JP2019193651A - Hasa-redoxin complex having oxidation activity and methods of making the same - Google Patents

Abstract

To provide a HasA-redoxin complex (heme iron active-type HasA) that asymmetrically oxidizes only one of the enantiomers of a secondary alcohol in the absence of coenzyme and exhibits an asymmetric oxidization activity in an aqueous solvent system in the presence of oxygen, and to provide methods for making an immobilized form thereof.SOLUTION: Disclosed herein is a method comprising: synthesizing HasA gene of a fluorescent bacterium Pseudomonas protegens Pf-5; inserting the synthesized HasA gene into an expression vector; transforming Escherichia coli with the expression vector to form a transformant; culturing the transformant to produce an expression product; purifying the expression product using a Ni affinity column to obtain HasA of P. protegens Pf-5; stirring the HasA in an aqueous solvent containing oxygen nano-babbles to form a HasA-redoxin complex; and immobilizing the HasA-redoxin complex to a carrier, where the carrier is a spherical porous kaolinitic ceramic carrier.SELECTED DRAWING: Figure 1

Description

  The present invention can catalyze an asymmetric oxidation reaction capable of stereoselectively oxidizing each enantiomer of racemic secondary alcohol and / or a HasA-redoxin complex capable of catalyzing non-selective oxidation. The present invention relates to a body (sometimes referred to as “heme iron-activated HasA”), an immobilized body thereof, and a method for producing the same.

  In various fields including pharmaceutical and food fields, there is an increasing need for a technique for separating optical isomers and fractionating one useful optical isomer. For example, research has been conducted on methods for producing pharmaceuticals and fragrances using an asymmetric hydrogenation (reduction) enzyme having a function of selectively redoxing or deracemizing one of optical isomers.

  A bioreactor is a system that uses the action / mechanism of such an enzyme for production and analysis of useful substances, but the reaction element that remains in the reactor is an enzyme having a metal ion as an active center, that is, an electron transfer / It has been understood as a function based on movement.

  Heme (iron porphyrin complex) enzymes are expected to be used in organic synthesis reactions. In particular, cytochrome P450 can hydroxylate inactive organic substrates (see Non-Patent Document 1) or epoxidation of styrene molecules. It has been reported that it can be made (see Non-Patent Document 2) or demethylated (see Non-Patent Document 3).

  Iron has an affinity for gases such as oxygen and can easily transfer electrons in a reducing environment at a neutral pH. Therefore, iron binds to proteins in the form of iron ions and iron prosthetic groups (iron-sulfur clusters and heme) and is used as the active center for various redox reactions including oxygen transport, energy production, and nucleic acid synthesis. Has been.

  As functions of hemoproteins, oxygen transport by oxygen (binding oxygen molecules) and electron transfer by cytochrome c (redox centers) are well known. Among its functions, the cholera-derived protein HutZ is known to have 1) the function of taking “heme” from hemoglobin into the cell body, and 2) the function of decomposing heme using oxygen (Non-patent Document 4). See).

The "heme" to another as Tokomu protein within the cells depriving from hemoglobin, pathogenic bacteria (Pseudomonas aeruginosa and Mycobacterium tuberculosis) from Hemofoa; is (HasA H eme A cquisition S ystem A) ( Non-Patent Document 5 See). In order to impart an asymmetric oxidation reaction to the hemoprotein HasA that does not have an oxidation catalytic activity, it must be a HasA-redoxin complex, and the redoxin that is an iron-sulfur cluster becomes an active center, resulting in a Fenton reaction. Yeah.

For the metabolic regulation of the iron and iron prosthetic groups, iron metabolism regulatory protein (IRP: I ron R egulatory P rotein) is, if the intracellular iron concentration is low only iron responsive element (IRE: I ron R esponsive E lement) and act to increase the concentration of intracellular free iron ions (see Non-Patent Document 6).

On the other hand, the iron - (there is a 4Fe-4S, 2Fe-2S type with a structure constituted by iron and sulfur) sulfur clusters are formed by mitochondrial ISC (I ron- S ulfur C luster assembly) machinery, cytoplasmic It is carried by cytoplasmic iron-sulfur cluster assembly (CIA) machinery and inserted into the IRP. It has been reported that the desorption of iron-sulfur clusters to IRP is a mechanism that senses changes in intracellular iron concentration and controls iron metabolism (see Non-Patent Document 7).

Furthermore, inhibitory transcriptional regulator of heme biosynthesis (IRR: I ron R esponsive R egulator) is by a signaling molecule iron concentration in the cell binds to heme, molecular oxygen binds to the heme A mechanism has also been reported in which reactive oxygen species are produced, and further promotes oxidative modification of proteins to eliminate transcriptional repression (see Non-Patent Document 8).

  Cytochrome P450 (P450), which is widely present in the biological world, is a group of powerful heme (iron porphyrin complexes) enzymes that hydroxylate inert organic substrates related to drug metabolism, detoxification, hormone biosynthesis, etc. Expected to be used in synthetic reactions. Bacteria-derived P450 has higher specific activity than animal and plant-derived P450, and substrate specificity to the substrate is high. However, there is a demerit that cannot cope with a wide range of substrate oxidation.

  For example, P450BSβ derived from Bacillus subtilis is known to hydrogenate long chain fatty acids using hydrogen peroxide, and P450cam of Pseudomonas putida-derived camphor hydroxylase and P450BM-3 derived from giant fungus exhibit very high activity. Therefore, it has been widely studied (see Non-Patent Document 1). P450 also has a demerit that it cannot be applied to oxidation reactions of other substrates.

  Moreover, as P450 reaction, there exists epoxidation reaction of styrene by P450BS (beta) origin derived from Bacillus subtilis, for example (refer nonpatent literature 2).

  Furthermore, soluble epoxide hydrolase (sEH) uses epoxyeicosatrienoic acid (EET), which is an endogenous physiologically active substance, as a substrate. EET is a fatty acid in which arachidonic acid cut from membrane phospholipid is epoxidized by cytochrome P450 (P450). That is, P450 is an EET synthase. For example, it is known to catalyze the following demethylation reaction (see Non-Patent Document 3).

Thus, P450, which is a group of heme (iron porphyrin complex) enzymes, is well known as a substrate oxidation of iron electron transfer system by reactive oxygen species, while P450 related to asymmetric dehydrogenase of secondary alcohol. The report has not yet been confirmed.

  This time, the presence of a HasA-redoxin complex that catalyzes an asymmetric oxidation reaction of a secondary alcohol, which cannot be derived from the P450 reaction, was confirmed. Although HasA is also an iron porphyrin complex, it generally does not have P450-like oxidation activity because it does not have a cysteine-coordinated heme structure. However, since redoxin (iron-sulfur cluster) is represented by the Fenton reaction (see Non-Patent Document 9), an iron electron transfer system by active oxygen species has been recognized, so the asymmetric oxidation catalyst of the HasA-redoxin complex is It can be recognized. Therefore, it can be expected to reduce the cost of catalyst production and the subsequent drug discovery process as a new enzyme considering the construction of a sustainable society.

  Protein expression by the E. coli expression system is a relatively straightforward technique for HasA. Production process: 1) Gene synthesis from gene information derived from fluorescent fungus HasA, 2) Plasmid construction, 3) Optimization of expression conditions after conducting small-scale E. coli expression test (host, induction / culture conditions, etc.), 4) Scale-up expression, 5) Can be produced by histag purification. For example, commissioned synthesis at Wako Pure Chemical Industries, Ltd. can also be handled.

  Expression of the gene protein can be performed using cultured cells, Escherichia coli, yeast, koji mold, Bacillus sp. Research on expression techniques using Escherichia coli has a long history (early 1970), and development of various related technologies is progressing, so it has been applied to the expression of various types of proteins. Although it is general-purpose but cannot be expressed by secretion, there is a demerit that the protein expressed in the microbial cell is accumulated in the microbial cell and the production amount is limited.

  Secretory microorganisms such as Koji mold and Bacillus genus secrete the target protein expressed inside the cell one after another, and thus have a feature of high expression level. It is used as an expression technique for proteins such as industrial enzymes.

  However, since secretory microorganisms secrete a variety of proteins, purification for extracting the target protein is complicated. Moreover, these secretory microorganisms have the property of actively degrading and utilizing proteins present outside the cells. Therefore, proteins that can be adapted to this expression method are limited to proteins that are difficult to be degraded. In other words, there has been no technology that has a high level of secretory expression while having versatility that allows expression of a wide variety of proteins.

  On the other hand, in the production of peas, there is a technique of spraying fluorescent bacteria, and the purpose of growth promotion and herbicidal effect is achieved (see Non-Patent Document 10). Fluorescent bacteria (a type of rhizobia) invade the host to become symbiotic organisms, and secondary metabolism of host-derived compounds produced by photosynthesis creates herbicides and the like. Therefore, there is a possibility that fluorescent bacteria in peas can produce HasA in cells in the sense of acquiring iron essential for survival.

  Therefore, HasA-redoxin complex expression having an asymmetric oxidation reaction can be produced in two ways: production by a microbial expression system and pea extraction method. In the case of microorganism-expressed HasA, 1) two steps of forming a HasA-redoxin complex in the presence of oxygen after gene expression, and 2) air oxidation of pea protein encapsulated in calcium alginate gel, then HasA-redoxin complex It is the method regarding the pea extraction method which extracts and refines.

Japanese Patent No. 3294860 International Patent Publication WO2010 / 134642

“J. Am. Chem. Soc. 120, (1998) 11044-11048” (R Hydroxylation of Carboxylic Acids with Molecular Oxygen Catalyzed by the R Oxidase of Peas (Pisum satiVum): A Novel Biocatalytic Synthesis of Enantiomerically Pure (R)- 2-Hydroxy Acids) "Waldemar Adam et al." “Tetrahedron 60, (2004) 525-528” (Alkene epoxidation catalyzed by cytochrome P450 BM-3139-3) “Edgardo T. Farinas et al.” “Chem. Res. Toxicol. 9 (1996) 365-373” (Involvement of Cytochrome P450 3A4 Enzyme in the N-Demethylation of Methadone in Human Liver Microsomes) “Christelle Iribarne et al.”

“Chem. Commun. 48, (2012) 6741-6743” (A heme degradation enzyme, HutZ, from Vibrio cholerae) “Takeshi Uchida et al.” “J. Inorg. Biochem. 138, (2014) 31-38” (Spectroscopic studies on HasA from Yersinia pseudotuberculosis) “S. Ozaki et al.” “Chem. Lett., 43, (2014) 1680-1689” (Unique Heme Environmental Structures in Heme-regulated Proteins Using Heme as the Signaling Molecule) “K. Ishimori et al.”

“Annual Review of Biochemistry 74 (2005) 247-281” (STRUCTURE, FUNCTION, AND FORMATION OF BIOLOGICAL IRON-SULFUR CLUSTERS) “Deborah C. Johnson et al.” “Biochemistry, 50 (2011), 1016-2102” (Unusual Heme Binding in the Bacterial Iron Response Regulator Protein: Spectral Characterization of Heme Binding to the Heme Regulatory Motif) “Haruto Ishikawa et al.” “Inorg. Chem. 53 (2014), 6473-6481” (Electronic Structure and Formation of Simple Ferryloxo Complexes: Mechanism of the Fenton Reaction) “Alban S. Petit et al.”

“Nat. Biotechnol. 23 (2005), 873-878” (Complete genome sequence of the plant commensal Pseudomonas fluorescens Pf-5) “Paulsen, I. T. et al.”

  One of the objects of the present invention is to catalyze an asymmetric oxidation reaction capable of stereoselectively oxidizing each enantiomer of racemic secondary alcohol and / or catalyze non-selective oxidation. It is to provide a possible HasA-redoxin complex.

  The present invention is based on the discovery that HasA (iron porphyrin complex) -redoxin complex can catalyze asymmetric oxidation of secondary alcohol substrates. That is, the present invention can provide a reactive oxygen species-iron electron transfer system in the presence of oxygen by linking HasA having no cysteine-coordinated heme structure to redoxin. Based on the discovery that the body can be stereoselectively asymmetrically oxidized.

  Another object of the present invention is to provide a HasA-redoxin complex excellent in environmental aspects and safety and cost of a drug discovery process, and a suitable production method thereof.

  As a result of intensive studies, the present inventors have newly discovered a HasA-redoxin complex capable of recovering S-alcohol by catalyzing R-alcohol asymmetric oxidation of racemic secondary alcohol, and a method for producing the same. The HasA-redoxin complex is obtained by encapsulating pea protein in calcium alginate gel, air oxidation and elution in warm water and purifying with a centrifugal filter or by expressing the fluorescent bacteria HasA gene in microorganisms. Obtainable.

  The present invention can include, for example, the following aspects.

[1] A method for producing a HasA-redoxin complex having reactivity with hydrogen peroxide in the absence of NAD (H) and / or NADP (H) from a plant, comprising the following steps: The manufacturing method characterized by the above-mentioned.
A first step of encapsulating plant-derived tissue with calcium alginate gel, followed by air oxidation;
A second step of centrifugally precipitating the oxidized product oxidized in the first step;

  [2] After the centrifugal precipitation treatment in the second step, the precipitate is dried to obtain a HasA-redoxin complex, or the supernatant is further subjected to centrifugal filtration (molecular weight 10 kDa; for example, Vivaspin 2-10 K). The production method according to [1], wherein a high-purity dried-HasA-redoxin complex-dried product is obtained.

[3] The manufacturing method according to [1] or [2], wherein the first step includes the following processes (a1) to (a3).
(A1) A generalization treatment in which the pulverized material derived from plant tissue (leaves, stems, roots) is directly included in the gel,
(A2) treatment for oxidizing the gel with air;
(A3) Treatment for eluting the HasA-redoxin complex from the gel after the oxidation treatment.

[4] The production according to [2], wherein the centrifugal precipitation treatment in the second step is a precipitation treatment involving gravity; and the second step includes the following treatments (b1) to (b3): Method.
(B1) From the aqueous solution of the oxidation product obtained in the first step, the HasA-redoxin complex is subjected to high-purity HasA-redoxin complex through re-centrifugal filtration (Vivaspin 2-10 K) of the centrifugal supernatant fraction. Treatment to dry and recover the body,
(B2) a process of re-dissolving the precipitated protein complex in a 50 mM glycine NaOH (pH 9.0) buffer solution, centrifugally precipitating and recovering the precipitate;
(B3) Treatment in which the pH of the 50 mM glycine NaOH is pH 9.0 to pH 11.0.

[5] The production method according to [2], wherein the precipitation recovery process in the second step is membrane filtration; and the second step includes the following processes (c1) to (c3).
(C1) treatment for separating and recovering the solid portion of the HasA-redoxin complex from the aqueous solution of the oxidation product obtained in the first step using glass beads or membrane treatment;
(C2) A process of further redissolving the separated and recovered product in a 50 mM glycine NaOH (pH 9.0) buffer, and separating and recovering the solid part using glass beads or membrane treatment,
(C3) A process of dehydrating and drying the separated and recovered product.

  [6] The method for producing a HasA-redoxin complex according to any one of [1] to [4], wherein the centrifugally precipitated HasA-redoxin complex obtained in the second step is powdered.

  [7] A plant-derived HasA-redoxin complex; and a protein having reactivity with hydrogen peroxide even in the absence of NAD (H) and / or NADP (H) Complex.

  [8] The HasA-redoxin complex according to [7], which has a function attributable to an organelle having asymmetric oxidation activity and / or catalase activity.

  [9] The HasA-redoxin complex according to [8], wherein the redoxin is present in a membrane protein and / or in the vicinity thereof and forms a complex with one or more iron sulfur clusters of IRP, IRE, and IRR.

  [10] The HasA according to [8], wherein the redoxin is derived from a membrane protein capable of forming a complex with a fluorescent bacterium, Pseudomonas aeruginosa, tuberculosis bacterium, or malaria bacterium HasA and having localized catalase activity. -Redoxin complex.

  [11] The HasA- described in [8], wherein the redoxin is present in a membrane protein and / or in the vicinity thereof, and has formed a complex with one or more iron-sulfur clusters of ferredoxin, adrenodoxin, and rubredoxin in HasA. Redoxin complex.

[12] Any of [7] to [11] having a vibration peak of iron-sulfur oxide (S—O and Fe—O) at least in the 950 cm ^{−1 to} 1250 cm ⁻¹ region in the FT-IR spectrum. The HasA-redoxin complex according to item 1.

  [13] The HasA-redoxin complex according to [12], wherein the region is a peak derived from iron-sulfur oxide (SO or Fe-O) generated by air oxidation of the amino acid cysteine in redoxin.

[14] As an amino acid sequence from the N-terminal to the 33rd amino acid, the following sequence:
MSX ^a SSISTX ^b YATNTVAQYLX ^a DWX ^b AYFGDLNHRE
(X ^a is probably Cys, X ^b is unknown); and a component having reactivity with hydrogen peroxide in the absence of NAD (H) and / or NADP (H) The HasA-redoxin complex according to any one of [7] to [13].

  [15] The HasA-redoxin complex according to [13], wherein the HasA-redoxin complex supported by the sequence is derived from a complex consisting of heme capture protein (HasA), ferredoxin, adrenodoxin, and rubredoxin.

  [16] The HasA-redoxin complex according to [15], wherein the HasA is derived from fluorescent bacteria, Pseudomonas aeruginosa, and / or Mycobacterium tuberculosis and derived from microorganisms expressed HasA including Escherichia coli.

  [17] The HasA-redoxin complex according to [16], which requires a period (2 to 3 days) of generating a HasA-redoxin complex in the presence of dissolved oxygen for the expression of the asymmetric oxidation reaction of the microorganism-expressed HasA.

  [18] The HasA-redoxin complex according to any one of [7] to [17], wherein the HasA-redoxin complex is coated with a polymer compound.

  [19] The HasA-redoxin complex according to any one of [7] to [17], wherein the HasA-redoxin complex is crosslinked.

  [20] Selectively obtaining one enantiomer of the racemic alcohol by allowing the HasA-redoxin complex described in any one of [7] to [19] to act on the racemic alcohol as a substrate. A process for producing an optically active alcohol characterized by

  [21] The HasA-redoxin complex according to [16], wherein the asymmetric oxidation reaction of the microorganism-expressed HasA requires a period of formation of a HasA-redoxin complex (2 to 3 days) in the presence of dissolved oxygen.

  [22] The HasA-redoxin complex according to any one of [7] to [21], wherein the HasA-redoxin complex is coated with a polymer compound.

  [23] The HasA-redoxin complex according to any one of [7] to [21], wherein the HasA-redoxin complex is subjected to a crosslinking treatment.

  [24] Selectively obtaining one enantiomer of the racemic alcohol by allowing the HasA-redoxin complex according to any one of [7] to [23] to act on the racemic alcohol as a substrate. A process for producing an optically active alcohol characterized by

  [25] The production of the optically active alcohol according to [24], wherein one enantiomer of the racemic alcohol is selectively asymmetrically oxidized to a ketone to selectively obtain the other enantiomer that is not involved in the reaction. Method.

  [26] The method for producing an optically active alcohol according to [25], wherein the precipitated HasA-redoxin complex according to [10] is used and water is used as a solvent in the reaction system.

  [27] The optically active alcohol according to [24], wherein the cross-linked HasA-redoxin complex according to [23] is used, and a glycine NaOH buffer (50 mM, pH 9.0) is used as a solvent in the reaction system. Production method.

  [28] Any solvent selected from the group consisting of 3% or less of DMSO, IPA, and ethanol as a cosolvent of an aqueous solvent system in the reaction system using the PEG-treated HasA-redoxin complex according to [22] The method for producing an optically active alcohol according to [24], wherein the protein complex does not impair the catalytic activity even when used.

  [29] The optical activity according to [12], wherein the cross-linked HasA-redoxin complex according to [23] is used and DMSO (<2% (v / v)) is used as a cosolvent in the reaction system. A method for producing alcohol.

  [30] The method for producing an optically active alcohol according to [24], wherein a catalytic reaction is performed in a reaction system of cap-free, nano-oxygen bubble water, or an oxygen supply system.

  [31] The method for producing an optically active alcohol according to [24], wherein the active center of the HasA-redoxin complex in the reaction system is an asymmetric oxidation reaction in redoxin accompanied by iron electron transfer by reactive oxygen species.

  [32] When the reactivity with the hydrogen peroxide is 15 mg of the HasA-redoxin complex (particle size 50 μm) in a test tube having a diameter of 18 mmφ, 1.0 mL of 5% hydrogen peroxide water is added thereto. Furthermore, the HasA-redoxin complex according to [7], which has a level of reactivity that generates bubbles that are visible to the naked eye based on the generation of oxygen.

  [33] 20 mg of the complex (particle size 50 μm) in an aqueous solvent system (4 mL) at room temperature 40 ° C., substrate racemic 6-methoxy-1- (2-naphthyl) ethanol (Rac-1) 0. The HasA-redoxin complex according to [7] having a substrate reactivity of 10 Unit / g (per hour) or more as a specific activity calculated by an average rate at a reaction time of 15 hours when the reaction is performed at 8 mM. body.

  [34] 20 mg of the complex (particle size 50 μm) in an aqueous solvent system (4 mL) at room temperature 40 ° C., substrate racemic-6-methoxy-1- (2-naphthyl) ethanol (Rac-1), 0 The HasA-redoxin complex according to [7], wherein the enantioselectivity that gives the naproxen precursor (s-1) is 95ee% or more when the reaction is carried out at 8 mM.

  [35] An organic solvent (toluene, acetonitrile, glutaraldehyde, etc.) of 1% to 2% of the solvent weight is used in the polymer compound (PEG) coating treatment after the centrifugal precipitation treatment in the second step [1] The manufacturing method as described in.

[36] The reactivity with hydrogen peroxide is 1 μM-H ₂ O ₂ aq. In a reaction for 5 minutes in a substrate 2 mM hydrogen peroxide (1 mL) as measured by a digital hydrogen peroxide concentration meter. The HasA-redoxin complex according to [7], wherein an amount capable of degrading is 200 μg or less.

  The present invention can also include the following aspects.

[A33] An immobilized HasA-redoxin complex comprising a carrier for immobilization and a HasA-redoxin complex immobilized on the carrier.

[A34] The immobilized HasA-redoxin complex according to [A33], wherein the carrier is a micronized and / or porous carrier.

[A35] The immobilized HasA-redoxin complex according to any one of [A33] or [A34], wherein the porous carrier is any one of an inorganic carrier, an organic carrier, and an inorganic-organic hybrid carrier.

[A36] The immobilized HasA-redoxin complex according to any one of [A33] to [A35], wherein the HasA-redoxin complex is immobilized on a porous carrier by adsorption.

[A37] The method according to any one of [A33] to [A36], wherein the “conversion rate to the product ketone” measured by a measurement method including the following steps (a) to (c) is 45% or less. Immobilized HasA-redoxin complex.

  (A) 5 mL of fluorescent bacteria HasA solution (concentration: 1.8 mg / mL) expressed by E. coli is placed in a centrifuge tube, and then a “carrier sample” (1000 mg) to be screened is dropped into the centrifuge tube and shaken. Using a machine, stir at “8-shape shaking” at 500 to 700 times / min for 48 hours to immobilize the fluorescent bacterium HasA on the carrier sample.

  (B) Collect “HasA solution residue” after the immobilization operation.

  (C) Substrate rac-1 (0.4 mg) was added to the HasA residue (0.2 mL / ion-exchanged water 4 mL) and reacted with stirring with a stirrer for 50 hours, and then the ketone conversion rate produced (%) Is measured.

[A38] When the immobilized HasA-redoxin complex is subjected to the first reaction, and then the complex is recovered, and when the “percentage of 50 h produced ketone” is measured for the recovered complex, The immobilized HasA-redoxin complex according to any one of [A33] to [A37], wherein the value of “percentage of 50 h-generated ketone” is 30% or more.

Examples of the features of the present invention include the following five points.
(A) Since the hemophore (HasA) gene is known, the Convention on Biological Diversity (CBC) entered into force on December 29, 1993 to which 193 countries including Japan joined, and 2010 Do not belong to microbial genetic resources related to the Nagoya Protocol related to Access and Benefit-Sharing (ABS) in October.
(B) Since it is a higher plant-derived HasA-redoxin complex, it should be used effectively as an asymmetric oxidation catalyst for unused biological resources (biomass).
(C) Effective utilization of the microorganism-expressed HasA for the asymmetric oxidation reaction of secondary alcohol after forming a HasA-redoxin complex in the presence of oxygen.
(D) The HasA-redoxin complex catalyzes an asymmetric oxidation reaction of a secondary alcohol through a resynthesis system of active oxygen species in the presence of oxygen.
Therefore, according to the present invention, a HasA-redoxin complex having an asymmetric oxidation reaction of a secondary alcohol produced from a plant and / or microbial expression is provided.

As described above, according to the present invention, a plant-derived HasA-redoxin complex and / or a microorganism-expressed HasA-redoxin complex imparted with an activity for catalyzing a practical asymmetric oxidation reaction is provided.
According to the present invention, the HasA-redoxin complex of the present invention (hereinafter referred to as ME) is further put into the environment by a suitable production method relating to powdering of such plant-derived HasA-redoxin complex. It can be provided as a novel asymmetric oxidation catalyst that can realize low-cost production gently.

It is a conceptual diagram showing the one aspect | mode of the purification method of plant origin HasA-redoxin complex (henceforth PP-HasA). It is a conceptual diagram showing the one aspect | mode of the manufacturing method of microorganisms expression HasA-redoxin complex (henceforth HasApf). It is an example of the graph and table | surface which show the result of the structural element analysis of PP-HasA and HasApf. It is a graph which shows an example of the result of FTIR of PP-HasA and HasApf. Preparative fractions (supernatant, precipitate (PC precipitate)) obtained by centrifuging the HasA-redoxin complex aqueous solution (hereinafter referred to as PC) obtained in the first step and the precipitates thereof. It is a graph which shows the asymmetric oxidation activity of the sample which carried out FD drying after cellulose coating (CM-Cellulose coating PC) or glutaraldehyde bridge | crosslinking (CLPC).

Each supernatant fraction obtained by centrifugation in FIG. 1; SDS (PC (A), ME (B), B (C) after treatment with Vivaspin 2-10K, and C (D) after asymmetric oxidation reaction It is a photograph which shows an example of a PAGE result. In the nano oxygen bubble aqueous solvent (5.0 mL) of 0.8 mM substrate racemic-1- (2-naphthyl) ethanol (hereinafter referred to as Rac-2) by the fluorescent bacteria HasA gene (HasApf) expressed in E. coli in FIG. It is a graph which shows asymmetric oxidation reaction. It is an example of the graph which shows the change of DO in the asymmetric oxidation reaction by Escherichia coli expression HasApf. It is a graph which shows an example of the difference of ESR spectrum of PP-HasA (a) and HasApf (b). It is a conceptual diagram showing the one aspect | mode in the asymmetric oxidation activity intensity | strength of each cell identification and fluorescent-bacteria HasA (PP-HasA) refinement | purification process in a pea protein (PP).

FIG. 7 shows the result of 33 amino acids detected in the N-terminal sequence of band 2 in FIG. 6 and the gene information of the fluorescent bacterium HasA obtained by the subsequent BLAST analysis. Moreover, an example of Cys is shown from the amino acid sequence of redoxin. FIG. 7 shows the result of 33 amino acids detected in the N-terminal sequence of band 2 in FIG. 6 and the gene information of the fluorescent bacterium HasA obtained by the subsequent BLAST analysis. Moreover, an example of Cys is shown from the amino acid sequence of redoxin. Asymmetric oxidation specific activity (Unit (mg · min) for substrate Rac-2 (0.8 mM) in distilled water or nano oxygen bubble water-solvent (5.0 mL) of dry PC, CMME, AGME obtained in the first step )). It is an example of the graph which showed the influence which acts on the asymmetric oxidation with respect to substrate Rac-2 (0.8 mM) in nano oxygen bubble water-solvent (5.0 mL) of DMSO co-solvent. It is a schematic diagram regarding proper use of both enantiomers for the substrate Rac-2 (1.2 mM) in nano oxygen bubble water-solvent (5.0 mL) using CMME and AGME asymmetric oxidation reaction. It is a figure which shows the one aspect | mode regarding the molecular function of a heme capture protein (HasA) and a heme receiving protein (HasR), and the figure which shows the genome sequence (627 bp) of fluorescent-bacteria HasA (HasApf) expressed by colon_bacillus | E._coli. It is a figure which shows the genome sequence (5369bp) of pET28a vector.

It is an example of the schematic diagram which shows secondary alcohol asymmetric oxidation reaction by the reactive oxygen species-resynthesis system of HasA-redoxin complex. It is an example of the schematic diagram which shows the synthesis | combination of the naproxen precursor using the protein complex of this invention; and the further naproxen synthesis | combination, and the conventional naproxen synthesis flow. It is an SDS-PAGE result after purification of an Escherichia coli expressed HasA gene. Substrate rac-2 (0.4 mg) / water solvent (4 mL) asymmetric oxidation reaction over time with HasApf (0.36 mg): optical purity (-●-:% ee), S-2 (-□-: 0.2 mg), R-2 (-O-: 0.2 mg), and the resulting ketone (-[Delta]-). It is a UV measurement result (after 0h, 24h, 48h, 72h) in the asymmetric oxidation reaction of fluorescent bacteria HasApf (0.36 mg) / substrate rac-2 (0.4 mg) / water solvent (4 mL).

It is an ESR measurement result after 0 hours and 50 hours after the asymmetric oxidation reaction in fluorescent bacteria HasApf / substrate rac-2 (rac-2) / water solvent (4 mL). It is an ESR measurement result 100 hours after asymmetric oxidation reaction in fluorescent bacteria HasApf / substrate rac-2 (rac-2) / water solvent (4 mL). It is a transparency comparison after 2 days shake adsorption of HasA solution (dark brown) / toyonite. FIG. 3 shows the results of 1)% ketone produced by the residue of HasA solution after adsorption of toyonite, 2)% of ketone produced by various toyonite, and 3) optical purity.

It is a pore distribution of Toyonite. It is a SEM photograph of Toyonite Toyonite. There are four types of toyonite. It is a state of the substrate concentration of Toyonite HasApf immobilization support (200 and 200A) and the state of continuous reuse.

It is a state of the substrate concentration of Toyonite HasApf immobilization support (200P and 200M) and continuous reuse. It is an asymmetric conversion mechanism of HasApf. It is a route of heme metabolism.

FIG. 1 is a schematic diagram 1 of a cyclic deracemization reaction. FIG. 2 is a schematic diagram 2 of a silic deracemization reaction.

  Hereinafter, the present invention will be described more specifically with reference to the drawings as necessary. In the following description, “part” and “%” representing the quantity ratio are based on mass unless otherwise specified.

(Overview of HasA-redoxin complex)
The outline of the production method, physical properties, and reactivity of the HasA-redoxin complex of the present invention is as follows (the details will be sequentially described later). In one preferred embodiment of the present invention, the protein complex (PC) of the present invention is a protein complex containing at least iron, and in the FT-IR spectrum, at least 1 in the region of 1085 ± 50 cm ^−1. It has a number of peaks and remarkably bubbles in the reaction with hydrogen peroxide, releasing oxygen. The complex of the present invention does not require the presence of NAD (H) in its expression of hydrogen peroxide activity (i.e., the complex of the present invention exhibits hydrogen peroxide activity even in the absence of NAD (H). Expression is possible). This is because redoxin (iron-sulfur cluster) works as an active center for the generation of reactive oxygen species based on the Fe electron transfer system and functions as an electron donor to the hydrogen peroxide like NAD (H). Can be estimated.

<Production method>
HasA is obtained from a plant-derived protein by a method for producing HasA, which will be described later, or by genetic engineering techniques. Then, the HasA can be subjected to a predetermined treatment (for example, gel inclusion → air oxidation → elution into an aqueous solution) to obtain the HasA-redoxin complex of the present invention.

<Physical properties>
The protein complex of the present invention has at least one peak in the region of 1085 ± 50 cm ⁻¹ in its FT-IR spectrum. Note that in the preferred embodiment of the present invention, HasA- redoxin complexes of the present invention is a HasA- redoxin complexes containing at least iron and the sulfur in its FT-IR ^{spectrum, 950cm -1 ~1250cm} -1 In this region, there are strong peaks of S—O and Fe—O vibrations. The amino acid sequence from the N-terminal to the 33rd amino acid of the HasA-redoxin complex is the following sequence.
MSCSISST * YATNTVAQYL CDW * AYFGDL NHRE,

<Reactivity-catalase activity>
(Method for measuring catalase activity-1)
As described above, the HasA-redoxin complex of the present invention has catalase activity in addition to asymmetric oxidation activity. As described above, this catalase activity has a property of reacting with hydrogen peroxide (H ₂ O ₂ ) to release oxygen. The AG / CM-ME of the present invention having a particle size of 50 μm is suitable depending on the “bubble generation” level based on oxygen generation when 15 mg is put into a test tube having a diameter of 18 mmφ and 1.0 mL of 5% hydrogen peroxide water is added. (See Table 3 below).

  In the confirmation of the “bubble generation” level, it is sufficient if “bubble generation” based on oxygen generation is visible with the naked eye. More specifically, the “bubble generation” level (or “height” in the bubble generation test tube) is preferably 2 mm or more. Further, the “height” of the bubble generation is more preferably 5 mm or more (more preferably 8 mm or more, patent 10 mm or more).

(Method for measuring catalase activity-2)
On the other hand, the catalase activity can also be suitably evaluated by measuring the consumption amount of “hydrogen peroxide” in the above reaction (see Table 3 described later).

  More specifically, as shown in the examples described later, the residual amount of “hydrogen peroxide” after the reaction is measured using a commercially available “pack test” (manufactured by Kyoritsu Riken). Can do. This measurement method can be suitably used, for example, for “screening” of candidate substances.

(Measurement method of catalase activity-3)
Further, as shown in the examples described later, a commercially available “digital hydrogen peroxide concentration meter” and the residual amount of “hydrogen peroxide” after the reaction were measured, and based on this, the AG / CM-ME in the present invention was measured. The specific activity for the amount of hydrogen peroxide decomposition reaction can be measured.

Specific activity of the iron-containing HasApf-redoxin complex of the present invention with respect to the hydrogen peroxide decomposition reaction in the case of using a “digital hydrogen peroxide concentration meter” (in the reaction for 5 minutes in the substrate 2 mM-hydrogen peroxide (1 mL)) The amount of AG / CM-ME that can decompose 1 μM-H ₂ O ₂ is preferably 200 μg, more preferably 150 μg (particularly 130 μg). In the embodiment in which the iron-containing HasApf-redoxin complex of the present invention has an “AG / CM-ME amount”, the specific activity (1 μM-H ₂ O ₂ can be decomposed with respect to the hydrogen peroxide decomposition reaction). The amount of AG / CM-ME is preferably 100 μg, more preferably 80 μg (particularly 60 μg).

<Reactivity-Asymmetric oxidation activity>
<Asymmetric oxidation activity-Measurement method 1>
20 mg of the HasA-redoxin complex (particle size 50 μm) of the present invention was used in an aqueous solvent system (4 mL) at a room temperature of 40 ° C. and a substrate racemic-6-methoxy-1- (2-naphthyl) ethanol (Rac-1). When the reaction is carried out under the condition of 0.8 mM, it has a substrate reactivity of 10 Unit / g (per hour) or more as the specific activity calculated by the average rate at a reaction time of 15 hours.

<Asymmetric oxidation activity-measurement method 2>
20 mg of the HasA-redoxin complex (particle size 50 μm) of the present invention was used in an aqueous solvent system (4 mL) at a room temperature of 40 ° C. and a substrate racemic-6-methoxy-1- (2-naphthyl) ethanol (Rac-1). When the reaction is carried out under the condition of 0.8 mM, the enantioselectivity giving the naproxen precursor (s-1) is 95ee% or more at a reaction time of 15 hours.

(Method for producing HasA-redoxin complex)
Hereinafter, a method for producing a PEG aggregation precipitation-protein complex in one preferred embodiment of the present invention will be described.

  In this embodiment, a method for producing a HasA-redoxin complex includes a packaging process in which a gel bead is obtained by encapsulating a plant-derived water-soluble crude protein in a gel, an air oxidation process in which the gel bead is air-oxidized, and an air-oxidized gel bead. A first step comprising an elution treatment for elution into an aqueous solution to obtain a HasA-redoxin complex (PC) aqueous solution; a concentration treatment for precipitating the HasA-redoxin complex (PC) by applying gravity to the aqueous PC solution; The precipitate is allowed to coexist with the polymer compound in an aqueous solution, and further gravity is applied to precipitate the HasA-redoxin complex (ME), and the supernatant fraction is centrifuged (molecular weight 10 kDa; for example, Vivaspin 2-10 K). The filtrate is dried to obtain a pure pin HasA-redoxin complex, or the precipitate fraction is immediately subjected to freeze-drying (FD) treatment to form a polymer compound. A second step of the coating -HasA- redoxin complex is characterized by including. Further, the present invention is characterized in that a HasA-redoxin complex can be prepared by expressing a HasA gene in E. coli and shaking for 2 to 3 days in nano oxygen bubble water.

(1st process; comprehensive processing)
In this embodiment, in the inclusion process, first, a plant-derived water-soluble crude protein is included in a gel to obtain a hydrophobic carrier.

  Here, the plant-derived water-soluble crude protein is a crude protein obtained from a plant, and either a commercially available product or a direct extract from a plant resource may be used. Examples of commercially available products include soybean protein (trade name “pea protein PP-CS”) sold by Organo Food Tech Co., Ltd.

  Examples of such “plant resources” include cereals such as buckwheat, amaranth, rice, wheat, barley, corn, oat, rye, straw, millet, millet, pigeon, sorghum; These seeds, leaves, stems, roots, flowers, and fruit plant tissues including beans such as soybeans and general grasses and weeds are also included. As a method for extracting crude protein from plant resources, the method disclosed in Patent Document 1 can be appropriately employed.

The fluorescent bacterium Pseudomonas fluorescens Pf-5 is used as a spraying agent in the agricultural field because it suppresses the growth of other microorganisms and produces compounds such as antibacterial substances to promote host growth. These rhizobia are transmitted to progeny early embryos as symbiotic bacteria held in cells and tissues in plants. Therefore, hemophor HasA (iron porphyrin complex) produced by the fluorescent bacterium Pseudomonas fluorescens Pf-5 is not hit by BLAST analysis but can be obtained from “plant resources”. Proteins involved in metabolic regulation of iron and iron prosthetic groups: i.e., iron metabolism regulatory protein (IRP: I ron R egulatory P rotein), iron responsive element (IRE: I ron R esponsive E lement), heme biosynthesis inhibitory Transcriptional regulators (IRR), ferredoxin, adrenodoxin, rubredoxin, and the like can also be said to be biomass that can be obtained from “plant resources”.

  As a specific method for obtaining the entangled gel of the plant tissue pulverized product, for example, it is incorporated in a fine lattice of a gel such as alginate, starch, konjac, polyacrylamide gel and polyvinyl alcohol (lattice type) or semi-transparent. There is a packaging method (microcapsule type) covering with a membranous film, but in order to separate the HasA-redoxin complex from plant tissue, the packaging method using a calcium salt of alginic acid is inexpensive and environmentally friendly, and The comprehensive operation and the recovery operation are most preferable in terms of simplicity.

  More specifically, for example, a plant tissue (seed, leaves, stems, roots, flowers, fruits) pulverized product is dissolved in distilled water, and then an aqueous sodium alginate solution is added and stirred until uniform, and the resulting mixture is obtained. A crude protein gel can be prepared by dropping the solution into an aqueous calcium chloride solution. Here, the concentration of the aqueous calcium chloride solution is preferably 0.5% to 1.0% (preferably 0.6%).

(First step; air oxidation treatment)
In a preferred embodiment of the present invention, in the air oxidation treatment, the gel is separated from the aqueous solution using a colander and air-oxidized by allowing to stand in air. The air oxidation treatment is performed for about 30 minutes to 3 hours, more preferably for 1 hour, whereby the final HasA-redoxin complex of the present invention can be obtained in a higher yield. This treatment promotes Heme destruction by oxygen and HasA-redoxin (iron-sulfur cluster) complex formation in the crude protein. In addition, since cysteine in redoxin binds to oxygen and changes to sulfur oxides (Fe-O and S-O), as well as the "exudation effect in aqueous solution", "asymmetric oxidation reaction" Therefore, the air oxidation treatment is the key to the production method of the HasA-redoxin complex of the present invention and is indispensable.

  In the air oxidation process of the crude protein-encapsulating gel, it is preferably 30 ° C. or lower, and more preferably 20 ° C. or lower, with attention being paid to suppressing bacterial growth. The treatment needs to be performed promptly until the hot water extraction, but if it is absolutely necessary, the inclusion gel can be refrigerated at 10 ° C. or less and around 4 ° C., but the deterioration of the active strength cannot be prevented.

(First step; elution treatment)
In a preferred embodiment of the present invention, the elution treatment is obtained by eluting the HasA-redoxin complex by shaking the air-oxidized plant tissue-encapsulated gel with warm water. As a means for eluting, it is preferable to shake / stir the gel beads in warm water. The water is preferably warm water, and the water temperature is preferably 30 to 50 ° C. As the number of rotations of shaking, it can be appropriately employed within a range in which the gel beads can shake. The shaking time is preferably about 2 to 4 days, and more preferably 3 days (72 hours). As the shaking means, a constant temperature shake incubator, a stirring temperature control tank, or the like can be used.

  As the elution of the HasA-redoxin complex proceeds from the gel, a unique sulfur odor begins to be produced, and bacterial growth in the process of hot water extraction at 30 ° C. to 40 ° C. cannot be prevented, but before the sulfur odor turns into a rotten odor. It is preferable to perform the concentration (centrifugation) operation and the FD drying operation more quickly. If the bacterial growth is left to produce a rotten odor, it causes 1) a decrease in activity intensity and 2) a loss of stereoselectivity. In order to prevent the production of spoiled odor, the gel can be refrigerated at 10 ° C. or lower and around 4 ° C., but its activity strength cannot be prevented from decreasing. Hereinafter, an aqueous solution formed by eluting the HasA-redoxin complex from the gel is referred to as PC.

(Second step; concentration treatment)
In a preferred embodiment of the present invention, in the concentration treatment, gravity is applied to the PC aqueous solution obtained in the first step to remove impurities remaining in the aqueous solution, thereby obtaining a precipitate of the HasA-redoxin complex. Is the purpose. As a means, the sediment which has been naturally allowed to stand and the earth's gravity sinks is further subjected to physically strong gravity with a centrifuge, the aqueous solution fraction is removed, and the HasA-redoxin complex having concentrated asymmetric oxidation activity The method of obtaining is mentioned. If the method can quickly concentrate the HasA-redoxin complex, there is no need to stick to this centrifugation method.

  For the purpose of further washing and concentrating the HasA-redoxin complex obtained by this concentration treatment, 50 mM glycine NaOH (pH 9.0 to pH 11.0) buffer was further added and centrifuged several times. I can do it. In the washing treatment, an aqueous solution (hereinafter referred to as ME) obtained by re-dissolving a buffer of 50 mM glycine NaOH (pH 9.0 to pH 11.0) added to the HasA-redoxin complex-precipitate obtained by the concentration treatment, By applying gravity again and removing the aqueous solution fraction, the activity is increased and washing is performed. Further, when it is desired to obtain a pure HasA-redoxin complex completely separated from the cell membrane, the ME centrifugal supernatant-water solution is centrifuged with an ultrafiltration membrane (for example, Vivaspin 2-10 K). Can be obtained simply by drying and concentrating the filtered aqueous solution.

(Second step: PEG coating treatment of HasA-redoxin complex)
In a preferred embodiment of the present invention, in the PEG coating treatment of the HasA-redoxin complex, the precipitate obtained by the concentration treatment (such as centrifugation) is redissolved in a 50 mM glycine NaOH (pH 9.0) buffer. In addition, 0.5% to 1% PEG (MW1000 (AGME) or MW4000 (CMME)) is allowed to coexist with the product, and left for about 30 minutes to 3 hours, preferably 2 hours, followed by concentration treatment (centrifugation, etc.) Thus, the R- / S-selective asymmetric oxidation catalyst is preferably obtained by freeze-drying (FD) treatment. With PEG coating, not only 1) conversion of stereoselectivity but also 3) improved reaction activity (0.6 mU → 0.8 mU) are realized.

  The specific activities of CMME and AGME are 0.6 mU (mg · min) and 0 when Rac-1 / -2 (1.2 mM) is allowed to act in a distilled water (5.0 mL) solvent system. The specific activity increases to 0.7 mU (mg · min) and 0.9 mU (mg · min) when the solvent is nano oxygen bubble water. Therefore, 0.1% concentration of PEG coating can 1) change the stereoselectivity of the complex, and 2) use nano-oxygen bubbled water as a solvent to improve specific activity.

  The amount of PEG added to the ME aqueous solution can be appropriately selected depending on the scale of production equipment and the molecular weight of PEG used. For example, when PEG (MW: 4000) is used, about 0.1% to 1.0% by weight of PEG, preferably 0.5% by weight, based on the weight of the ME aqueous solution obtained by the concentration treatment. Is preferably used. For example, Rac-1 / having a suitable activity strength is obtained by re-dissolving in 2 g of the ME aqueous solution with PEG (10 g), collecting the centrifugal precipitate, and subsequently freeze-drying under reduced pressure (FD). -2 can be processed into a HasA-redoxin complex having an R-alcohol selective oxidation reaction.

  A high-purity product of the HasA-redoxin complex can be suitably obtained by simply filtering from the ME centrifugal supernatant using a centrifugal filter with an ultrafiltration membrane. On the other hand, the ME precipitation fraction is a HasA mixture composed of cell membrane fragments such as membrane proteins containing cell membranes (for example, ABC transfer, HasR, etc). Furthermore, it can be predicted that a sufficient structure of the HasA-redoxin complex that catalyzes the asymmetric oxidation reaction can be maintained by exhibiting an activity 200 times or more that of the fluorescent bacterium HasA expressed by the microorganism. By coating ME with PEG, the substrate in the aqueous solvent can easily come into contact with the ME active center (HasA-redoxin complex) due to its surfactant effect, contributing to the improvement of specific activity, and the nano oxygen bubble Further improvement can be achieved by using water as a solvent.

(Second step; powdering treatment)
In a preferred embodiment of the present invention, the means for processing into a powder form includes methods such as vacuum freeze drying, hot air drying, pressure drying, reduced pressure drying, and natural drying. Among these, vacuum freeze-drying is preferable because moisture can be effectively and completely removed in a short time. In the case of performing vacuum freeze-drying, for example, after freezing at about −50 ° C. for 1 hour or more, preferably completely, the system is evacuated and the temperature is raised to about 50 ° C. to sublimate moisture. When pulverizing, air (oxygen, nitrogen, carbon dioxide, etc.) should be dried as much as possible in the dryer and dried at about 50 ° C., and the HasA-redoxin complex air (oxygen, nitrogen, carbon dioxide, etc.) Treatment to avoid reaction with is the key to maintaining activity. In the drying process, the HasA-redoxin complex turns black when it reacts with oxygen.

  The dried HasA-redoxin complex (AGME and CMME) does not affect the specific activity even when pulverized with many pulverizing models including a ball mill. In addition, the specific activity of CMME can be maintained for more than half a year at room temperature storage, for more than two years for refrigerated storage, and for more than three years for refrigeration / argon gas encapsulation.

(HasA-redoxin complex)
In a preferred embodiment of the present invention, HasA- redoxin complexes of the present invention is a HasA- redoxin complexes containing at least iron and sulfur, in the FT-IR ^spectrum, the area of 950cm ^-1 ~1250cm -1 And have strong peaks of S—O and Fe—O vibrations. The amino acid sequence from the N-terminal to the 33rd amino acid of the HasA-redoxin complex is the following sequence:

  MSCSISST * YATNTVAQYL CDW * AYFGDL NHRE,

  The 3rd and 21st Cys (C) comes from redoxin, while exhibiting 93% synergism with the fluorescent fungus HasA. In addition, it is characterized in that it bubbles significantly in the reaction with hydrogen peroxide and releases oxygen.

(Preferred genetic engineering method)
The operation of the first step in FIG. 2 first synthesizes a Pseudomonas protegens Pf-5 gene sequence (HasA; accession No. YP — 262445.1, or WP — 011063600; FIG. 15). The synthesized gene (HasA) is inserted into an expression vector (pET28a (+); FIG. 15). The produced expression vector (pET28a / HasA) is transformed into an E. coli strain (3 types: BL21, Rosetta2, SHuffle (NEB)) to produce a transformant. Using each of the obtained three transformants, expression culture conditions were examined for each of them (2 types of medium: LB, TB, induction temperature 2 points: 30 ° C., 20 ° C.), and a part of the obtained expression product was obtained. The expression is confirmed by Western blotting using an anti-His tag antibody. Next, the culture condition in which the expression level is the highest in the soluble fraction is set as the optimum condition, and an expression product corresponding to a 100 mL culture solution is prepared. The obtained expression product is used for purification with a Ni affinity column. After confirming the purified product by Western blotting, the concentration and purity are measured. Purified fluorescent bacterium HasA is stirred for 3-4 days in nano oxygen bubble water-solvent to create a HasA-redoxin complex capable of catalyzing asymmetric oxidation reaction.

  FIG. 3 shows ion dried (IC) and ICP emission spectroscopy (ICP-ACS) of dried pea-derived HasA-redoxin complex (PP-HasA) and dried microorganism bacterium HasA (HasApf). The results for nutrients (wt%; carbon (C), hydrogen (H), nitrogen (N), sulfur (S), oxygen (O)) and metal (ppm; iron (Fe)) are shown. From this component analysis, from the point that the iron / sulfur content is almost approximate, 1) purified with high purity of PP-HasA (3 ppm / 0.05%) and HasApf (8 ppm / 0.09%), Furthermore, PP-HasA (5, 6%) has an oxygen content 7 times that of HasApf (39%), suggesting that 2) it is an asymmetric oxidation reaction involving iron electron transfer by reactive oxygen species. ing.

FIG. 4 shows the difference in molecular structure when PP-HasA and HasApf are dried and then subjected to FT-IR. That is, PP-HasA having an oxygen content of 7 times shows Fe—O and S—O vibration characteristic of cysteine thiolate coordination hem between 950 cm ^{−1 and} 1250 cm ⁻¹ . On the other hand, no vibration in this range is observed in HasApf expressed by microorganisms because of Hys.Tyr-6 coordinated heme. However, after mixing and stirring (3 to 4 days) with HasOpf in a nano-oxygen bubble water solvent, dissolved oxygen cleaves the heme in HasA and forms a complex with non-heme redoxin (iron-sulfur cluster). The characteristic cysteine thiolate coordination heme peak occurs.

  FIG. 5 shows PC suspension, centrifuged PC supernatant (Supernatant), centrifuged PC precipitate (Precipitate), carboxy-cellulose coated PC precipitate (CM-Cellulose coating PC), and glutaraldehyde. It is the figure which put together the result of the asymmetric oxidation activity which crosslinked PC precipitate (CLPC) showed. From this result, while a suitable activity was observed, the reaction was not preferred with a centrifugal PC precipitate. As shown in FIG. 10, by re-dissolving the PC precipitate in 50 mM glycine NaOH (pH 9.0 to pH 11.0) buffer, the ME precipitate is also subjected to PC suspension, centrifuged PC supernatant, And the activity more than the same as cellulose coating PC can be provided. This is the significance of the PC → ME process.

  FIG. 6 shows PC supernatant after centrifugation (A), ME supernatant after centrifugation (B), B (C) after centrifugal filtration (Vivaspin 2-10 K), and Rac-2 oxidation / hexane. It is an SDS-Page result in each sample for C (D) after extraction. The results showed that the ME supernatant (B) can be easily formed into a 39 kDa PP-HasA-redoxin complex (PP-HasA) by centrifugal filtration (Vivaspin 2-10 K). Furthermore, after asymmetric oxidation and after extraction with hexane, sample C changes to 21 kDa Pseudomonas aeruginosa HasA (N-terminal sequence result), so the HasA-redoxin complex (39 kDa) is a substrate Rac-2 oxidation / hexane. Extraction showed that the reaction stopped after separating into HasA and redoxin.

  FIG. 7 shows that “HasApf (20 μM)” in which a fluorescent bacterium HasA was expressed in Escherichia coli was mixed with a substrate racemic-1- (2-naphthyl) ethanol (Rac-2; 0.8 mM) in a nano-oxygen bubble water solvent (5 mL). After approximately 2 days (48 hours have passed), asymmetric oxidation has begun to occur, and after 3 to 4 days (72 to 96 hours), only R-2 is asymmetrically oxidized to the ketone. The time course for which unreacted S-2 is obtained with a yield of 50% (optical purity> 99% ee) is shown. This result supports the explanation relating to the formation of the cysteine thiolate-coordinated heme peak in FIG. The heme absorption wavelength 410 nm of the spectrophotometer gradually decreased from the first to the second day (48 hours elapsed), and then disappeared completely.

FIG. 8 shows the dissolved oxygen (DO) concentration in the nano-oxygen bubbled water solvent when air bubbling was started after 24 hours for the process “fluorescent fungus HasApf (21 kDa) → HasA-redoxin complex (39 kDa) in FIG. From this result, the fluorescent bacterium HasApf (21 kDa) in the nano-oxygen bubble water solvent takes in oxygen, and settles down to a DO concentration of 0.5 mg / L or less in the first few hours. When the oxygen supply is started, a rise is observed once around the DO concentration of 7.0 mg / L, but as shown in Fig. 8, the DO concentration closes at around 0.5 mg / L. Oxidative cleavage "and subsequent formation of a" HasA-redoxin complex ", and further, a resynthesis system of reactive oxygen species by this complex (Fe ²⁺ + This suggests a Fenton reaction of O ₂ → Fe ³⁺ —O—O ⁻ → Fe ⁴⁺ ═O (oxidizing Rac−1 / −2) → Fe ²⁺ + H ₂ O).

  FIG. 9A shows an ESR spectrum of a pea-derived HasA-redoxin complex (PP-HasA). This result exhibits a 4.3 Gauss non-heme characteristic characteristic of iron sulfur clusters (redoxins). Furthermore, as a non-polar hem type, the S—O and Fe—O vibrations of FIG. 4 are supported. In other words, it suggests a resynthesis system for reactive oxygen species.

  FIG. 9B shows “HasApf” in which the fluorescent bacterium HasA is expressed in E. coli. The 6-coordinate ESR spectrum in which amino acids His-32 and Tyr-72 in the HasA protein are coordinated to heme (6 Gauss). Accordingly, the “oxidative cleavage of heme” by oxygen absorption in FIG. 8, the subsequent formation of “HasA-redoxin complex”, and the resynthesis system of reactive oxygen species by this complex are supported.

  FIG. 10 examines the reason why a fluorescent bacterium-derived HasA-redoxin complex is obtained from peas. In other words, microorganisms that contaminate the raw pea protein are 1) aerobic spore bacteria that form heat-resistant spores and are widely distributed in nature, and 2) are well separated from human and animal skin. Catalase-positive Gram-positive cocci and other materials (sodium alginate powder, calcium chloride powder, distilled water did not contain bacteria. For the above reasons, the fluorescent bacteria HasA eluted from peas is , A fluorescent bacterium Pseudomonas, a symbiotic bacterium that is used as a spraying agent in the agricultural field to suppress the growth of other microorganisms and promote the growth of the host by producing compounds such as antibacterial substances. Therefore, since the fluorescent bacterium HasA is not a pea gene, it does not hit even when BLAST analysis is performed.

  FIG. 11 shows the result of 33 amino acids obtained by N-terminal sequencing of sample D (band 2) of FIG. 6, and the gene sequence and amino acids of “fluorescent fungus HasA” hit with 93% homology from the BLAST analysis. An array is shown. FIG. 15 shows a change in the molecular structure in which HasA takes heme from hemoglobin and delivers the heme to the membrane protein HasR. From the N-terminal sequence result, Cys is detected in the N-terminal 3rd amino acid and 21st amino acid, and the 21st amino acid of rubredoxin (6 kDa), for example, ferredoxin (9 kDa) is Cys. It can be considered as a result detected as a redoxin complex. FIG. 16 shows an image in which the HasA-redoxin complex is catalyzed by an asymmetric oxidation reaction based on a resynthesis system of reactive oxygen species.

  FIG. 12 is a table summarizing specific activities of PC dried products, CMME, and AGME.

  In the present invention, “unit” means a unit of enzyme activity, and one unit represents the amount of enzyme (unit: μmol / h) capable of changing 1 μmol of substrate per hour. To do. “Specific activity” indicates the activity per unit mass of the enzyme. The specific activity per gram of enzyme was expressed in unit Unit / g. From this result, the specific activity calculated from the instantaneous speed of ME was approximately 15 to 36 Unit / g (/ hour). Similarly, the specific activity calculated from the average rate at a reaction time of 12 hours was 14 Unit / g (/ hour).

From the results of FIG. 12, at Unit (mg / min), the specific activity of CMME is 0.6 ± 0.02 mU, and the specific activity of AGME is 0.8 ± 0.02 mU. The substrate concentration was 0.06%. Furthermore, when the solvent was changed to nano oxygen bubble water, the specific activity increased to 0.7 ± 0.02 mU. This reaction is, HasA- redoxin complex by active oxygen species resynthesis system _{^{(Fe 2+ + O 2 → Fe}} 3+ -O-O - → Fe 4+ = O (oxidizing Rac-1 / -2) → Fe ^This is a data result indicating that there is a Fenton reaction based on ( ₂ + H ₂ O).

FIG. 14 is a schematic diagram showing the proper use of both enantiomers by AGME and CMME of the substrate Rac-1 / -2 (1.2 mM to 3.6 mM) in an aqueous solvent (5 mL). In this reaction system, DMSO is used as a co-solvent, and the influence on ME activity is summarized in FIG. As a result, it was confirmed that the co-solvent of less than 3% is not affected by this reaction. CMME has catalase activity. Catalase has a property of reacting with hydrogen peroxide (H ₂ O ₂ ) to release oxygen. When 15 mg of PEG-PC of the present invention having a particle size of 50 μm is put into a test tube having a diameter of 18 mmφ and 1.0 mL of 5% hydrogen peroxide water is added, bubbles of about 0.2 cm to 3.5 cm are generated due to generation of oxygen. appear. In addition, the height of this bubble is proportional to the iron concentration in PC as shown in Table 11, and in comparison with CLPC, a proportional correlation with the asymmetric oxidation reaction time can be confirmed.

(Method for producing optically active alcohol)
In a preferred embodiment of the present invention, the method for producing an optically active alcohol is characterized in that AGME and CMME are allowed to act on a racemic alcohol as a substrate to selectively obtain one enantiomer of the racemic alcohol. .

  In addition, a method for producing an optically active alcohol characterized in that one enantiomer of racemic alcohol is selectively asymmetrically oxidized to a ketone to selectively obtain the other enantiomer not involved in the reaction. Provided by the present invention.

(Estimated mechanism)
According to the knowledge of the present inventor, it is presumed that, in a preferred embodiment of the present invention, a HasA-redoxin complex derived from a suitable plant or produced from microbial expression is provided for the following reason. .

  The plant-derived crude protein of an inexpensive biological material is included in the calcium alginate gel, so that an environment that can be oxidized in the air in the presence of calcium can be obtained. (I) Fenton in the HasA-redoxin complex The iron sulfur cluster having a reaction causes a property change to the water solubility of cysteine thiolate by oxygen absorption, and (ii) the cationically charged PC changes the aqueous solution to the alkali side, and (iii) shakes in warm water. Thus, PC that induces an asymmetric oxidation reaction is preferably eluted and purified.

  The presence of dissolved Ca salt and oxygen eluted in warm water changes the property of redoxin into cysteine thiolate by absorption of oxygen in the HasA-redoxin complex, that is, iron-sulfur oxide (Fe-O and / or S-). It promotes the change to water solubility (PP → PC) accompanying the generation of O), and helps to facilitate extraction in warm water. The points of improvement in catalytic activity, yield improvement and yield improvement of PC are “exudation” with the point that the gel is suitably oxidized in air, and the appropriate dissolved oxygen amount and Ca ion concentration when it is dissolved in warm water. It is to make it easier.

  In the production method of the present invention, it is possible to suitably obtain PC by taking advantage of the characteristics of these “bleeding effect” accompanying air oxidation of plant proteins and “elution effect of PC having asymmetric oxidation reaction”. It is not the purpose of fixing with calcium alginate gel, but it is possible to reduce the cost of PC that acts like an enzyme by “reversal idea” such as the purpose of suitably extracting PC with asymmetric oxidation reaction with water and extracting it with water. And it is characterized by environmentally friendly and effective extraction.

(Related literature on presumed mechanisms)
Regarding the support of the “presumed mechanism” of the present invention described above, there is a heme (iron porphyrin complex) enzyme group related to drug metabolism, detoxification, hormone biosynthesis, and the like. For example, the following documents can be cited. Non-Patent Document 1 introduces cytochrome P450 that decomposes long-chain carboxylic acids into carbon dioxide and water by epoxidation and hydroxylation as iron-binding proteins. As an example of an organic synthesis reaction using cytochrome P450 as a biocatalyst, a report on a hydroxylation reaction has been made here.

  As the epoxidation reaction by P450, for example, there is an epoxidation reaction of styrene by P450BSβ derived from Bacillus subtilis, and further an epoxidation reaction of alkenes using P450bm-3 of Non-Patent Document 2.

  Furthermore, soluble epoxide hydrolase (sEH) uses epoxyeicosatrienoic acid (EET), which is an endogenous physiologically active substance, as a substrate. EET is a fatty acid in which arachidonic acid cut from membrane phospholipid is epoxidized by cytochrome P450 (P450). That is, P450 is an EET synthase. For example, it is known to catalyze the following demethylation reaction (see Non-Patent Document 3).

  On the other hand, the heme (iron porphyrin complex) enzyme P450 is well known as a substrate oxidation of an iron electron transfer system by reactive oxygen species, but there is no report regarding an asymmetric dehydrogenase of a secondary alcohol. This patent is based on the finding of a reaction that catalyzes an asymmetric oxidation reaction using a heme protein-redoxin complex other than P450.

As a protein to take the "hem" from hemoglobin (see Non-Patent Document 4) cholerae derived HutZ, Pseudomonas aeruginosa and Mycobacterium tuberculosis derived Hemofoa (HasA; H eme A cquisition S ystem A) ( see Non-Patent Document 5) It has been known. The proteins involved in metabolic regulation of iron and iron prosthetic groups, iron-sulfur cluster (4Fe-4S, 2Fe-2S type) of iron metabolism regulatory proteins (IRP: I ron R egulatory P rotein) ( see Non-Patent Document 7 ), iron responsive element (IRE: see I ron R esponsive E lement) involvement is known (non-Patent Document 6).

As proteins involved in uptake of heme and iron, it encourages the progress of oxidative modification of proteins to eliminate the transcriptional repression, repressive transcription regulator of heme biosynthesis: even mechanism (IRR I ron R esponsive R egulator ) Report (See Non-Patent Document 8).

  In addition, in a structure such as an iron-sulfur cluster (4Fe-4S, 2Fe-2S type), the amino acid cysteine forms a disulfide bond associated with air oxidation and develops a Fenton reaction (see Non-Patent Document 9). In this case, the meaning to be included in the alginate Ca gel is the formation of iron-sulfur oxide (Fe—O and S—O) accompanying the air oxidation of the amino acid cysteine contained in the plant protein, and the “bleeding effect” thereby, Is based on the “reversal idea” that makes use of the “elution of PC having an asymmetric oxidation reaction”, and is characterized by the low-cost, environmentally friendly and effective extraction of PC having an asymmetric oxidation reaction. There is.

(Various studies)
Hereinafter, various studies on the pea-derived fluorescent bacterium HasApf complex (ME, purified HasApf) or the microbial expression-fluorescent bacterium HasApf (HasApf expressed) in one preferred embodiment of the present invention will be described.

(Various studies: Pea extraction of the present invention-fluorescent fungus HasApf (PP-HasA), microbial expression-constituent element analysis of fluorescent fungus HasApf (HasApf))
Using pure PP-HasA and HasApf prepared from FIGS. 1 and 2, using ion chromatography (IC), atomic emission spectrophotometry (ICP-AES), fluorescent X-ray / energy dispersive X-ray spectroscopy, etc. FIG. 3 information based on the constituents was obtained. As a result, the oxygen content of PP-HasA was 7 times equivalent to HasApf, and the structure of cysteine thiolate-iron was supported. In addition, both contain iron and sulfur, supporting the existence of complexes with iron sulfur clusters.

(Various studies; FT-IR analysis of PP-HasA and HasApf of the present invention)
FIG. 4 (red: PP-HasA) and (blue: HasApf) show the differences by FT-IR for pea extraction-fluorescent fungus HasApf and microbial expression-fluorescent fungus HasApf. To 950cm ^-1 ~1250cm ^-1, iron - sulfur oxides absorption of Fe-O / S-O vibration showing a is shown remarkably, in the PP-HasA, cysteine thiolate - there is iron; that, HasApf- redoxin (Or iron-sulfur cluster) Absorption that supports the complex.

As shown in FIG. 7 (HasApf) and FIG. 15 (PP-HasA), the asymmetric oxidation activity of HasApf proceeds from the third day (after 50 hours), and then takes 24 hours as in PP-HasA. Then, S-1- (2-naphthyl) ethanol is resolved at a 50% yield optical purity> 99% ee. The R form is converted to a ketone with a yield of 50%. That is, 0 hours to 50 hours in FIG. 7 are stages of 1) heme cleavage by oxygen in dissolved oxygen and 2) formation of HasApf-redoxin. The spectrophotometer absorption 410 nm is 1) the point where it disappears after 50 hours, and 2) the remarkable absorption as shown in FIG. 4 (red: PP-HasA) at 950 cm ^{−1 to} 1250 cm ⁻¹ FT-IR after 50 hours. It is confirmed by the point that was seen.

(Various studies; Identification of proteins that catalyze asymmetric oxidation reactions)
The ME supernatant fraction (sample B) in FIG. 6 can be prepared pure PP-HasA (39 kDa) simply by subsequent centrifugal filtration (Vivaspin 2-10K) treatment and concentration / drying of the filtrate after filtration. Furthermore, the asymmetric oxidation reaction of P-39 with Rac-2 (0.8 mM) / distilled water (5.0 mL), and the result of SDS-PAGE of the sample C solution remaining after hexane extraction is the result of the 21 kDa peak of sample D. give.

  FIG. 11 shows 33 amino acids obtained by sequencing the deep sample D-band 2 that is characteristic of SDS-PAGE by SDS-PAGE followed by N-terminal sequencing using a protein sequencer. From the result of the BLAST analysis, the enzyme involved in the activity was identified as HasA derived from fluorescent bacteria (93% error range: HasAp gene product [Pseudomonas fluorescens Pf-5]). The molecular weight was 20.853 Da, which was consistent with the SDS-PAGE result. From the above, based on the fluorescent bacterium HasA (21 kDa), a protein complex (39 kDa) containing an iron-sulfur cluster (redoxin) as an active center was estimated as a protein that catalyzes an asymmetric oxidation reaction.

(Various studies; Fe-O inspection by ESR)
FIG. 9 shows ESR results of (a) PP-HasA and (b) HasApf. (B) Microbial expression of HasApf—The fluorescent bacterium HasApf is a low-spin state six-coordinated heme protein HasA in which histidine (His) and tyrosine (Tyr) are coordinated as shown in Non-Patent Document 5. It was. On the other hand, (a) PP-HasA of pea extraction-fluorescent fungus HasApf contains 1) 7-fold equivalent of oxygen content, 2) non-heme type with no absorption at 410 nm spectrophotometer, and redoxin (iron-sulfur cluster) specific Absorption characteristic of 4.3 Gauss was obtained. From these results, an oxidation reaction by reactive oxygen species (Fe (II) + O ₂ → Fe (III) -OO ⁻ → Fe (IV) = O (similar to oxygen-driven P450 of Non-Patent Documents 1 to 3) It was supported that it was activated by oxidizing rac-1 / -2) → Fe (II) + H ₂ O).

  As shown in FIG. 10, the fluorescent bacterium HasA does not exist as a contaminant in the material, but exists as a symbiotic bacterium in pea cells. Therefore, the pea-fluorescent bacterium can produce HasA at a stage when it is included in the calcium chloride gel, and is extracted with warm water after air oxidation. However, fluorescent bacteria produce not only HasA but also iron-sulfur clusters (IRP, IRE, IRR, etc.) that promote / control expression. Therefore, iron and sulfur are detected in the microbial expression of the fluorescent bacterium HasA as shown in FIG.

  As shown in FIG. 1, the fluorescent bacterium HasA is complexed with redoxin after air oxidation, eluted as a membrane protein containing the HasApf-redoxin complex, and can be easily concentrated and precipitated by centrifugation. The precipitate is coexisted with a polymer compound in an aqueous solution and processed into FD by treatment with FD, so that it can be processed into a powder AG / CM-ME having improved storage stability, and becomes a catalyst capable of asymmetrically oxidizing a secondary alcohol.

  AG / CM-ME is an oxygen and iron-dependent electron transfer system, does not require the addition of a cofactor NAD (P), and does not require selection of a buffer solution or pH conditions. An optically active alcohol can be easily obtained by stirring in an aqueous solvent and in the presence of oxygen (without a cap).

  AG / CM-ME can synthesize naproxen precursor (S-1,> 99% ee) from Rac-1. According to FIG. 17, the drug “naproxen” can be easily synthesized. The organic synthesis process from S-1 can be 1) Bromination of hydroxyl group (—OH) (—Br) → 2) Nitrilation (—CN) → 3) Organic synthesis of carboxylic acid (—COOH).

  FIG. 17 (b) shows a flow chart according to the conventional method of naproxen synthesis. Naproxen is an organic compound classified as an aromatic carboxylic acid and is a type of non-steroidal anti-inflammatory drug (NSAID) used as an analgesic, antipyretic and anti-inflammatory drug.

  The ME of the present invention can be presumed to be an organelle, particularly a mitochondrial membrane protein, and is considered to be a membrane protein having an iron electron transfer chain function based on the fluorescent bacterium HasA and an iron sulfur cluster (redoxin) involved in its expression. It is done.

  FIG. 1 shows the flow of step 1. From the air oxidized alginate gel-encapsulated pea protein, the 2.6% calcium-coated PC of FIG. 12 was eluted, 0.5 mM glycine NaOH (pH = 9-11) and polyethylene glycol (eg PEG 4000), and After the ME is treated by treatment with 0.5 mM organic solvent (glutaraldehyde, acetonitrile, etc.), the asymmetric oxidation reaction of various secondary alcohol molecules occurs due to the action of the ME iron electron transfer system. .

  FIG. 2 relates to a method for producing HasApf obtained by expressing a HasA gene derived from a fluorescent bacterium in a microorganism such as Escherichia coli. Even when expressed by a HasA gene derived from Pseudomonas aeruginosa or Mycobacterium tuberculosis, it is the same as HasA derived from a fluorescent bacterium. It is possible to realize the asymmetric oxidation reaction shown in FIG.

As shown in FIG. 8, the alginate Ca gel-encapsulated plant-derived protein suitably induces air oxidation in the presence of oxygen after air oxidation. The Fe—O / S—O vibration peak appearing in the region of 950 cm ⁻¹ to 1250 cm ⁻¹ obtained by FT-IR measurement indicates iron-sulfur oxide, and the redoxin in the HasApf-redoxin complex Derived from oxide of iron-sulfur cluster, it functions as an active center. Furthermore, the iron sulfur cluster oxide produces an effect of being eluted in warm water.

  The composition of the HasApf-redoxin complex that catalyzes the asymmetric oxidation reaction shown in the present invention is (1) an iron-sulfur cluster (redoxin) involved in electron transfer and (2) HasA as its foundation. It is characterized by a secondary alcohol dehydrogenation reaction in the presence of oxygen. Therefore, the present invention provides a reaction system that is clearly different from the conventional secondary alcohol dehydrogenase.

  Furthermore, there are two types of MEs that have an S-selective oxidation reaction and that work on R-selective oxidation. As shown in FIG. 14, the ME shown in the present invention produces two types of enantiomers having different stereoselectivities by properly using the PEG molecular weight to be coated in the first step (AG / CM-ME). is doing. Therefore, by combining the two types of ME, it is possible to synthesize both enantiomers (S body and R body).

  From the above, this discovery is compared with the conventional secondary alcohol dehydrogenase of zinc and cofactor (NAD (P) or FAD) electron transfer system, including the conventional complicated and troublesome enzyme purification and isolation steps. The asymmetric oxidation reaction is 1) the point that no cofactor (NAD (P) or FAD) is required, 2) the oxygen-dependent asymmetric oxidation reaction, 3) not pH-dependent, water or nano 4) Furthermore, it is advantageous in terms of cost and operation because it can be used properly for both enantiomers, and it is highly selective because it is highly selective. Provided is an asymmetric organic synthesis reagent capable of obtaining an active alcohol (yield 50%, 99% ee or more).

(Immobilization of HasA-redoxin complex)
The HasA-redoxin complex of the present invention (heme iron-activated HasA) can be immobilized on various carriers as required. The complex thus immobilized can be easily separated from the reaction solution and the enzyme after the immobilized complex is subjected to the reaction, the complex can be easily reused, and the continuous reaction. It has advantages such as easy use in a tank (fluid tank, column, etc.).

  The “carrier” to be used for the immobilization is not particularly limited as to whether it is porous as long as the HasA-redoxin complex of the present invention can be immobilized on the carrier. The porous material may be any of organic, inorganic, and organic-inorganic hybrid, for example. For example, an inorganic carrier is preferable from the viewpoints of easy pores optimal for immobilizing proteins and the like, high activity expression of the immobilized enzyme, ensuring long-term reaction stability, no pressure deformation, and high chemical resistance. On the other hand, the organic carrier is easy to be flexible (for example, in the form of a film). The organic-inorganic hybrid carrier can have the advantages of inorganic-organic as appropriate.

  The “immobilization” mechanism is not particularly limited, but from the viewpoint of mutual compatibility at the molecular level with the HasA complex of the present invention, the “immobilization” mechanism is preferably “adsorption”.

  The “carrier” to be used for the immobilization is preferably a carrier having a large surface area (for example, a particulate and / or porous carrier). Whether or not the carrier can be suitably used in the present invention can be suitably determined by screening using “adsorbability of fluorescent bacteria HasA” described later.

(Porous carrier)
As the porous carrier, for example, those having the following physical properties can be suitably used.
Specific surface area: ^{2 to} 38 m ² / g is preferable, 10 to 30 m ² / g is more preferable, and 15 to 25 m ² / g is particularly preferable (specific surface area = 20 m ² / g for Toyonite 200 series).
Average pore diameter: 30 to 130 nm is preferable, 50 to 110 nm is more preferable, and 70 to 100 nm is particularly preferable (for Toyonite 200 series, average pore diameter = 80 nm)
Average particle size: 105 to 210 μm is preferable, 125 to 190 μm is more preferable, and 145 to 170 μm is particularly preferable (for toyonite 200 series, average particle size = 155 μm)
Apparent bulk density: 0.18 to 1.18 g / mL is preferable, 0.38 to 0.98 g / mL is more preferable, and 0.58 to 0.78 g / mL is particularly preferable (Toyonite 200 series has an apparent bulk density. Density = 0.68 g / mL)

With respect to the method for immobilizing the HasA-redoxin complex on the porous carrier and the method for measuring each physical property of the porous carrier, for example, the following documents can be referred to.
・ Patent No. 2862509 (for protein immobilization method using toyonite)
Reference can be made to M. Kamori et al. ““ Kamori 509 (for protein immobilization using toyonite) ”. The organic-inorganic hybrid carrier can have the advantages of inorganic-organic as appropriate. 4) Furthermore, the use of both enantiomers is e-fixed lipase (2000), published by Journal of Molecular Catalysis B: Enzymatic, pages 9, 269-274. Toyonite pamphlet (English version, Japanese version), published by Toyo Denka Kogyo Co., Ltd.

(Suitable carrier)
In the embodiment in which the HasA-redoxin complex of the present invention is immobilized, after the immobilized HasA-redoxin complex is subjected to the first reaction, the complex is recovered, and the recovered complex is further recovered. It is easy to use for the second reaction. In this case, the recovered complex (that is, the HasA-redoxin complex to be subjected to two or more reactions) is the “percentage of 50 h-generated ketone” (%) described in Example 12 (and FIGS. 28 to 29) described later. ) To determine its usefulness.

More specifically, when the “percentage of 50 h-generated ketone” was measured for the recovered complex by the method described in Example 12 (and FIGS. 28 to 29), the value of “percentage of 50 h-generated ketone” was measured. However, it is preferable that it is 30% or more. This value is preferably 35% or more, more preferably 40% or more, and particularly preferably 45% or more.
(Carrier screening using the adsorptivity of HasA)
Basically, it is preferable to use the conditions of “Example 12” described later. That is, carrier screening is preferably performed by the following method. As will be apparent from the following description, this screening method can be carried out conveniently using a centrifuge tube (so-called “Spitz tube”; length: 18 mm, capacity: about 15 mL). For example, when “stirring with a shaker” is used, about 100 types of carrier samples can be screened at a time by stirring about 100 centrifuge tubes at a time.

(1) Escherichia coli expression-fluorescent bacterium HasA solution: An E. coli expression-fluorescent bacterium HasA solution (concentration: 1.8 mg / mL) is prepared by the method of Example 2 (method of microbial expression of HasA gene).
(2) Immobilization: 5 mL of the HasA solution obtained in the above operation (1) is placed in a centrifuge tube, and then the “carrier sample” (1000 mg) to be screened is dropped into the centrifuge tube and shaken for 20 to 40 hours. Using a machine (for example, Toyonite 200, 200A, 200P, 200M, manufactured by Toyo Denka Kogyo Co., Ltd.), under the conditions of “Shape of figure 8”, 500 to 700 times per minute (rpm). Stir for 24-48 hours. By this operation, the fluorescent bacterium HasA is immobilized on the carrier sample.
(3) Post-treatment of immobilized HasA:
The immobilized HasA obtained by the above operation (2) is dried in a low temperature wind or desiccator (for example, about 24 to 48 hours) and stored under dark and low temperature conditions suitable for HasA.

As described in Example 12, it was found that the adsorptivity of HasApf to the “toyonite” immobilization support proceeded favorably in the order of toyonite 200>T200P>T200A> T200M. The brown HasApf solution changed as follows (see FIG. 23).
-T200 ... HasA solution became transparent.
-T200A ... HasA solution became light brown.
-T200P ... HasA solution became light brown (almost transparent).
-T200M ... HasA solution was light brown (slightly dark eyes).

  That is, in the same manner as in “Example 12”, the possibility of using the carrier sample can be easily determined by “color”. In the above example, “toyonite 200, toyonite 200P and toyonite 200A” can be determined as “available” in this screening.

(Evaluation of carrier by ketone conversion rate)
In the evaluation of the above-mentioned “support sample”, according to the method and conditions shown in Example 12 (FIG. 24), adsorption of various “supports” — “activity intensity due to HasA solution residue” after 48 hours (conversion rate to produced ketone) ) Can be measured. An example of this measurement is shown by the leftmost bar graph in each bar graph group (e.g., the group for Toyonite-200) in FIG. For example, the above-mentioned “activity intensity due to HasA solution residue” (conversion rate to produced ketone) for Toyonite-200 can be read as about 13%. This conversion rate to the produced ketone can be measured by the following method.
(Leftmost bar graph)-Spitz tube (18 mm x 15 mL) was allowed to react with each HasA residue (0.2 mL / ion-exchanged water (4 mL / substrate rac-1 (0.4 mg) scale) for 50 hours with stirring. After that, the ketone conversion rate (%) produced is measured.

  The smaller the “conversion rate to the produced ketone” is, the more “more HasA” is immobilized on the carrier. In the present invention, the “conversion rate to the product ketone” is preferably 45% or less. Further, the “conversion rate to the product ketone” is preferably 40% or less, 35% or less, more preferably 30% or less, further 25% or less, further 22% or less, and further 20% or less. Further, it is preferably 20% or less, more preferably 18% or less, and particularly preferably 16% or less.

(Specific examples of carriers)
In immobilizing the HasA-redoxin complex of the present invention, for example, the following commercially available carriers (for example, carriers that can be used for immobilizing organic substances or proteins) can be suitably used.
・ Toyonite (Toyonite; Toyo Denka Kogyo Co., Ltd .; porous ceramic spherical carrier whose surface is modified with organic functional groups by granulating and firing the kaolinite material after acidic hydrothermal treatment)
Toyonite-200 (a carrier having a hydroxyl group as an organic functional group), 200A (a carrier having an amino group as an organic functional group), 200P (a carrier having a phenylamino group as an organic functional group), 200M (a carrier having a methacrylo group)

  Next, the present invention will be specifically described based on examples, but this is for the purpose of explanation and should not be construed as being limited thereto.

  In each Example, the HasApf-redoxin complex having an asymmetric oxidation reaction is described in Example 1 (extracting method of plant-derived fluorescent fungus HasA) and Example 2 (Method of expressing microorganisms of HasA gene). Obtained.

Example 1 (Method for extracting plant-derived fluorescent fungus HasA)
To 300 g of soybean protein protein (manufactured by Organo Food Tech Co., Ltd., trade name “pea protein PP-CS”) was added 3000 mL of distilled water, and 3000 mL of a 3% sodium alginate aqueous solution was added and stirred until uniform. While stirring the obtained crude protein-sodium alginate mixed solution, 4% calcium chloride aqueous solution was dropped using a separatory funnel to prepare gel beads.

  Next, the solution portion was discarded, and the gel beads were collected and allowed to stand in the air for 1 to 5 hours for air oxidation. 4000 mL of distilled water was added to the air-oxidized gel beads, and the mixture was shaken at 40 ° C. for 48 hours using a constant temperature shake incubator to elute the PC in the gel beads to obtain an aqueous PC solution.

  The PC aqueous solution was centrifuged at 10,000 rpm for 10 minutes using a centrifuge (manufactured by Hitachi Koki Co., Ltd., himac CR20G) to obtain PC precipitates (80 g to 120 g). To 100 g of this PC precipitate, 3-7% (3-7 g) PEG (1000) /0.5% guisin NaOH buffer (pH = 9-11, 700 mL) was added and stirred well. Weight% (3 to 7 g) acetonitrile or glutaraldehyde was allowed to coexist with the polymer compound coating and homogenized by stirring for 1 to 3 hours.

  After the homogenized ME aqueous solution was centrifuged, the precipitate was dried using a vacuum freeze dryer (manufactured by Nissei Co., Ltd., RLEII203) and pulverized with a ball mill. As the FD condition, after freezing at −50 ° C., the system was evacuated, the temperature was raised to 50 ° C., and water was sufficiently sublimated. In the purification process, the HasApf-redoxin complex of the present invention was suitably purified by centrifugal filtration (Vivaspin 2-10 K) of the ME supernatant after centrifugation and then FD drying the filtrate. The drying step is preferably performed in a state close to vacuum. This is because air oxidation due to heat-discoloration and accompanying activity decrease occur.

Example 2 (Method for expressing microorganisms of HasA gene)
The Pseudomonas protegens Pf-5 gene sequence (HasA; accession No. YP — 262445.1, or WP — 011063600; FIG. 15) is synthesized. If necessary, synthesize by optimizing the sequence for E. coli. Synthesis was performed by adding restriction enzyme sites at both ends of the target gene for transfer to an expression vector. It is also possible to request a contract manufacturer to deliver a synthetic gene inserted into the pUC-57 vector.

  The fluorescent bacterium HasA gene cleaved from pUC-57 after being treated with restriction enzyme (NEB) at 37 ° C. for 3 hours or more was isolated from the agarose gel and purified (DNA purification kit: BEX DNA generation kit I (PCR & GEL Purification)). It was introduced into the vector pET28a (Novagen; FIG. 15), pET-47b (Novagen), or pGEX (GE Healthcare). Ligation reagents (DNA Ligation Kit <Mighty Mix> (TaKaRa)), Escherichia coli (Competent high DH5α (TOYOBO)), antibiotics (Ancipillin or Kanamycin), plasmid extraction kit (QIAprep Spin MiniQ) )).

  The ligation between the vector and the fluorescent bacteria HasA gene was performed using a sequence reagent (ABI Prism 3130xl Genetic Analyzer (Applied Biosystems)) using a sequencing reagent (BigDye Terminators v1.1 using a Cycense Sequencing Kit (Applied)).

  The constructed expression vector is transformed into E. coli BL (DE3; Novagen) or BL21, Rosetta2, SHuffle (NEB) and cloned. The clone is picked up and cultured overnight at 37 ° C. in 2 mL of TB medium (with antibiotics). 1 mL of the preculture solution is added to 10 mL of TB medium (with antibiotics) and cultured at 37 ° C. As a sample before induction of expression, a part of the cells is modified. When OD = near 1.0, IPTG was added to a final concentration of 0.1 mM, followed by culturing at 30 ° C. for 16 h.

  Thereafter, the expressed E. coli is collected by centrifugation and lysed by 1 mL of E. coli lysis buffer (BugBuster Protein Extraction Reagent (Novagen)) or ultrasonic disruption. Centrifuge the E. coli lysate and collect the supernatant of the soluble fraction. The precipitate of the insoluble fraction is solubilized with 1 mL of denaturing buffer (8M Urea, PBS). After solubilization, centrifugation is performed and the supernatant is recovered as an insoluble fraction.

  The sample before expression induction, the soluble fraction after expression induction, and the insoluble fraction after expression induction were mixed in an equal amount with 2 × sample buffer, respectively, and heat-treated at 99 ° C. for 5 minutes to obtain a migration sample. SDS-PAGE was performed for about 70 minutes at 20 mA with a 5-20% gradient gel. As a result of the electrophoresis, it was confirmed that the fluorescent bacterium HasA was suitably expressed.

  The cells were inoculated from a glycerol stock of E. coli and cultured overnight at 37 ° C. in 10 mL of TB medium (with antibiotics). 10 mL of the preculture was added to 100 mL of TB medium (with antibiotics) and cultured at 37 ° C. When OD = near 1.0, IPTG was added to a final concentration of 0.1 mM, followed by culturing at 30 ° C. for 16 h.

  The expressed E. coli was collected by centrifugation and lysed by washing with 10 mL of E. coli elution buffer (BugBuster Protein Extraction Reagent (Novagen)) or ultrasonically. Centrifuge the E. coli lysate and collect the soluble supernatant. Add 1 mL of resin (TALON Metal Affinity Resin (Takara Bio)) to the Ni affinity column and equilibrate. The supernatant is added to the equilibrated resin to bind the His-Tag fusion protein. Add 10 mL wash buffer to the column to wash the resin. 3 mL of elution buffer was added to the column to elute the His-Tag fusion protein.

  At that time, an equilibration / washing buffer (50 mM sodium phosphate, 300 mM NaCl, pH 8) and an elution buffer (150 mM meaning, 50 mM sodium phosphate, 300 mM NaCl, pH 8) were used.

  Obtained as fluorescent fungus HasA (concentration: 630 μM) in 3 mL of elution buffer. A stirring bar is placed in an 18 mmφ test tube, nano oxygen bubble water (5 mL) with adjusted HasA concentration (20 μM) is added, and stirring is performed at 700 rpm without plugging for 3 days or more. Heme decays (410 nm absorption disappears), and a HasA-redoxin complex having an asymmetric oxidation reaction is formed (g = 6 → g = 4.3). A sample obtained by naturally drying fluorescent bacteria HasA (concentration: 630 μM) can be used for measurements such as FTIR and EST.

  In addition, the fluorescent bacterium HasA production process from the above-mentioned expression of E. coli also corresponds to Pseudomonas aeruginosa-derived HasA and Mycobacterium tuberculosis-derived HasA. In other words, E. coli derived from Pseudomonas aeruginosa and Mycobacterium tuberculosis can be obtained by stirring at 700 rpm in a nano-oxygen bubbled water (5 mL) solvent for 3 days or longer at 700 rpm with a HasA concentration (20 μM). Expresses a homogeneous oxidation reaction.

  Microbial expression-Constituent components of fluorescent fungus HasA (HasApf) are in the distribution shown in FIG. 3 (constituent components), Fe—O / S—O vibration in FIG. 4 (FTIR), and nonheme absorption in FIG. 9 (ESR). Reference can be made from physicochemical molecular structure measurements such as (active oxygen species: Fe—O, g = 4.3).

Example 3 (FT-IR analysis of (a) PP-HasA and (b) HasApf of the present invention)
FIG. 4 shows the results of investigation using FT-IR (ST Japan, Inc., portable ATR apparatus (A2 Technologes, ML version), ATR method). (A) PP-HasA is a dried product of the filtrate obtained by centrifugal filtration (Vivaspin 2-10 K) of the ME supernatant obtained in the first step. In addition, (b) HasApf is a protein obtained by expressing Escherichia coli a fluorescent bacterium HasA gene by the above-described method.

Example 3 (ESR analysis of (a) PP-HasA and (b) HasApf of the present invention)
FIG. 9 shows a measuring apparatus of an electron spin resonance (ESR) apparatus (JESFA200 type manufactured by JEOL), measurement conditions: Magnetic field (322.5 ± 250 mT), Modulation Field (0.6 mT), Time Constant (0.3 sec). ), Microwave Power (1 mW), Sweep Time (8 min), Temperature (−162 ° C.). (A) PP-HasA was dried under the conditions of evaporating moisture at room temperature, drying the obtained solid content in a vacuum, separating it into an ESR sample tube, and vacuum degassing to prepare a sample. (B) For drying of HasApf, after evaporating water at room temperature, the obtained solid content was collected in an ESR sample tube and vacuum degassed to prepare a sample.

As a result of ESR, (a) PP-HasA confirmed the absorption of Fe ³⁺ at g = 4.3, and (b) HasApf confirmed the absorption at g = 6.0 in the heme iron state. As for the fluorescent bacteria HasA gene expression HasA pf, a liquid of 630 μM (APPROX) /1.0 mL, His-Tag MW: 2306) was air-dried.

Example 4 (SDS-PAGE of active fraction)
About 10 μL of each of the samples A to D below, molecular weight was determined using a precast gel (SDS-PAGE mini) manufactured by Tefco Corporation. CBB staining solution was used for staining. Centrifugation was performed at 10,000 rpm for 10 minutes using a Himac CR20G manufactured by Hitachi Koki Co., Ltd.
A) PC aqueous solution eluted from the PP gel of Example 1.
B) Centrifugal supernatant fraction of ME solution obtained by re-dissolving the centrifugal precipitate in 0.5 mM glycine NaOH buffer (pH = 9-11).
C) A filtrate fraction obtained by subjecting the sample B to centrifugal filtration (Vivaspin 2-10 K).
D) Residue of sample C after asymmetric oxidation with substrate Rac-2 and hexane extraction.

  The results are shown in FIG. Band 1 (39 kDa) changed to band 2 (21 kDa) after the asymmetric oxidation reaction and / or after hexane extraction. That is, the asymmetric oxidation activity is in the HasA-redoxin complex of band 1 (39 kDa), and after the asymmetric oxidation reaction and / or hexane extraction, the activity is changed to HasA of band 2 (21 kDa) that has lost its activity. Yes.

Example 5 (Protein identification of band 2)
SDS band 2 obtained in Example 4 was cut off, and amino acid sequence analysis was performed with a protein sequencer (manufactured by Shimadzu Corporation, PPSQ-21A). The results are shown in Table 1.

  Furthermore, the amino acid sequences in Table 1 were analyzed (BLAST) using an identification database for proteins. For this BLAST, enter the amino acid sequence you want to search from the “protein blast” in the BLAST analysis page (http://blast.ncbi.nlm.nih.gov/) of the National Center for Biotechnology Information (NCBI) You can get it. Of the BLAST search results, those hit with an error range of 93% or more are shown in Table 2.

  From the above results, it was derived that the band 2 was a fluorescent bacterium HasA.

Example 6 (second step, biochemical properties of PC and ME)
In FIG. 1, the first step “air oxidation treatment” “elution treatment” “centrifugal sediment recovery” (PC elution / PC sediment recovery), second step “PC sediment coating treatment” “centrifugal sediment recovery” ( In the ME precipitation recovery stage, 1) activity intensity monitoring of various fractions (FIG. 5), 2) monitoring of changes in dissolved oxygen concentration (DO) (FIG. 8), and further, the third step “AG / CM− This is a study of the effect of ME ″ on the activity of the 3) DMSO addition concentration (FIG. 13).

  FIG. 5 shows PC suspension, centrifuged PC supernatant (Supernatant), centrifuged PC precipitate (Precipitate), carboxy-cellulose coated PC precipitate (CM-Cellulose coating PC), and glutaraldehyde crosslinked. It is the figure which put together the result of the asymmetric oxidation activity which the PC precipitate (CLPC) showed. From this result, while a suitable activity was observed, the reaction was not preferred with a centrifugal PC precipitate. As shown in FIG. 10, by re-dissolving the PC precipitate in 50 mM glycine NaOH (pH 9.0 to pH 11.0) buffer, the ME precipitate is activated like a cellulose-coated PC. Even if the detailed change cannot be traced, the biochemical significance of the PC → ME treatment has been confirmed.

  As shown in FIG. 8, the dissolved oxygen content (DO) of the PC supernatant fraction, PC precipitate, ME supernatant fraction, and ME precipitate in the jar fermenter was 0.2 mg / hour in several hours. L and drop to near zero. After 24 hours, even if oxygen supply is started, DO is stabilized at around 0.6 mg / L. From the above, the HasA-redoxin complex in PC and ME always works to lower the dissolved oxygen concentration, but does not fall below 0.6 mg / L.

  For the purpose of exploring the biochemical properties of additional HasA-redoxin conjugates, FIG. 13 shows the co-solvent DMDO (IPA, IPA, Rac-2 (1.2 mM × 3) in nanooxygen bubble water solvent (5 mL). (Ethanol) concentration (2%, 4%, 6%, 8%) is a graph showing the outline of the influence on AG / CM-ME (50 mg) asymmetric oxidation activity. “1.2 mM × 3” means a concentration of 30 μL of a 20,000 ppm substrate solution after 0 hour (1.2 mM), after 15 hours (2.4 mM), and after 30 hours (3.6 mM). It shows the influence of DMDO (IPA, ethanol) concentration when added up to.

  From FIG. 13, the AG / CM-ME oxidation activity is not affected by the co-solvent DMDO from 0% to 3%, but the asymmetric oxidation activity is reduced to less than half when the concentration is 6% or more. From these results, when the concentration of the organic solvent (DMSO, IPA, ethanol, diethyl ether, hexane, etc.) is 3% or more, the HasA-redoxin complex is decomposed into HasA and redoxin and loses its activity. . Therefore, when continuously reusing AG / CM-ME, it is necessary to pay attention to the organic solvent concentration. Even when any solvent selected from the group consisting of 3% or less of DMSO, IPA, and ethanol is used, the protein complex does not impair the catalytic activity.

Example 8 (Fluorescent bacteria HasA in pea protein)
FIG. 10 examines the reason why a fluorescent bacterium-derived HasA-redoxin complex is obtained from peas. In other words, the microorganisms that contaminate the raw pea protein were not in other materials (sodium alginate powder, calcium chloride powder, distilled water. The above reason is described in detail as follows.

  As shown in FIG. 10, microbial contamination in the raw pea protein (PP) is as follows: 1) aerobic spore bacteria that are thermospore-forming spores that are widely distributed in nature, and 2) humans and animals. Two types of catalase-positive gram-positive cocci that are well separated from the skin were confirmed. Alginate gel-encapsulated pea protein (PP gel), PC eluate, and ME solution all showed ∞, while the number of cells in each precipitate fraction after FD drying showed ND.

  The fluorescent bacterium HasA eluted from peas is used as a spraying agent in the agricultural field because it suppresses the growth of other microorganisms and produces compounds such as antibacterial substances to promote host growth. The fluorescent bacterium Pseudomonas fluorescens Pf-5 is symbiotic as a symbiotic bacterium, is assimilated into the host, is retained in cells and tissues in plants, and is inherited by progeny. Therefore, the reason why HasA does not BLAST in the pea gene is that the detected fluorescent bacterium HasA is derived from a pea symbiotic fluorescent bacterium. Therefore, the fluorescent bacterium confirmed from SDS-Page (FIG. 6) and the N-terminal sequence (FIG. 11) has validity, and further, the fluorescent bacterium HasA is isolated from the host pea in the presence of oxygen. By forming a redoxin complex, it is shown that it newly contributes to the secondary metabolic system of substance conversion like cytochrome P450.

Example 7 (Study of components of membrane protein (ME) of SDS-page)
In addition, LCMS-IT-TOF (SHIMAZU: mode; nanoESI +: MS range: MS1 (m / z 400-1500) MS2 (m / z) for other SDS bands of Example 4 (FIG. 6: bands 3 to 4) 50-1500) data-dependent scan; flow rate: 300 nL / min; flow solvent: A = 0.1% formic acid / 2% acetonitrile, B = 0.1% formic acid / 80% acetonitrile; gradient: 5- 40% B / 0-30 min, 40-100% B / 30-40 min, 100% B / 40-60 min) and the results of protein identification by MASCOT analysis are shown in Table 4 (band 5 only), Table 2 For protein identification of other bands of PMF method (BIFLEX III (Bruker Daltonics: mode; positive / reflector: matrix; α-cyano-4-hydroxy cinnamic acid (CHCA); target plate; peptide calibration standards; angiotensin II ( [M + H] + = 1046.542), angiotensin I ([M + H] + = 1296.685), substance ([M + H] + = 1347.736), bom besin ([M + H] + = 1619.823), ACTH1-17 ([M + H] + = 2093.087), ACTH18-39 ([M + H] + = 2465.199)) and MASCOT analysis Judged.

  The ABC-Transporter of band 4 in Table 2 is a membrane protein that serves as a retreat for HasA, and can be easily centrifuged with the membrane (fatty acid and glycerin). For the above reasons, it is considered that the precipitate was recovered as a HasA-redoxin complex formed by air oxidation and a membrane tissue containing HasA. Water-soluble HasA separated from the membrane protein is present in the supernatant fraction, and can be formed into a pure HasA-redoxin complex by combining the centrifugal filtration step (molecular weight 10 kDa; Vivaspin 2-10 K) of FIG. there were.

  Furthermore, the ligand-binding receptor of band 3 in Table 2 is a membrane protein called HasR, which plays a function of receiving heme captured by HasA and taking it into the fungus body. Therefore, it can be precipitated and recovered as a membrane tissue containing HasA and further as an oxidized HasA-redoxin complex. Here, redoxin is a protein having an iron-sulfur cluster structure (ferredoxin, rubredoxin, etc.). For example, the aforementioned proteins (IRP, IRE, IRR) involved in HasA expression are also redoxins of HasA-redoxin complex. As included.

Example 8 (catalase activity of HasA-redoxin complex)
From the results of the inorganic element analysis shown in FIGS. 3, 4, and 9, the Fenton reaction system (Fe (II) + O ₂ → Fe (III) -OO ⁻ → Fe (IV) = O ( Since oxidizing rac-1 / -2) → Fe (II) + H ₂ O) was shown, the characteristics of the catalase activity of the cationic protein were examined. The results are summarized in Table 3.

In the catalase test, 15 mg of each sample was placed in an 18 mmφ test tube, and a 5% -H ₂ O ₂ aqueous solution (1 mL) was added to compare the foaming height. The asymmetric oxidation activity shown in Table 3 shows that the ME precipitate has about 2.5 times the intensity of catalase activity with the PC precipitate, and about 2.5 times that of the microorganism-expressed HasApf-dried product. The intensity of the colorase activity was shown. The measurement of the catalase activity was evaluated by visual recognition of “foaming” based on Example 8.

Example 8 (Measurement of Catalase Activity; Measurement by Visualization of “Bubbling”)
To the ME precipitate (15 mg), PC precipitate (15 mg) and microbial expression HasApf (15 mg) obtained in Example 7, 5% -H ₂ O ₂ (1 mL) was added in an 18 mmφ test tube, respectively. However, the ME precipitate solution foams significantly more intensely than the PC precipitate, and the volume of 1.0 mL of the solution is expanded five times by the foam volume, and the PC precipitate expands about four times, and after about 5 minutes, , Settled in the original bulk. On the other hand, in the microorganism-expressed HasApf, oxygen was generated about twice in the solution surface layer portion, and bubbling stopped after 5 minutes.
In the following examples, to calculate the H ₂ O ₂ remaining volume and H ₂ O ₂ consumption after the reaction, was examined the intensity of catalase activity.

Example 9 (Measurement of catalase activity; measurement with a hydrogen peroxide concentration meter)
PC colorase activity was measured using a hydrogen peroxide concentration meter PAL-39S (digital display) manufactured by Atago Co., Ltd., which can calculate the concentration from the strength of the refractive index of hydrogen peroxide contained in water. In addition, regarding the detail of the hydrogen peroxide concentration meter used here, the following URL can be referred as needed.
http://www.atago.net/japanese/products_pal_top.html

  Catalase activity was measured in each sample: 1) ME precipitate (15 mg), 2) PC precipitate (15 mg), or 3) microbial expression HasApf (15 mg) and 5% hydrogen peroxide ( 1 mL) was added and stirred (700 rpm) at 37 ° C. for 5 minutes. The reaction solution was centrifuged (3600 rpm, 10 min), and 0.3 mL of the supernatant was measured on the prism surface of a hydrogen peroxide concentration meter PAL-39S. At the same time, under the same conditions, 5% hydrogen peroxide solution (1 mL) and pure water (1 mL) are exchanged, and the measurement is carried out. For ME precipitate (15 mg) / water (1 mL), “Blank 1”, PC precipitate “Blank 2” was used for (15 mg) / water (1 mL), and “Blank 2” was used for the microorganism-expressed HasApf (15 mg) / water (1 mL).

From the above, the _H 2 _{O 2} consumption in the reaction system when using PEG-PC, and the _H 2 _{O 2} consumption in the reaction system when using CLPC, can be calculated as follows. Table 4 shows the results.
H ₂ O ₂ remaining amount after reaction (ME precipitate) = (actual value: 8.0%) − (blank 1 value: 2.2%) = 5.8%
Residual amount of H ₂ O ₂ after reaction (PC precipitate) = (actual value: 8.2%) − (blank 2 value: 2.4%) = 6.0%
H ₂ O ₂ remaining amount after reaction (microbe expression HasApf) = (actual value: 8.4%) − (blank 3 value: 2.0%) = 6.4%
H ₂ O ₂ consumption (ME precipitate) = (residual amount of H ₂ O ₂ in BLANK system: 6.8%) − (residual amount of H ₂ O ₂ after ME precipitate reaction: 5.8%) = 1 .0% (14.7% consumption as a relative value)
H ₂ O ₂ consumption (PC precipitate) = (H ₂ O ₂ remaining amount in BLANK system: 6.8%) − (H ₂ O ₂ remaining amount after CLPC-PC reaction: 6.0%) = 0 .8% (11.8% consumption as a relative value)
H ₂ O ₂ consumption (microbe expression HasApf) = (residual amount of H ₂ O ₂ in BLANK system: 6.8%) − (residual amount of H ₂ O ₂ after CLPC-PC reaction: 6.4%) = 0 .4% (5.9% consumption by relative value)

  From the results of the hydrogen peroxide concentration meter PAL-39S shown in Table 4, the identity of the “bubble” generated in the experiment is oxygen, and the involvement of the enzyme of catalase activity that converts hydrogen peroxide into water and oxygen has been confirmed. It was. Therefore, the ME precipitate (15 mg) was consumed with about 10,000 ppm of catalase activity with respect to 5% hydrogen peroxide water, the PC precipitate (15 mg) with about 8000 ppm consumed catalase activity, and the microorganism-expressed HasApf (15 mg). ) Showed about 4000 ppm consumption of catalase activity.

Therefore, by calculating the amount of ME precipitate or PC precipitate or microbial expression HasApf that can change 1 μmol of substrate (H ₂ O ₂ : MW34) per minute, the activity (unit) of each catalyst is calculated as follows: It can be calculated as follows.
_6.8% -H ₂ O ₂ aq. _{= 68mg / mL-H 2 O} 2 = 2mM / mL-H 2 O 2

Of these, the consumption of each catalyst is as follows per 5 minutes with reference to the consumption% in the above "Table 4" .
* ME precipitate (15 mg) = 2 mM / mL × 0.147 = 294 μM / mL-H ₂ O _2, therefore unit = 15000 ÷ 294≈50 μg / μM .
* PC precipitate (15 mg) = 2 mM / mL × 0.118 = 236 μM / mL-H ₂ O _2, therefore unit = 15000 ÷ 236≈63.5 μg / μM .
* Microbe-expressed HasApf (15 mg) = 2 mM / mL × 0.059 = 118 μM / mL-H ₂ O ₂
Therefore, unit = 15000 ÷ 118≈127 μg / μM .

From the above results, the catalase activity of the ME precipitate (50 μg) capable of changing 1 μmol of the substrate (H ₂ O ₂ : MW34) per 5 minutes is the same as that of the PC precipitate (63.5 μg). It was found to be 3 times, 2.3 times the microbial expression HasApf (118 μg). This result. The results were almost equivalent to the observation results of the ME precipitate, the PC precipitate, and the microorganism-expressed HasApf bubbles shown in Table 3.

  As a result of each of the above examples, the catalase activity and / or the amount of redoxins (ferredoxin, rubredoxin, IRP, IRE, 2.3 times that of the microorganism-expressed HasApf and 1.3 times that of the PC precipitate compared to the ME precipitate) It has been suggested that these redoxins express asymmetric oxidation activity by forming a complex with the fluorescent bacterium HasA. The microorganism-expressed HasApf shows heme absorption in the spectrophotometer 410 nm, while forming a HasA-redoxin complex having an asymmetric oxidation reaction by coexisting and stirring in nano oxygen bubble water for about 50 hours; After 50 hours, the spectrophotometer has no peak at 410 nm and forms a nonheme-redoxin oxide (see FIGS. 4 and 9).

Example 10 (Effectiveness of continuous reuse of CMME)
As shown in FIG. 14, CMME (20 mg) and AGME (20 mg) were dissolved in distilled water (4 mL) / DMSO (<1.0% (v / v)) in the substrate Rac-2 (20,000 ppm, 30 μL). It was made to react by addition. After completion of the reaction, the supernatant was collected by centrifugation, distilled water (4 mL) was added to the precipitate, and the substrate Rac-2 (20,000 ppm, 30 μL) was added and reacted.

  The operation of continuous reuse is as follows: CMME (S-body synthesis), first time-16 hours, Second-28 hours, Third time-38 hours, AGME (R-body synthesis), First time-13 After 20 hours, after 2-20 hours, after 3-25 hours, hexane (4.0 mL) was added to each supernatant, and after homogenization, 10 μL of hexane was inserted into an optical resolution column for quantitative determination by HPLC measurement. Optical purity was performed. The results are shown in Table 5.

  From Table 5 and FIG. 14, it was found that continuous reuse up to 3 times was possible without compromising optical purity and yield. CMME can synthesize S-2 (> 99% ee / ˜50%) by stereoselective R-form oxidation, whereas AGME can synthesize R-2 (> 99% ee by stereoselective S-form oxidation. / -50%) could be synthesized. However, since the S-form oxidation of AGME continues to produce R-form oxidation to become all ketones, it is necessary to strictly observe the time for stopping the reaction.

As a result, both enantiomers can be combined and synthesized only by the difference in the PEG molecular weights to be coated such as CMME and AGME (CMME is PEG4000 coating, AGME is PEG1000 coating).
In the present invention, there is disclosed a method for treating a complex that exhibits not only a HasA-redoxin complex as an asymmetric oxidation catalyst but also a stereoselectivity in accordance with the purpose using a coating agent.

Example 11 (Enzyme-immobilized carrier-fluorescent fungus HasA production and its structural function)
The continuous reuse in the CMME and AGME reaction system has the disadvantage that the activity decreases drastically when the concentration of the organic solvent in the aqueous solution exceeds 3%. 1) Resistance to organic solvents, and 2) Fixing the effectiveness of continuous reuse. There was a need to find a chemical enzyme. Therefore, E. coli expression-fluorescent HasA solution (1.8 mg / mL) x 5-fold amount obtained from Example 2 (HasA gene microbial expression method) was added to an enzyme-immobilized carrier-Toyoden (1000 mg) manufactured by Toyo Denka Kogyo. By adsorbing and immobilizing, 1) resistance to organic solvents, 2) effectiveness of continuous reuse, and 3) easy separation after organic synthesis reaction could be achieved. The detailed procedure using toyonite is as follows.

  The amount of Escherichia coli expression-fluorescent fungus HasA prepared in Example 2 (HasA gene microorganism expression method) was approximately 36 mg (concentration of 1.8 mg / ml, liquid volume 20 ml) at the first time (600 mL flask culture scale), The second time (4000 mL flask culture scale) was about 200 mg (concentration of 1.8 mg / ml, liquid volume 110 ml). FIG. 18 shows SDS-PAGE after purification of the fluorescent bacterium HasA (HasApf).

  FIG. 19 shows the result of subjecting the substrate rac-1 (approximately 0.36 mg / 4 mL-ion-exchanged water) to an asymmetric oxidation reaction with the fluorescent bacterium HasApf (approximately 0.4 mg / 4 mL-ion-exchanged water) expressed in E. coli. Is shown. As a specific reaction operation method, 1 mL of fluorescent bacteria HasApf (1.8 mg / mL) expressed in E. coli was added to 20 mL of ion-exchanged water, and dispensed into 5 Spitz tubes each 4 mL. A 0.36 mg / 4 mL solution was adjusted as the concentration, and a stir bar was inserted in advance in a 40 ° C. environment.

  FIG. 19 shows that a substrate rac-1 / IPA (2-propanol) solution (40,000 ppm: 10 μL) was added to each Spitz tube to obtain a substrate concentration (approximately 0.4 mg) / 4 mL solution at 40 ° C. / This is the result of starting the reaction and optionally tracking the reaction. The difference from FIG. 7 is the fluorescent fungus HasApf concentration, and FIG. 19 is approximately twice the concentration in FIG. As a result, the reaction time is shortened by about 20 hours.

  FIG. 20 shows the result of measuring the UV spectrum after 0h, 24h, 48h and 72h after the start of the asymmetric oxidation reaction of FIG. The purpose is to know the state of the fluorescent bacterium HasApf / heme. As the reaction time of the produced ketone spectrum (350 nm) gradually increases, the absorption intensity indicated by the heme (porphyrin iron complex) spectrum (410 nm). From the point that there is almost no difference, it was confirmed that the change of HasApf / heme to the nonheme type accompanying the asymmetric oxidation reaction does not occur during the 72 h reaction period.

Therefore, the structural change of HasApf / heme during this asymmetric transformation was further confirmed by ESR spectrum. FIG. 21 shows the results of monitoring the ESR spectrum after 0 h, 50 h, and 100 h after the start of the asymmetric oxidation reaction of FIG. From the fact that ESR signals after 0 h (g = 2.8, 2.22, 1.72) can be confirmed, the fluorescent bacterium HasApf hem has a low 6-coordinated position of proximate histidine (proximal site) and terminal tyrosine (distal site). It was in a spin state, and it was confirmed that there was no open end structure in which adjacent cysteines and oxygen molecules could freely coordinate like cytochrome P450. The ESR signal (g = 2.8, 2.22, 1.72) disappeared from the state after 50 h, and the signal g = 2.8, 2.22, 1. of the oxidized heme in the low spin state. From the point of disappearance of 72, it was confirmed that a ligand giving a strong ligand field such as OH ⁻ was bonded to the top and bottom of the heme to cause distortion, and an oxidation reaction accompanying the generation of active oxygen species occurred.

Further, FIG. 22 shows ESR signals g = 4.3 and 2.0 100 hours after the start of the asymmetric oxidation reaction, leading to confirmation of oxoferryl species (Fe ^IV = O). , Hemofoa HasApf / heme, like the cytochrome -P450, the oxidation reaction mechanism involved in reactive oxygen species generation _{^{(Fe II + O 2 → Fe}} III -O-O - → Fe IV = O (oxidizing rac-1) → Fe II + H ₂ O) could be clarified. FIG. 9 also shows an EPR signal, and FIG. 20, FIG. 21, and FIG. 22 also estimate the state of distortion by binding to the top and bottom of the hemophor HasApf associated with the asymmetric oxidation reaction. Thus, it has been clarified that an oxidative catalytic function can occur even in HasA that is not in a penta-coordinated low-spin state high-spin state terminal open structure in which oxygen molecules can freely coordinate.

Example 12 (Enzyme-immobilized support-adsorbability of fluorescent bacteria HasA to toyonite)
Therefore, the effectiveness of continuous reuse using HasApf in which HasApf is further adsorbed and immobilized on immobilized carrier toyonite will be described in detail. First, since toyonite has a track record of adsorbing and immobilizing lipase asymmetric hydrolase, an outline of the toyonite immobilization carrier will be described. FIG. 25, FIG. 26, and FIG. 27 are quoted from the Toyonite pore distribution, SEM photographs, and types of toyonite from the company brochure.

  Toyonite is a porous ceramic spherical support whose kaolinite material is granulated and fired after acidic hydrothermal treatment, and whose surface is modified with an organic functional group (FIGS. 25 and 26). It is an enzyme-immobilized carrier that is excellent in stability, suitable for industrial scale production of pharmaceuticals and agrochemical intermediates, chemicals, etc., and is marketed by Toyo Denka Kogyo.

  Summary of Toyonite features: (1) Easy enzyme immobilization: Enzyme protein can be added by stirring into the crude enzyme solution due to the effects of fine pores (pore distribution and SEM photo) and organic functional groups on the support surface. Can be easily fixed. (2) High activity expression: By selecting the surface modification type according to each enzyme, the binding force with the enzyme can be increased, a large amount of enzyme can be immobilized, and high activity is expressed. Therefore, the bioreactor and related equipment can be made compact. (3) Ensuring long-term reaction stabilization: Since the organic functional group whose surface has been modified binds tightly to the enzyme, it can be used repeatedly for a long time with very little decrease in activity. (4) No pressure deformation: A spherical ceramic carrier excellent in strength does not undergo pressure deformation even when packed in a column. Therefore, there is no concern about consolidation on an industrial scale. (5) Strong chemical resistance: Since it is a kaolinite ceramic carrier, it is excellent in chemical resistance such as acid, alkali and organic solvent.

  Physical properties: Average particle size -155 ± 15μm, apparent bulk density -0.68 ± 0.15g / mL, chemical resistance-acid, alkali pH 2-10 can be used, heat resistance-120 ℃ heat sterilization is possible.

  Type of Toyonite: Toyonite has four types of organic functional groups, 200, 200-A, 200-P, and 200-M (FIG. 27), and can be selected according to the type of enzyme. Immobilization performance can be improved by modifying the surface with various functional groups depending on the nature of the enzyme. Type 1: Toyonite 200-carrier having a hydroxyl group as an organic functional group, Type 2: Toyonite 200A-carrier having an amino group as an organic functional group, Type 3: Toyonite 200P-carrier having a phenylamino group as an organic functional group, Type 4: Toyonite 200M- There are four types of carriers having methacrylo groups as organic functional groups.

  First, an example in which the adsorption property of HasApf to a toyonite-immobilized support (as a conclusion, 200> T200P> T200A> T200M) was examined as shown in FIG. Adsorption immobilization procedure, reaction operation method, etc.) are described in detail.

E. coli expression-fluorescent Bacteria HasA solution (1.8 mg / mL) 5-fold amount was adsorbed and immobilized on enzyme-immobilized support-Toyonite (1,000 mg) manufactured by Toyo Denka Kogyo.
(1) HasA solution: E. coli expression-fluorescent bacteria HasA solution (concentration: 1.8 mg / mL) is prepared according to the method of Example 2 (Method of microbial expression of HasA gene).
(2) Immobilization: Toyonite (1000 mg) is added to 5 mL of the HasA solution obtained by the above operation (1) and stirred for 20 to 40 hours. Stirring uses a stirring blade or a shaker. If a magnetic stirrer is used, the carrier particles may be destroyed and the stirring bar may be worn. After immobilization, an immobilized enzyme is obtained by filtration.
(3) Post-treatment of immobilized HasA: The immobilized HasA obtained by the above operation (2) was dried in a low temperature wind or a desiccator and stored under conditions suitable for each HasA.

As shown in FIG. 23, the adsorptivity of HasApf to the toyonite-immobilized support proceeded favorably in the order of toyonite 200>T200P>T200A> T200M. The brown HasApf solution changed as follows.
-T200 ... HasA solution became transparent.
-T200A ... HasA solution became light brown.
-T200P ... HasA solution became light brown.
-T200M ... HasA solution became light brown (slightly dark eyes).

Next, adsorption of various toyonites—after 48 hours, 1) activity intensity due to HasA solution residue, 2) activity intensity due to various toyonites, and 3) optical purity results are shown in FIG. FIG. 19 shows the original asymmetric oxidation reaction of the brown HasA solution. FIG. 24 shows the toyonite adsorption rate of HasApf. The specific explanation of the blue / red / yellow graph is as follows.
1. (Blue) -Spitz tube (18 mm × 15 mL) was charged with each HasA residue (0.2 mL / ion-exchanged water (4 mL / substrate rac-1 (0.4 mg) scale) for 50 hours with a stir bar reaction. It is a result.
2. (Red)-% of ketone produced by stirring with a Spitz tube (18 mm × 15 mL) on a HasA-immobilized support (10 mg portion / ion exchange water (4 mL) / substrate rac-1 (0.4 mg) scale for 50 hours) It is a result.
3. The optical purity of S-alcohol in (green) -HasA immobilization support (for 10 mg: red scale) is shown.

  From the above results, it was shown that HasApf is suitably adsorbed and immobilized on a toyonite-immobilized carrier and can be used as an immobilizing agent in the same manner as when toyonite is a lipase. Regarding the HasApf adsorptivity of toyonite, the order of T200> T200P> T200A> T200M was observed from the change in the color of the HasApf solution turbidity. This difference in adsorptivity is due to the effect of the four types of organic functional groups of toyonite. Further, the substrate concentration was changed from 0.8 mM to 1.2 mM and 2.0 mM, and the reaction solution was continuously extracted after extraction with hexane (organic solvent). By repeating the reuse up to 4 times, it was decided to confirm the effects of 1) resistance to organic solvents and 2) continuous reuse.

Example 13 (Enzyme Immobilization Carrier-Effectiveness of Continuous Reuse Using Toyonite Test)
28 and 29 show the substrate rac-1 (0.8 mM, 1.2 mM, 2.0 mM; equivalent to 0.24 to 0.48 mg) / toyonite in a test tube (18 mm × 15 mL). Stirring (700 rpm) with immobilized HasApf carrier (30 mg) / ion-exchanged water (4 mL) in an environment of 40 ° C., followed by extraction with hexane (4 mL) after a reaction time of 50 h or 72 h, the ratio of S and R isomers The results of confirming the optical purity (% ee) and the conversion rate (%) to the resulting ketone are shown.

  The purpose of this study was to solve the CMME / AGME system that was deactivated at an organic solvent concentration of 3% or more in an aqueous solution with a HasApf-immobilized toyonite carrier. That is, 1) resistance to organic solvent, 2) immobilized enzyme effective for continuous reuse, and 3) a test aimed at enabling easy separation of post-reaction treatment.

  As shown in FIG. 28 and FIG. 29, about the immobilized HasApf after hexane extraction, the substrate rac-1 (0.8 mM, 1.2 mM, 2.0 mM; equivalent to 0.24 to 0.48 mg) ) Was added, and it was found that inactivation of enzymes and the like was not confirmed, and that continuous reuse was possible four or more times. At the same time, the results when the substrate concentration is changed from 0.8 mM to 1.2 mM or 2.0 mM are summarized as follows.

As a result of evaluating four types of Toyonite 200, Toyonite 200A, Toyonite 200P, and Toyonite 200M as HasApf immobilization carriers,
1. It was shown that by adsorbing HasApf on a toyonite-immobilized support, the asymmetric oxidation activity can be reproduced at least four times or more after hexane extraction, and it is resistant to organic solvents.
2. From the results referring to the substrate concentration of 1.2 mM / 72 h, the order of the activity intensity of each immobilized carrier is
“T200> T200P = T200A> T200M” and HasA adsorptivity “T2
00>T200P>T200A> T200M ”in this order.
It could be determined that the sA activity was dependent on the adsorption rate on toyonite.
3. It was found that Toyonite 200 had the highest HasA adsorption rate.

In the substrate concentration system (1.2 mM, 72 h), although it is slightly inferior to Toyonite 200, 200A, 200P, and 200M can be obtained with optical purity (> 95% ee), so that a suitable racemic alcohol resolving agent is obtained. It turned out that it becomes. However, because Toyonite 200M has a low amount of HasA adsorption, the activity decreases as the substrate concentration increases. Therefore, as an in-depth consideration, the following can be added.
4. It was found that the amount of HasA adsorbed is a factor indicating the activity strength, and the effect of the type of organic functional group of the toyonite immobilization support depends on the adsorptivity.
5. The substrate concentration of 1.2 mM is the optimal concentration as with PP-HasA, while the immobilized Has
Apf has the advantages of 1) resistance to organic solvents, 2) effectiveness of continuous reuse, and 3) easy separation after the reaction.
6. The amount of HasApf used in the asymmetric oxidation reaction is as shown in FIG. 19 (0.36 mg).
The amount of immobilized HasApf in FIGS. 28 and 29 was (0.09 mg: 10 mg), and it was confirmed that the same activity could be covered with an enzyme amount approximately four times as large.
7. Furthermore, while the substrate concentration for FIG. 19 (0.36 mg) is 0.8 mM / 4 mL-water system, the immobilized HasApf amount (0.09 mg: 10 mg) in FIGS. 28 and 29 is 1
. It was confirmed that it was a 2 mM / 4 mL-water system and could contribute to the separation of the substrate racemic alcohol about 1.5 times.

  From the above results, as shown in FIG. 30, HasApf has a reaction mechanism in which 1) dissolved oxygen is converted into reactive oxygen species by heme and 2) only one of the substrate racemic secondary alcohols is stereoselectively oxidized. 3) It can be suitably immobilized on a kaolinite substance-derived immobilization carrier (toyonite). Further, as shown in FIG. 31, the heme metabolism of HasApf / heme, which is the active center, has been studied as a system in which heme oxygenase (HO) or (MhuD: Mycobacterium tuberculosis enzyme) is catalyzed in an oxygen-dependent manner. On the other hand, a functional system that catalyzes an asymmetric oxidation reaction is the first case discovered this time.

  As promising data to support these, there is the knowledge that g = 2.8, 2.22, 1.72 disappears in the ESR spectrum of FIG. 21 and FIG. 22, and cysteine is present in heme like conventional cytochrome P450. The data of the present invention refers to a new concept that only the coordinated pentacoordinate type can contribute to toxic degradation as well as the active heme protein capable of coordinating oxygen.

Example 14 (Cyclic-Deracemization Reaction Using PP-HasA and NaBH4)
PP-HasApf (0.1 g, 0.5 g, 1.0 g, 3.0 g) was used as a biocatalyst in an aqueous solvent system (400 mL), and the reaction environment was made to act on the substrate rac-2 (100 mg) at 40 ° C. It was. Next, sodium borohydride (NaBH4: 50 mg) was arbitrarily added to the reaction system, and 1) the deracemization was studied, and at the same time, 2) the optimum reaction scale of the substrate / catalyst / solvent was confirmed.

  First, the results of examining the optimum amount of PP-HasApf complex (0.1 g, 0.5 g, 1.0 g, 3.0 g) are shown below.

  From the results, it was found that 100 mg of the substrate could be divided and synthesized with 0.5 g or more of PP-HasApf. Therefore, it was derived that complete optical resolution can be achieved if the PP-HasApf amount is 500 mg or more with respect to the substrate (100 mg). In addition, when it was examined as ion-exchanged water (400 mL → 300 mL → 250 mL → 200 mL) at a PP-HasApf amount of 500 mg / substrate 100 mg, it was confirmed that if it was 250 mL or more, it could be completely optically resolved. However, since the reaction takes about 50 hours in a 40 ° C. environment, the data was unified from 400 mL to data considering that the amount of 400 mL of water solvent decreases to nearly 300 mL.

Next, as shown in FIG. 32, cyclic deracemization reaction is caused by periodically administering sodium borohydride (NaBH ₄ : 50 mg). That is, as shown in FIG. 33, HasApf asymmetrically oxidizes only R-1 to ketone, and the resulting ketone is uniformly reduced to racemic S and R forms by NaBH ₄ , and the cycle is continued by continuing the cycle. In particular, the chemical yield of S-alcohol is improved.

As a result of earnest and examination, it was clarified that PP-HasApf (100 mg) can be deracemized and synthesized with a yield of 90% or more and an optical purity of 98% ee or more by combining the following reaction procedure.
(Reaction procedure)
1 Substrate (45 mg) / Water (400 mL) / PP-HasA (0.5 g to 3.0
g) at 40 ° C. → 16 hours 2 Substrate (55 mg) added → 8 hours 3 NaBH4 (50 mg) added → 5 hours 4 NaBH4 (50 mg) added → 5 hours 5 NaBH4 (50 mg) added → Completed after 8 hours.

The results of the cyclic deracemization reaction are summarized as follows: when the addition of NaBH4 (50 mg) is completed once, when the substrate rac-2 (100 mg) is allowed to act, PP-HasApf (0 0.5 g), a yield of about 70% is obtained, but NaBH ₄ (50 mg) × 3 times does not have enough titer, and 91% ee leads to a yield of about 80%. On the other hand, when the PP-HasApf amount is increased to 1.0 to 2.0 g, the titer becomes sufficient, and even with NaBH4 (50 mg) × 3 times, a yield of 90% or more is achieved with 99% ee or more. Led to need.

From the above results, PP-HasApf was newly found that a cyclic deracemization reaction with NaBH ₄ is possible, and the yield of the target optically active alcohol can be improved only by this addition. The same cyclic deracemization reaction with aBH ₄ has also been confirmed for the HasApf-immobilized carrier, and there is no problem if the results of PP-HasApf are replaced with the HasApf-immobilized carrier.

A schematic description of FIGS. 18 to 33 is as follows.
Figure description:
FIG. 18: SDS-PAGE results after purification of E. coli expressed HasA gene. Substrate rac-2 (0.4 mg) / water solvent (4 mL) asymmetric oxidation reaction over time with HasApf (0.36 mg): optical purity (-●-:% ee), S-2 (-□-: 0.2 mg), R-2 (-O-: 0.2 mg), and the resulting ketone (-[Delta]-).
FIG. Results of UV measurement in asymmetric oxidation reaction of fluorescent bacteria HasApf (0.36 mg) / substrate rac-2 (0.4 mg) / water solvent (4 mL) (after 0 h, 24 h, 48 h, 72 h).

FIG. ESR measurement results after 0 hours and 50 hours after asymmetric oxidation reaction in fluorescent bacteria HasApf / substrate rac-2 (rac-2) / water solvent (4 mL).
FIG. ESR measurement result after 100 hours of asymmetric oxidation reaction in fluorescent bacteria HasApf / substrate rac-2 (rac-2) / water solvent (4 mL).
FIG. FIG. 24. Comparison of transparency after adsorption of HasA solution (dark brown) / toyonite after 2 days of shaking 1) Ketone% produced by HasA solution residue after toyonite adsorption, 2) Ketone% produced by various toyonite, 3) Optical purity results

FIG. 26. Toyonite pore distribution map SEM photograph of Toyonite FIG. 27.4 is a diagram showing the difference in organic functional groups of four types of Toyonite.
FIG. Substrate concentration of Toyonite HasApf immobilized carrier (200 and 200A) and state of continuous reuse

FIG. Substrate concentration of Toyonite HasApf immobilized carrier (200P and 200M) and state of continuous reuse FIG. Asymmetry conversion mechanism of HasApf FIG. Heme metabolism pathway

FIG. Schematic diagram of a cyclic deracemization reaction 1
FIG. Schematic diagram of the cyclic deracemization reaction 2

  As described above, according to the present invention, HasA can catalyze an asymmetric oxidation reaction capable of stereoselectively oxidizing each enantiomer of racemic secondary alcohol, and can catalyze non-selective oxidation. -A redoxin complex (heme iron active type HasA), its immobilization body, and its manufacturing method can be provided.

  The HasA-redoxin complex (heme iron activated HasA) having an asymmetric oxidation reaction of the present invention and its immobilized form are optical isomers such as optically active alcohols synthesized in the fine chemical field such as pharmaceuticals, perfumes and foods. It is a novel asymmetric oxidation catalyst that is inexpensive to manufacture, is environmentally friendly and can be produced easily.

Claims (6)

A method for producing an immobilized HasA-redoxin complex comprising a carrier for immobilization and a HasA-redoxin complex immobilized on the carrier,
Gene synthesis of HasA, which is the gene sequence of the fluorescent bacterium Pseudomonas protegens Pf-5; accession No. YP — 262445.1 or WP — 011063600,
Inserting the synthesized fluorescent bacterium HasA gene into an expression vector;
Transforming the expression vector into E. coli to produce a transformant;
Culturing the transformant to produce an expression product;
Purifying the expression product with a Ni affinity column to obtain fluorescent bacteria HasA;
Stirring the fluorescent fungus HasA in a nano oxygen bubble aqueous solvent to form a HasA-redoxin complex;
Immobilizing the HasA-redoxin complex on a carrier;
And the carrier is a porous kaolinite ceramic spherical carrier whose surface is modified with an organic functional group.   The method of claim 1, further comprising PEG coating the HasA-redoxin conjugate. The support has a specific surface area of ² to 38 m ² / g, an average pore size of 30 to 130 nm, an average particle size of 105 to 210 μm, and an apparent bulk density of 0.18 to 1.18 g / mL. The method according to claim 1 or 2.   The method according to any one of claims 1 to 3, wherein the carrier is Toyonite (registered trademark) 200, 200P, 200A, or 200M. The following steps (a) to (c):
(A) Putting 5 mL of the fluorescent bacteria HasA concentration 1.8 mg / mL into a centrifuge tube, and then dropping 1000 mg of the carrier sample to be screened into the centrifuge tube, using a shaker, Agitating at 500 to 700 times / min for 48 hours to immobilize the fluorescent bacterium HasA on the carrier sample;
(B) collecting the HasA solution residue after the immobilization; and (c) adding 0.4 mg of the substrate racemic-6-methoxy-1- (2- Naphthyl) After adding ethanol and reacting under stirring with a stirrer for 50 hours, the conversion rate to the generated ketone measured by the measuring method including the step of measuring the generated ketone conversion rate (%) is 45%. The method according to any one of claims 1 to 4, wherein:   The immobilized HasA-redoxin complex was subjected to the first reaction, and then the immobilized HasA-redoxin complex was recovered, and the percentage of 50 h-generated ketone was measured for the recovered immobilized HasA-redoxin complex. The method according to any one of claims 1 to 5, wherein the value of the percentage of the 50 h-generated ketone is 30% or more.