Perform at 37 ° C. The pH is adjusted using an inorganic or organic acid, an alkaline solution or the like, and may be set to pH 5.0 to 9.0, preferably pH 6.5 to 7.5.

  On the other hand, in the method for producing a nitrile hydratase protein according to the present invention, the recovery of the nitrile hydratase protein from the culture is first performed by cell lysis treatment, ultrasonic disruption treatment or grinding treatment using an enzyme such as cellulase or pectinase. The cells or cells are destroyed by such as. Next, insoluble matters are removed by filtration or centrifugation to obtain a crude protein solution. The obtained crude protein solution is applied to salting out, various chromatography (eg gel filtration chromatography, ion exchange chromatography, affinity chromatography, etc.), SDS-polyacrylamide gel electrophoresis, etc. alone or in appropriate combination. And can be isolated and purified.

  When the nitrile hydratase protein according to the present invention is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like to obtain a culture supernatant. . Thereafter, a general biochemical method used for protein isolation / purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., alone or in appropriate combination, is used to obtain a culture supernatant By applying to the above, the nitrile hydratase protein according to the present invention can be isolated and purified.

Moreover, the production of the nitrile hydratase protein according to the present invention can be carried out by adopting a cell-free protein synthesis system that does not use any living cells in addition to the protein synthesis system using the transformant as described above. The cell-free protein synthesis system is a system that synthesizes nitrile hydratase protein in an artificial container such as a test tube using a cell extract. The cell-free protein synthesis system used in the present invention includes a cell-free transcription system that synthesizes RNA using DNA as a template. In this case, the cell extract to be used is preferably derived from the aforementioned host cell. The cell extract may be concentrated or diluted, or may be used as it is, and is not limited. The cell extract can be obtained, for example, by ultrafiltration, dialysis, polyethylene glycol (PEG) precipitation or the like. Such cell-free protein synthesis can also be performed using a commercially available kit. Examples include reagent kits PROTEIOS ^™ (Toyobo), TNT ^™ System (Promega), PG-Meta ^™ (Toyobo), RTS (Roche Diagnostics), and the like. The nitrile hydratase protein produced by the cell-free protein synthesis system can be purified by appropriately selecting means such as chromatography as described above.

5. Method for Producing Amide Compound In the method for producing an amide compound according to the present invention, an amide compound converted from the nitrile compound is obtained by contacting the culture of the transformant according to the present invention or a treated product thereof with the nitrile compound. be able to. The method for culturing the transformant according to the present invention is the same as the method described above in the method for producing a nitrile hydratase protein according to the present invention. Further, the “culture” has the same definition as described above in the method for producing a nitrile hydratase protein according to the present invention. On the other hand, “treated product” means a product obtained by subjecting a culture to one or more treatment steps so as to isolate and purify the nitrile hydratase protein according to the present invention. As a processed material, what was obtained by the manufacturing method of the nitrile hydratase protein which concerns on this invention is mentioned, for example.

  The culture or the treated product thereof may be held on a suitable carrier and contacted with a nitrile compound as an immobilized enzyme.

  The nitrile compound used as the substrate may be any as long as the nitrile hydratase protein according to the present invention can be converted into the corresponding amide compound by acting on the nitrile group of the nitrile compound. The nitrile compound used as the substrate is not limited, but mandelonitrile is preferable. In particular, the nitrile hydratase according to the present invention is racemic in neutral or basic polar media for mandelonitrile and its derivatives, such as mandelonitrile and its derivatives resulting from a mixture of benzaldehyde and its derivatives and hydrocyanic acid. The nitrile group of mandelonitrile and its derivatives can be stereospecifically hydrated to produce optically active S-(+)-mandelamide or a derivative thereof.

  In the method for producing an amide compound according to the present invention, a culture or a treated product thereof is brought into contact with a nitrile compound so that the amide compound is produced. Here, the state of “contact” means that the nitrile hydratase protein according to the present invention in the culture or its treated product functions as an enzyme and the nitrile hydratase protein and the nitrile compound are enzymatically reacted. Good. “Contact” includes, for example, mixing separated and purified nitrile hydratase and a nitrile compound, adding a nitrile compound to a culture vessel of a cell expressing a nitrile hydratase gene, and subjecting the cell to the presence of the nitrile compound. And culturing the mixture with a nitrile compound. Conditions under which the nitrile hydratase protein according to the present invention functions as an enzyme are, for example, pH 5.0 to 9.0, preferably pH 6.5 to 7.5, and temperature is 0 ° C. to 40 ° C., preferably 10 ° C. to 30 ° C. Due to the above, it is preferable to contact the nitrile hydratase protein according to the present invention and the nitrile compound under the present conditions.

  In the method for producing an amide compound according to the present invention, the concentration of the nitrile compound in the reaction solution is not particularly limited, and examples thereof include 0.1 to 5% by weight, preferably 0.5 to 2.0% by weight. The concentration of the nitrile hydratase protein according to the present invention in the culture contacted with the nitrile compound concentration or the treated product thereof is not particularly limited as long as the enzyme reaction occurs. In addition, since the enzyme reaction also depends on the concentration of the substrate and the enzyme, the enzyme reaction can be efficiently performed by optimizing the concentration of the nitrile compound as the substrate and the concentration of the nitrile hydratase protein according to the present invention as the enzyme. Can proceed.

  In the method for producing an amide compound according to the present invention, the amide compound is recovered by removing the bacterial cells or cells by centrifugation or the like when the culture or a mixture of the treated product and the nitrile compound contains the bacterial cells or cells. Then, it can be recovered and purified by applying it to ordinary methods such as filtration, concentration, various types of chromatography, extraction, activated carbon treatment and distillation alone or in appropriate combination. Or you may collect | recover and refine | purify a mixture by applying directly to normal methods, such as filtration, concentration, various chromatography, extraction, activated carbon treatment, and distillation, individually or suitably combining.

  As described above, according to the method for producing an amide compound according to the present invention, an amide compound can be efficiently produced from a nitrile compound.

  Hereinafter, examples of the present invention will be described in detail.

[Example 1] Identification of nitrile hydratase gene
(1) Adjustment of chromosomal DNA derived from HT40-6 strain Rhodococcus sp. HT40-6 (FERM BP-5231) in MYK medium (0.5% polypeptone, 0.3% bactoeast extract, 0.3% bactomalt extract, 1% Glucose, 0.2% K ₂ HPO ₄ and 0.2% KH ₂ PO ₄ , pH 7.0) were cultured with shaking at 30 ° C. for 72 hours in 100 ml.
After cultivation, the cells were collected and suspended in 4 ml of a Saline-EDTA solution (0.1 M EDTA and 0.15 M NaCl (pH 8.0)). Next, 8 mg of lysozyme was added to the suspension, shaken at 37 ° C. for 1-2 hours, and then frozen at −20 ° C.

  Next, 10 ml of Tris-SDS solution (1% SDS, 0.1 M NaCl, and 0.1 M Tris-HCl (pH 9.0)) was added to the frozen suspension while gently shaking, and proteinase K (Merck) was added. (Final concentration 0.1 mg) was added and shaken at 37 ° C. for 1 hour to obtain a mixture.

  Next, an equal amount of TE-saturated phenol (TE: 10 mM Tris-HCl and 1 mM EDTA (pH 8.0)) was added to the mixture and stirred, followed by centrifugation. After centrifugation, the upper layer was collected, twice the amount of ethanol was added, the precipitated DNA was wound up with a glass rod, and rinsed in the order of 90%, 80%, and 70% ethanol to remove the remaining phenol.

The obtained DNA was dissolved in 3 ml of TE buffer. Next, ribonuclease A solution (100 ° C., heat-treated for 15 minutes) was added to the solution to 10 μg / ml, and shaken at 37 ° C. for 30 minutes. After adding proteinase K and shaking at 37 ° C for 30 minutes, add equal amounts of TE saturated phenol (TE: 10 mM Tris-HCl and 1 mM EDTA (pH 8.0)) and centrifuge to separate the upper and lower layers. It was.
Obtained after repeating the operation from adding equal amounts of TE saturated phenol (TE: 10 mM Tris-HCl and 1 mM EDTA (pH 8.0)) to separating the upper layer and the lower layer twice. Further, an equal amount of chloroform (containing 4% isoamyl alcohol) was added to the upper layer and centrifuged to separate the upper layer and the lower layer (hereinafter, the above operation was referred to as “phenol treatment”). Thereafter, twice the amount of ethanol was added to the upper layer, and the DNA was wound up and collected with a glass rod to obtain chromosomal DNA.

(2) Preparation of probe for nitrile hydratase gene PCR was performed using the chromosomal DNA obtained in (1) above as a template and the reaction solution composition and primers shown in Table 5 below.

  Primers NH-01 and NH-02 were both prepared with reference to the amino acid sequence of a region having high homology in the known nitrile hydratase gene. In NH-01 (SEQ ID NO: 5), s represents g or c (location: 6 and 15), and r represents g or a (location: 9, 12, and 18). W represents a or t (location: 17). Furthermore, in NH-02 (SEQ ID NO: 6), s represents g or c (location: 3, 6 and 12), and r represents g or a (location: 9 and 16). K represents g or t (location: 10), and n represents inosine (location: 18).

  After preparing the reaction solution, 30 cycles of a reaction in which one cycle was 93 ° C .: 30 seconds, 55 ° C .: 30 seconds and 72 ° C .: 3 minutes were performed. After completion of PCR, the amplified DNA was recovered using a GFX PCR DNA band and GelBand Purification kit (Amersham Biosciences). The recovered DNA was separated by 0.7% agarose gel electrophoresis, and then a DNA fragment of about 200 bp thought to encode a part of the nitrile hydratase gene was obtained. The DNA fragment thus obtained was labeled using a DIG DNA Labeling Kit (manufactured by Roche Diagnostics) as a probe.

(3) Preparation of DNA library
To 200 μl of chromosomal DNA derived from HT40-6 strain, 40 μl of 10-fold concentration restriction enzyme buffer, 145 μl of sterilized water and 15 μl of restriction enzyme Sau3AI were added and reacted at 37 ° C. for 6 hours, and then DNA was collected by ethanol precipitation. Next, the recovered DNA was subjected to agarose electrophoresis, and a DNA fragment in the vicinity of 7 kb was excised from the gel and recovered using a GFX DNA recovery kit (Amersham Biosciences). This DNA fragment was inserted into the BamHI site of E. coli vector pUC18 using a ligation kit (Takara Shuzo Co., Ltd.) to prepare a recombinant DNA library. The Escherichia coli vector pUC18 used for ligation was purchased from Takara Bio.

(4) Preparation of transformants and selection of recombinant DNA E. coli strain JM109 was inoculated into 1 ml of LB medium (1% bactotryptone, 0.5% bacto yeast extract and 0.5% NaCl), and incubated at 37 ° C. for 5 hours. Pre-cultured. After pre-culture, 100 μl of the culture was added to 50 ml of SOB medium (2% bactotryptone, 0.5% bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 1 mM MgSO ₄ and 1 mM MgCl ₂ ) and cultured at 18 ° C. for 20 hours. . After culturing, E. coli was collected by centrifugation, 13 ml of cold TF solution (20 mM PIPES-KOH (pH 6.0), 200 mM KCl, 10 mM CaCl ₂ and 40 mM MnCl ₂ ) was added, and the mixture was allowed to stand at 0 ° C. for 10 minutes. It was centrifuged again. After removing the supernatant, the precipitated E. coli was suspended in 3.2 ml of cold TF solution, 0.22 ml of dimethyl sulfoxide was further added, and the mixture was allowed to stand at 0 ° C. for 10 minutes. To 200 μl of the competent cells thus prepared, 10 μl of a solution (DNA library) containing a recombinant plasmid containing the DNA fragment derived from the HT40-6 strain prepared in (3) above was added and left at 0 ° C. for 30 minutes. . Next, the mixture was subjected to a heat shock at 42 ° C. for 30 seconds, cooled at 0 ° C. for 2 minutes, and then SOC medium (2% bactotryptone, 0.5% bacto yeast extract, 20 mM glucose, 10 mM NaCl, 2.5 mM KCl). , 1 mM MgSO ₄ and 1 mM MgCl ₂ ) 0.8 ml were added, followed by shaking culture at 37 ° C. for 60 minutes. After shaking culture, 200 μl of the culture solution was plated on LB agar medium containing 100 μg / ml of ampicillin and cultured at 37 ° C. for 18 hours.

  From the transformant colonies grown on the agar medium, transformants having the nitrile hydratase gene derived from the HT40-6 strain were selected using the colony hybridization method. That is, the transformant grown on the agar medium was transferred onto a nylon membrane (Biodyne A: manufactured by Nihon Pall Co., Ltd.), the cells were dissolved and the DNA was fixed, and then the nylon membrane was prepared according to (2) above. After treatment with a probe (about 200 kb fragment), colonies containing the desired recombinant DNA were selected using DIG Luminescent Detection Kit (Roche Diagnostics).

(5) Preparation of recombinant plasmid The transformant selected in (4) above was cultured overnight at 37 ° C in 100 ml of LB medium, and the plasmid was extracted using FlexiPrep Kit (Amersham Biosciences). . The recombinant plasmid thus obtained was named pMS057. FIG. 1 is a schematic diagram showing the structure of recombinant plasmid pMS057. In FIG. 1, “NHaseα” means a nitrile hydratase α subunit gene, and “NHaseβ” means a nitrile hydratase β subunit gene.

(6) Determination of base sequence The base sequence of the recombinant plasmid pMS057 was determined using CEQ2000XL (Beckman Coulter). As a result, the base sequence represented by SEQ ID NO: 7 was obtained. The nucleotide sequence shown in SEQ ID NO: 7 includes a nitrile hydratase α subunit protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 9, and a nitrile hydra consisting of the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 8. An open reading frame was found that encodes the Tase β subunit protein and activator region (SEQ ID NO: 10).

  Table 6 below shows a comparison between the amino acid sequence encoded by the obtained strain-derived nitrile hydratase gene and the amino acid sequence of a known microorganism-derived nitrile hydratase.

  As shown in Table 6, the nitrile hydratase gene derived from the HT40-6 strain is, for example, a known nitrile hydratase derived from Rhodococcus rhodochrous J1 (L type) and any of α subunit and β subunit at the amino acid sequence level. Furthermore, it was found that the nitrile hydratase gene derived from the HT40-6 strain was a novel nitrile hydratase gene.

[Example 2] Production of Rhodococcus genus bacterial transformant Using the plasmid pMS057 obtained in Example 1 as a template, PCR was performed using the reaction solution composition and primers shown in the following table.

  PCR was performed using Thermalcycler personal (Takara Shuzo Co., Ltd.) for 30 cycles of one cycle of 94 ° C: 30 seconds, 65 ° C: 30 seconds, and 72 ° C: 3 minutes.

  After completion of PCR, 5 μl of the reaction solution was subjected to electrophoresis on a 0.7% agarose gel to detect a 3 kb PCR product.

  After confirming that the PCR product was detected, the reaction solution was purified with GFX PCR DNA band and GelBand Purification kit (manufactured by Amersham Biosciences) and cleaved with restriction enzymes XbaI and Sse8387I. The PCR product subjected to the restriction enzyme treatment was subjected to electrophoresis on a 0.7% agarose gel, and a band around 3 Kb was recovered. The collected PCR product was ligated to the XbaI-Sse8387I site of vector pSJ034 using Ligation Kit (Takara Shuzo) to prepare a recombinant plasmid. The resulting recombinant plasmid was named pSMJ057. FIG. 2 is a schematic diagram showing the structure of recombinant plasmid pSMJ057. In FIG. 2, “NHaseα” means the nitrile hydratase α subunit gene, and “NHaseβ” means the nitrile hydratase β subunit gene. The SK92-B1 nitrilase regulator means a regulatory gene of the nitrilase gene derived from Rhodococcus erythropolis SK92-B1 strain.

  The vector pSJ034 is a plasmid already known to be able to express a nitrile hydratase gene in Rhodococcus bacteria. Vector pSJ034 can be prepared from vector pSJ023 according to the method disclosed in JP-A-10-337185. pSJ023 has been deposited as a transformant ATCC12674 / pSJ023 (FERM BP-6232) at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (Chuo 1-1-1, Higashi 1-1-1, Tsukuba, Ibaraki Prefecture).

  The nucleotide sequence of the gene inserted into the recombinant plasmid pSMJ057 was confirmed and confirmed to be a nitrile hydratase gene derived from HT40-6.

(2) Production of Rhodococcus genus bacterial transformants Cells in the logarithmic growth phase of Rhodococcus rhodochrous ATCC 12674 were collected using a centrifuge and washed three times with ice-cold sterile water. And suspended in sterilized water. 1 μl each of the plasmid (pSMJ057) prepared in (1) and 10 μl each of the cell suspension were mixed, and each was ice-cooled. A suspension of each plasmid and each bacterial cell was placed in a cuvette and subjected to electric pulse treatment at 2.0 KV and 200 OHMS using a gene introduction device Gene Pulser (BIO RAD). The electric pulse treatment solution was allowed to stand for 10 minutes under ice-cooling, and heat shocked at 37 ° C. for 10 minutes. Then add 500μl of MYK medium (0.5% polypeptone, 0.3% bacto yeast extract, 0.3% bacto malt extract, 0.2% K2HPO4, 0.2% KH2PO4) to the cuvette, let stand at 30 ° C for 5 hours, and then add 50μg / ml kanamycin. It was applied to a MYK agar medium and cultured at 30 ° C. for 3 days. The plasmid of the obtained colony was confirmed, and two types of transformants (Rhodococcus rhodochrous ATCC12674 / pSMJ057) were obtained.

[Example 3] Measurement of nitrile hydratase activity Rhodococcus rhodochrous ATCC12674 / pSMJ057 obtained in Example 2 was added to GGPK medium (1.5% glucose, 1% sodium glutamate, 0.1% bactoeast extract, 0.05% K2HPO4 , 0.05% KH2PO4, 0.05% MgSO4 · 7H2O, kanamycin 50 μg / ml, pH 7.2) and inoculated into 100 ml, and cultured with shaking at 30 ° C. for 72 hours.

As a comparative example, Rhodococcus sp. HT40-6 was inoculated into the following medium and cultured aerobically at 30 ° C. for 96 hours.
Medium (pH7.4)
Glucose 27g
Polypepton 4g
Yeast extract 2g
Ammonium sulfate 0.2g
Ammonium nitrate 2g
MgCl ₂ 0.2g
CaCl ₂ 40mg
MnSO ₄・ 4H ₂ O 4mg
FeCl ₃・ 7H ₂ O 0.7mg
ZnSO ₄・ 7H ₂ O 0.1mg
ε-Caprolactam 4g
CoCl ₂・ 6H ₂ O 30mg
30mM phosphate buffer (pH7.4) 1000ml

Each cell was washed three times with 20 mM phosphate buffer (pH 7.5) by centrifugation. The precipitated cells were suspended in 1.5 ml of the above buffer containing 20 mM mandelonitrile, and the reaction was performed while shaking at 30 ° C. for 30 minutes. After completion of the reaction, each reaction solution was centrifuged to remove the cells, and then the mandelamide content in the supernatant was measured by liquid chromatography (column; SHODEX ODSF511A, carrier; 0.2MH ₃ PO ₄ : acetonitrile = 4: 1, Analysis with a monitor (208 nm). The optical purity of the produced mandelamide was measured using an optical resolution column (CHIRALCEL CA-1, Daicel Chemical Industries, Carrier; 100% ethanol). The results are shown in Table 8.
The activity (U) was defined as the specific activity per dry cell (DC) as the amount of enzyme that produced 1 μmol of product per minute.

  As shown in the above examples, the transformant ATCC12674 / pSMJ057 has a very high catalytic ability. By using this transformant, optically active S-(+)-mandelamide is efficiently converted from mandelonitrile. Can be manufactured.

FIG. 6 is a structural diagram of plasmid pMS057. FIG. 6 is a structural diagram of plasmid pSMJ057.

Sequence number 5: Synthetic DNA
Sequence number 6: Synthetic DNA
SEQ ID NO: 7: pMS057 (Example 1)
SEQ ID NO: 11: synthetic DNA
SEQ ID NO: 12: synthetic DNA

Claims (9)

A nitrile hydratase protein comprising the following protein (a) and protein (b):
(a) A protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
(b) A protein comprising the amino acid sequence set forth in SEQ ID NO: 4. A protein having the function of a nitrile hydratase α subunit consisting of the amino acid sequence set forth in SEQ ID NO: 2. A gene encoding the protein according to claim 2. A protein having the function of a nitrile hydratase β subunit consisting of the amino acid sequence set forth in SEQ ID NO: 4. A gene encoding the protein according to claim 4. A recombinant vector containing the gene according to claim 3 and / or the gene according to claim 5. A transformant comprising the recombinant vector according to claim 6. A method for producing a nitrile hydratase protein, comprising culturing the transformant according to claim 7 and recovering the nitrile hydratase protein from the culture. A method for producing an amide compound, comprising contacting the culture of the transformant according to claim 7 or a treated product thereof with a nitrile compound to recover the amide compound.