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WO2017038107A1 - Complexe hasa-redoxine présentant une réaction d'oxydation, et son procédé de production - Google Patents

Complexe hasa-redoxine présentant une réaction d'oxydation, et son procédé de production Download PDF

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Publication number
WO2017038107A1
WO2017038107A1 PCT/JP2016/052213 JP2016052213W WO2017038107A1 WO 2017038107 A1 WO2017038107 A1 WO 2017038107A1 JP 2016052213 W JP2016052213 W JP 2016052213W WO 2017038107 A1 WO2017038107 A1 WO 2017038107A1
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hasa
redoxin
complex
reaction
treatment
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宏行 永岡
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Sanyo Foods Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture

Definitions

  • 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.
  • 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.
  • 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 affinity with oxygen and other gases, and can easily transfer electrons in a reducing environment at 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 an active center for various redox reactions including oxygen transport, energy production, and nucleic acid synthesis. Has been.
  • Non-patent Document 4 As functions of heme proteins, oxygen transport by oxygen (binding oxygen molecules) and electron transfer by cytochrome c (redox center) 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).
  • iron metabolism regulatory protein IRP: I ron R egulatory P rotein
  • IRP iron metabolism regulatory protein
  • IRE iron responsive element
  • 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).
  • inhibitory transcriptional regulator of heme biosynthesis is by a signaling molecule iron concentration in the cell binds to heme, molecular oxygen binds to the heme
  • IRR I ron R esponsive R egulator
  • Cytochrome 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.
  • P450BS ⁇ derived from Bacillus subtilis is known to hydrogenate long chain fatty acids using hydrogen peroxide
  • P450cam of camphor hydroxylase derived from Pseudomonas putida 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.
  • P450 reaction for example, there is an epoxidation reaction of styrene with P450BS ⁇ derived from Bacillus subtilis (see Non-Patent Document 2).
  • soluble epoxide hydrolase uses epoxyeicosatrienoic acid (EET), which is an endogenous physiologically active substance, as a substrate.
  • EET is a fatty acid obtained by epoxidizing arachidonic acid cut from membrane phospholipid with cytochrome P450 (P450). That is, P450 is an EET ⁇ synthase.
  • P450 is an EET ⁇ synthase.
  • it is known to catalyze the following demethylation reaction (see Non-Patent Document 3).
  • 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.
  • 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.
  • redoxin iron-sulfur cluster
  • Fenton reaction see Non-Patent Document 9
  • 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 simple 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.
  • the expression of the gene protein can be performed using cultured cells, Escherichia coli, yeast, koji, Bacillus, or the like. 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.
  • secretory microorganisms secrete a variety of proteins
  • purification for extracting the target protein is complicated.
  • 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.
  • Fluorescent bacteria a type of rhizobia
  • HasA-redoxin complex expression having an asymmetric oxidation reaction
  • production by a microbial expression system and pea extraction method.
  • HasA 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.
  • 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 to catalyze non-selective oxidation. It is to provide a HasA-redoxin complex.
  • the present invention is based on the discovery that HasA (iron porphyrin complex) -redoxin complex can catalyze an asymmetric oxidation reaction of a secondary alcohol substrate. 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, safety and cost of a drug discovery process, and a suitable production method thereof.
  • HasA-redoxin complex capable of recovering S-alcohol by catalyzing the asymmetric oxidation of racemic secondary alcohol with R-alcohol, and a method for producing the same.
  • the HasA-redoxin complex is obtained by encapsulating pea protein in calcium alginate gel, air-oxidized and then eluting 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.
  • 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 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.
  • HasA-redoxin complex according to [7] which has a function attributable to an organelle having asymmetric oxidation activity and / or catalase activity.
  • 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 amino acid cysteine in redoxin.
  • SO or Fe-O iron-sulfur oxide
  • HasA-redoxin complex according to [13], wherein the HasA-redoxin complex supported from the sequence is derived from a complex consisting of a heme capture protein (HasA), ferredoxin, adrenodoxin, and rubredoxin.
  • HasA heme capture protein
  • ferredoxin ferredoxin
  • adrenodoxin adrenodoxin
  • rubredoxin heme capture protein
  • HasA-redoxin complex according to [15], wherein the HasA is derived from a fluorescent bacterium, Pseudomonas aeruginosa, and / or Mycobacterium tuberculosis, and derived from a microorganism-expressed HasA including E. coli.
  • HasA-redoxin complex according to [16], wherein the asymmetric oxidation reaction of the microorganism-expressed HasA requires a period of formation of the HasA-redoxin complex in the presence of dissolved oxygen (2 to 3 days).
  • HasA-redoxin complex according to any one of [7] to [17], wherein the HasA-redoxin complex is coated with a polymer compound.
  • HasA-redoxin complex according to any one of [7] to [17], wherein the HasA-redoxin complex is subjected to a crosslinking treatment.
  • a mirror image of the racemic alcohol is selectively obtained 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
  • HasA-redoxin complex according to [16], which takes a period (2 to 3 days) of the generation of the HasA-redoxin complex in the presence of dissolved oxygen for the asymmetric oxidation reaction of the microorganism-expressed HasA.
  • HasA-redoxin complex according to any one of [7] to [21], wherein the HasA-redoxin complex is coated with a polymer compound.
  • HasA-redoxin complex according to any one of [7] to [21], wherein the HasA-redoxin complex is subjected to a crosslinking treatment.
  • the reactivity with hydrogen peroxide is 1 ⁇ M-H 2 O 2 aq.
  • 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 decomposing is 200 ⁇ g or less.
  • the present invention can also include the following aspects.
  • HasA-redoxin complex comprising a carrier for immobilization and a HasA-redoxin complex immobilized on the carrier.
  • 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.
  • Examples of the features of the present invention include the following five points.
  • HasA hemophore gene
  • CBC Convention on Biological Diversity
  • B Since it is a higher plant-derived HasA-redoxin complex, it must be used effectively as an asymmetric oxidation catalyst for unused biological resources (biomass).
  • C The microorganism-expressed HasA is effectively used for the asymmetric oxidation reaction of secondary alcohol after forming a HasA-redoxin complex in the presence of oxygen.
  • 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, there is provided a HasA-redoxin complex having an asymmetric oxidation reaction of a secondary alcohol produced from a plant and / or microbial expression.
  • 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 the powdering of the plant-derived HasA-redoxin complex. It can be provided as a novel asymmetric oxidation catalyst that can realize low-cost production gently.
  • FIG. 1 is a conceptual diagram showing one embodiment of a method for purifying a plant-derived HasA-redoxin complex (hereinafter referred to as PP-HasA).
  • PP-HasA plant-derived HasA-redoxin complex
  • 1 is a conceptual diagram showing one embodiment of a method for producing a microorganism-expressed HasA-redoxin complex (hereinafter referred to as HasApf).
  • 2 is an example of a graph and a table showing the results of constituent 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.
  • PC precipitate obtained by centrifuging the HasA-redoxin complex aqueous solution (hereinafter referred to as PC) obtained in the first step and the precipitate
  • PC hasA-redoxin complex aqueous solution
  • 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.
  • FIG. 2 is an example of a graph showing the effect of DMSO co-solvent on asymmetric oxidation of substrate Rac-2 (0.8 mM) in nano oxygen bubble water-solvent (5.0 mL). It is a schematic diagram regarding the 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.
  • FIG. 2 is an example of a schematic diagram showing a secondary alcohol asymmetric oxidation reaction by a reactive oxygen species-resynthesis system of a HasA-redoxin complex. It is an example of the schematic diagram which shows the synthesis
  • Toyonite 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.
  • HasApf 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.
  • the protein complex (PC) of the present invention is a protein complex containing at least iron and has at least 1 in the region of 1085 ⁇ 50 cm ⁇ 1 in its FT-IR spectrum. 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.
  • HasA is obtained from a plant-derived protein by a method for producing HasA, which will be described later, or by genetic engineering techniques. Next, 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.
  • a predetermined treatment for example, gel inclusion ⁇ air oxidation ⁇ elution into an aqueous solution
  • 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.
  • the HasA-redoxin complex of the present invention is a HasA-redoxin complex containing at least iron and sulfur, and its FT-IR spectrum shows 950 cm ⁇ 1 to 1250 cm ⁇ 1. This region has 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 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 ⁇ m is suitable for the “bubble generation” level based on oxygen generation when 15 mg is put into a test tube having a diameter of 18 mm ⁇ and 1.0 mL of 5% hydrogen peroxide water is added. (See Table 3 below).
  • 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).
  • 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).
  • 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.
  • the amount of AG / CM-ME that can decompose 1 ⁇ M-H 2 O 2 is preferably 200 ⁇ g, more preferably 150 ⁇ g (particularly 130 ⁇ g).
  • the iron-containing HasApf-redoxin complex of the present invention is “AG / CM-ME amount”
  • its specific activity (1 ⁇ M-H 2 O 2 can be decomposed with respect to the hydrogen peroxide decomposition reaction).
  • the amount of AG / CM-ME is preferably 100 ⁇ g, more preferably 80 ⁇ g (particularly 60 ⁇ g).
  • ⁇ Asymmetric oxidation activity-Measurement method 2 20 mg of the HasA-redoxin complex of the present invention (particle size: 50 ⁇ m) in an aqueous solvent system (4 mL) at room temperature of 40 ° C., substrate racemic-6-methoxy-1- (2-naphthyl) ethanol (Rac-1)
  • the enantioselectivity giving the naproxen precursor (s-1) is 95ee% or more at a reaction time of 15 hours.
  • HasA-redoxin complex Method for producing HasA-redoxin complex
  • 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.
  • 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 then dried to obtain a pure pin HasA-redoxin complex, or the precipitate fraction is immediately freeze-dried and dried (FD).
  • 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.
  • a plant-derived water-soluble crude protein is included in a gel to obtain a hydrophobic carrier.
  • 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 plant resources may be used.
  • commercially available products include soybean protein (trade name “pea protein PP-CS”) sold by Organo Food Tech Co., Ltd.
  • plant resources examples include cereals such as buckwheat, amaranth, rice, wheat, barley, corn, oat, rye, straw, millet, millet, pigeon, sorghum; red beans, kidney beans, peas, mung bean These seeds, leaves, stems, roots, flowers, and fruit plant tissues including beans such as soybeans and general grasses and weeds are also included.
  • 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”.
  • hemophor HasA iron porphyrin complex
  • Iron metabolism regulatory protein IRP: I ron R egulatory P rotein
  • iron responsive element I ron R esponsive E lement
  • IRR heme biosynthesis inhibitory Transcriptional regulators
  • 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.
  • a packaging method microcapsule type covering with a membranous film, but in order to separate the HasA-redoxin complex from plant tissue, a packaging method using a calcium salt of alginic acid is inexpensive and environmentally friendly. The comprehensive operation and the recovery operation are most preferable in terms of simplicity.
  • 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.
  • the concentration of the aqueous calcium chloride solution is preferably 0.5% to 1.0% (preferably 0.6%).
  • 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 the Heme destruction of HasA in the crude protein and the formation of a HasA-redoxin (iron-sulfur cluster) complex.
  • the air oxidation treatment is the key to the production method of the HasA-redoxin complex of the present invention and is indispensable.
  • the crude protein-encapsulating gel 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 the point of 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.
  • the elution treatment is obtained by eluting the HasA-redoxin complex by shaking the air-oxidized plant tissue-encapsulated gel with warm water.
  • 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.
  • 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).
  • a constant temperature shake incubator, a stirring temperature control tank, or the like can be used.
  • the gel As the elution of the HasA-redoxin complex progresses 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.
  • the aqueous solution formed by eluting the HasA-redoxin complex from the gel is hereinafter referred to as PC.
  • the PCA aqueous solution obtained in the first step is subjected to gravity to remove impurities remaining in the aqueous solution, and a precipitate of the HasA-redoxin complex is obtained.
  • the sediment which has been allowed to stand naturally and sinks to the earth's gravity is further subjected to physically strong gravity with a centrifuge, and the aqueous fraction is removed to concentrate the HasA-redoxin complex having concentrated asymmetric oxidation activity.
  • the method of obtaining is mentioned. There is no need to stick to this centrifugation method as long as the method can rapidly concentrate the HasA-redoxin complex.
  • 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.
  • an ultrafiltration membrane for example, Vivaspin 2-10 K
  • CMME and AGME 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 adopted 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.
  • 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 (FD).
  • -2 can be processed into a HasA-redoxin complex having an R-alcohol selective oxidation reaction.
  • a high-purity product of HasA-redoxin complex can be suitably obtained by simply filtering from the ME centrifugal supernatant using a centrifugal filter with an ultrafiltration membrane.
  • 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).
  • a sufficient structure of the HasA-redoxin complex that catalyzes the asymmetric oxidation reaction can be maintained by exhibiting 200 times or more the activity of fluorescent microorganism HasA expressed by microorganisms.
  • 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 the specific activity, and the nano oxygen bubble Further improvement can be achieved by using water as a solvent.
  • the ME active center HasA-redoxin complex
  • 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.
  • vacuum freeze-drying is preferable because moisture can be effectively and completely removed in a short time.
  • the system is evacuated and the temperature is raised to about 50 ° C. to sublimate moisture.
  • air oxygen, nitrogen, carbon dioxide, etc.
  • HasA-redoxin complex 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.
  • 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 complexes of the present invention is a HasA- redoxin complexes containing at least iron and sulfur, in the FT-IR spectrum, the region 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:
  • 3rd and 21st Cys (C) comes from redoxin, while showing 93% compatibility with fluorescent bacteria HasA. In addition, it is characterized in that it bubbles significantly in the reaction with hydrogen peroxide and releases oxygen.
  • 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.
  • FIG. 3 shows the dried pea-derived HasA-redoxin complex (PP-HasA) and the microorganism-expressed fluorescent fungus HasA (HasApf) by ion chromatography (IC) and ICP emission spectroscopy (ICP-ACS). The results for nutrients (wt%; carbon (C), hydrogen (H), nitrogen (N), sulfur (S), oxygen (O)) and metal (ppm; iron (Fe)) are shown.
  • 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, which shows 7 times the oxygen content, exhibits Fe—O and S—O vibrations characteristic of cysteine thiolate coordination hem between 950 cm ⁇ 1 and 1250 cm ⁇ 1 .
  • the HasApf expressed by microorganisms does not vibrate in this range because of the Hys.Tyr-6 coordinated heme.
  • dissolved oxygen cleaves the heme in HasA and forms a complex with nonheme redoxin (iron sulfur cluster). The characteristic cysteine thiolate coordination heme peak occurs.
  • FIG. 5 shows PC suspension (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.
  • FIG. 6 shows PC supernatant after centrifugation (A), ME supernatant after centrifugation (B), B (C) after centrifugal filtration (Vivaspinvas2-10 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).
  • 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 ⁇ M)” in which a fluorescent bacterium HasA is expressed in Escherichia coli is used as a substrate racemic-1- (2-naphthyl) ethanol (Rac-2; 0.8 mM) in a nano oxygen bubble aqueous solvent (5 mL).
  • Rac-2 racemic-1- (2-naphthyl) ethanol
  • FIG. 8 shows the concentration of dissolved oxygen (DO) 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.
  • DO dissolved oxygen
  • Oxidative cleavage followeded by the 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. 9 (a) 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.
  • PP-HasA pea-derived HasA-redoxin complex
  • FIG. 9B shows “HasApf” in which the fluorescent bacterium HasA is expressed in E. coli.
  • FIG. 10 shows the reason why a fluorescent bacteria-derived HasA-redoxin complex is obtained from peas.
  • 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 did not contain bacteria.
  • 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 results, Cys is detected in the 3rd and 21st amino acids of the N-terminal, and the HasA-- such as rubredoxin (6 kDa) and, for example, the 21st amino acid of 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.
  • unit means a unit of enzyme activity, and one unit represents the amount of enzyme (unit: ⁇ mol / h) capable of changing 1 ⁇ mol of substrate per hour.
  • 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).
  • FIG. 14 is a schematic diagram showing the proper use of both enantiomers by AGME and CMME for the substrate Rac-1 / -2 (1.2 mM to 3.6 mM) in an aqueous solvent (5 mL).
  • DMSO is used as a co-solvent
  • the influence on ME activity is summarized in FIG.
  • CMME has catalase activity.
  • Catalase has a property of reacting with hydrogen peroxide (H 2 O 2 ) to release oxygen.
  • 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. .
  • 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.
  • one enantiomer of racemic alcohol is selectively asymmetrically oxidized to a ketone to selectively obtain the other enantiomer not involved in the reaction.
  • HasA-redoxin complex derived from a suitable plant or produced from microbial expression is provided for the following reason. .
  • An inexpensive biological material plant-derived crude protein 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.
  • 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.
  • 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 to 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.
  • 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.
  • cytochrome P450 As an example of an organic synthesis reaction using cytochrome P450 as a biocatalyst, a report on a hydroxylation reaction has been made here.
  • epoxidation reaction by P450 for example, there is an epoxidation reaction of styrene by P450BS ⁇ derived from Bacillus subtilis, and further an epoxidation reaction of alkenes using P450bm-3 of Non-Patent Document 2.
  • soluble epoxide hydrolase uses epoxyeicosatrienoic acid (EET), which is an endogenous physiologically active substance, as a substrate.
  • EET is a fatty acid obtained by epoxidizing arachidonic acid cut from membrane phospholipid with cytochrome P450 (P450). That is, P450 is an EET synthase.
  • P450 is an EET synthase.
  • it is known to catalyze the following demethylation reaction (see Non-Patent Document 3).
  • the heme (iron porphyrin complex) enzyme P450 is well known as a substrate oxidation of an iron electron transfer system by active oxygen species, but there is no report relating to an asymmetric dehydrogenase of a secondary alcohol.
  • This patent lies in the finding of a reaction that catalyzes an asymmetric oxidation reaction using a heme protein-redoxin complex other than P450.
  • Non-Patent Document 5 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.
  • Non-Patent Document 8 As proteins involved in uptake of heme and iron, encourage 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).
  • FIG. 4 shows the differences by FT-IR for pea extraction-fluorescent fungus HasApf and microbial expression-fluorescent fungus HasApf. From 950 cm ⁇ 1 to 1250 cm ⁇ 1 , the absorption of Fe—O / S—O vibration indicating iron-sulfur oxide is markedly shown, and cysteine thiolate-iron is present in PP-HasA; ie, HasApf-redoxin (Or iron-sulfur cluster) Absorption that supports the complex.
  • HasApf-redoxin Or iron-sulfur cluster
  • HasApf 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.
  • the S-1- (2-naphthyl) ethanol is resolved with an optical purity of> 99% ee with a yield of 50%.
  • the R form is converted to a ketone with a yield of 50%. That is, 0 to 50 hours in FIG. 7 is a stage of 1) heme cleavage and 2) formation of HasApf-redoxin by oxygen in dissolved oxygen.
  • the spectrophotometer absorption 410 nm is 1) the point that disappears after 50 hours, and 2) after 50 hours, 950 cm ⁇ 1 to 1250 cm ⁇ 1 FT-IR as shown in FIG. 4 (red: PP-HasA). It is confirmed by the point that was seen.
  • the ME supernatant fraction (sample B) in FIG. 6 can be prepared pure PP-HasA (39 kDa) only 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 N-terminal of the dense sample D-band 2 which is characteristic of SDS-PAGE by SDS-PAGE and then by protein sequencer.
  • an 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.
  • 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-coordinate heme protein HasA in which histidine (His) and tyrosine (Tyr) are coordinated as shown in Non-Patent Document 5. It was.
  • the fluorescent bacterium HasA does not exist as a contaminant in the material, but exists as a symbiotic bacterium in the pea cell. 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 and “low intracellular iron ion concentration”.
  • 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.
  • the fluorescent bacterium HasA is complexed with redoxin after air oxidation, eluted as a membrane protein containing the HasApf-redoxin complex, and can be concentrated and precipitated easily by centrifugation.
  • the precipitate is coexisted with a polymer compound in an aqueous solution and treated with FD to be processed into 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 that does not require the addition of a cofactor NAD (P) and does not require selection of 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).
  • 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 is 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) that is used as an analgesic, antipyretic and anti-inflammatory drug.
  • NSAID non-steroidal 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.
  • polyethylene glycol such as PEG 4000
  • 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.
  • the alginate Ca gel-encapsulated plant-derived protein suitably induces air oxidation in the presence of oxygen after air oxidation. Appearing in the region of 950cm -1 ⁇ 1250cm -1 obtained by FT-IR measurement, the peak of Fe-O / S-O vibration iron - is indicative of the sulfur oxides, HasApf- redoxin redoxin in the 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 structure of the HasApf-redoxin complex catalyzing the asymmetric oxidation reaction shown in the present invention is (1) an iron-sulfur cluster (redoxin) involved in electron transfer and (2) HasA as a foundation thereof. 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.
  • the ME shown in the present invention produces two types of enantiomers with 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).
  • 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.
  • an asymmetric organic synthesis reagent capable of obtaining an active alcohol (yield 50%, 99% ee or more).
  • HasA-redoxin complex heme iron-activated HasA
  • the HasA-redoxin complex heme iron-activated HasA of the present invention 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 hybrids, 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.
  • 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 for example, those having the following physical properties can be suitably used.
  • Specific surface area preferably 2 to 38 m 2 / g, more preferably 10 to 30 m 2 / g, particularly preferably 15 to 25 m 2 / g
  • toyonite 200 series has a specific surface area of 20 m 2 / g
  • Average particle size: 105 to 210 ⁇ m is preferable, 125 to 190 ⁇ m is more preferable, and 145 to 170 ⁇ m is particularly preferable (for Toyonite 200 series, average particle size 155 ⁇ m)
  • Apparent bulk density preferably 0.18 to 1.18 g / m, more preferably 0.38 to 0.98 g / m, and particularly preferably 0.58 to 0.78
  • 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.
  • the recovered complex that is, the HasA-redoxin complex to be subjected to two or more reactions
  • the recovered complex is the “percentage of 50 h produced ketone” (%) described in Example 12 (and FIGS. 28 to 29) described later. ) To determine its usefulness.
  • the value of “percentage of 50 h-produced 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.
  • this screening method can be carried out conveniently using a centrifuge tube (so-called “Spitz tube”; length: 18 mm, capacity: about 15 mL).
  • a centrifuge tube so-called “Spitz tube”; length: 18 mm, capacity: about 15 mL.
  • “stirring with a shaker” about 100 types of carrier samples can be screened at a time by stirring about 100 centrifuge tubes at a time.
  • E. coli expression-fluorescent fungus HasA solution E. coli expression-fluorescent fungus HasA solution (concentration: 1.8 mg / mL) is prepared according to the method of Example 2 (HasA gene microbial expression method).
  • 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.
  • the “conversion rate to 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.
  • 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)
  • Example 1 extract method of plant-derived fluorescent fungus HasA
  • Example 2 method of expressing microorganisms of HasA gene
  • Example 1 Method for extracting plant-derived fluorescent fungus HasA
  • soybean protein powder manufactured by Organo Food Tech Co., Ltd., trade name “pea protein PP-CS”
  • pea protein PP-CS 3% aqueous sodium alginate solution
  • the solution part 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.
  • 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).
  • a centrifuge manufactured by Hitachi Koki Co., Ltd., Himac® CR20G
  • PC precipitates 80 g to 120 g.
  • the precipitate was dried using a vacuum freeze dryer (manufactured by Nissei Co., Ltd., RLEII203) and pulverized with a ball mill.
  • a vacuum freeze dryer manufactured by Nissei Co., Ltd., RLEII203
  • the HasApf-redoxin complex of the present invention was suitably purified by centrifugally filtering the ME supernatant after centrifugation (Vivaspin 2-10 K) and then drying the filtrate by FD.
  • 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 reagent DNA Ligation Kit ⁇ Mighty Mix> (TaKaRa)
  • Escherichia coli Competent high DH5 ⁇ (TOYOBO)
  • antibiotics Ancipirin or Kanamycin
  • Plasmid extraction kit QIAprep SpinI MiniQ )
  • the linking of the vector and the fluorescent fungus HasA gene was performed using a sequence reagent (ABI Prism 3130xl Genetic Analyzer (Applied Biosystems)) using a sequencing reagent (BigDye Terminators v1.1 Cycle Sequencing Kit).
  • 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.
  • a part of the cells is modified.
  • OD near 1.0
  • IPTG was added to a final concentration of 0.1 mM, followed by culturing at 30 ° C. for 16 h.
  • 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.
  • E. coli lysis buffer BugBuster Protein Extraction Reagent (Novagen)
  • denaturing buffer 8M Urea, PBS
  • the sample before the expression induction, the soluble fraction after the expression induction, and the insoluble fraction after the expression induction were mixed with 2 ⁇ sample buffer in an equal amount, and heat-treated at 99 ° C. for 5 minutes to obtain an electrophoresis sample.
  • SDS-PAGE was performed for about 70 minutes at 20 mA with a 5-20% gradient gel.
  • the fluorescent bacterium HasA was suitably 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 allow the His-Tag fusion protein to bind. 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.
  • E. coli elution buffer BugBuster Protein Extraction Reagent (Novagen)
  • an equilibration / washing buffer 50 mM sodium phosphate, 300 mM NaCl, pH 8
  • an elution buffer 150 mM meaning, 50 mM sodium phosphate, 300 mM NaCl, pH 8) were used.
  • the fluorescent bacterium HasA production process from the above-mentioned expression of Escherichia coli also corresponds to Pseudomonas aeruginosa-derived HasA and Mycobacterium tuberculosis-derived HasA.
  • 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 ⁇ M). Expresses a homogeneous oxidation reaction.
  • HasA fluorescent fungus
  • Fig. 3 constituent components
  • FTIR Fe-O / SO vibration in Fig. 4
  • ESR non-heme absorption in Fig. 9
  • Reference can be made from physicochemical molecular structure measurements such as (active oxygen species: Fe—O, g 4.3).
  • FIG. 4 shows the results of investigation using FT-IR (ST Japan, Inc., portable ATR apparatus (A2 Technologes, ML version), ATR method).
  • 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.
  • HasApf is a protein obtained by expressing Escherichia coli a fluorescent bacterium HasA gene by the above-described method.
  • 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.).
  • ESR electron spin resonance
  • Example 4 SDS-PAGE of active fraction
  • the molecular weight was determined using a precast gel (SDS-PAGE mini) manufactured by Tefco. 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.
  • B) Centrifugal supernatant fraction of ME solution obtained by re-dissolving the centrifugal precipitate in 0.5 mM glycine NaOH buffer (pH 9-11).
  • 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.
  • BLAST 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 in the text box from “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.
  • the band 2 was a fluorescent bacterium HasA.
  • Example 6 (second step, biochemical properties of PC and ME)
  • 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”
  • activity intensity monitoring of various fractions FIG. 5
  • DO dissolved oxygen concentration
  • 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 (PC suspension), centrifuged PC supernatant (Supernatant), centrifuged PC precipitate (Precipitate), carboxy-cellulose coated PC precipitate (CM-Cellulose coating PC), and glutaraldehyde crosslinked.
  • PC suspension PC suspension
  • Supernatant centrifuged PC supernatant
  • Precipitate carboxy-cellulose coated PC precipitate
  • CM-Cellulose coating PC carboxy-cellulose coated PC precipitate
  • 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.
  • FIG. 10 by re-dissolving the PC precipitate in a 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
  • 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.
  • FIG. 13 shows the co-solvent DMDO (IPA, IPA, Rac-2 (1.2 mM ⁇ 3) in nano-oxygen bubbled water solvent (5 mL).
  • (Ethanol) concentration (2%, 4%, 6%, 8%) is a graph showing the outline of the effect on AG / CM-ME (50 mg) asymmetric oxidation activity.
  • “1.2 mM ⁇ 3” means a concentration of 30 ⁇ L 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.
  • FIG. 13 indicates that the AG / CM-ME oxidation activity is not affected by the auxiliary solvent DMDO from 0% to 3%, but the asymmetric oxidation activity is reduced to less than half when the concentration is 6% or more.
  • the HasA-redoxin complex is decomposed into HasA and redoxin when the concentration of organic solvent (DMSO, IPA, ethanol, diethyl ether, hexane, etc.) is 3% or more, 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.
  • FIG. 10 shows the reason why a fluorescent bacteria-derived HasA-redoxin complex is obtained from peas.
  • 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.
  • microbial contamination in the raw pea protein 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 entrapped 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.
  • Fluorescent fungus HasA eluted from peas is used as a spray 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 assimilated into the host, is retained in the cells and tissues in the plant, and is inherited by the offspring. 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.
  • the ABC-Transporter in 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 membrane tissue containing HasA and further as a HasA-redoxin complex formed by air oxidation. 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) in FIG. there were.
  • the band 3 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 microbial cells. Therefore, the membrane tissue containing HasA can be precipitated and recovered as an oxidized HasA-redoxin complex.
  • the redoxin is a protein having an iron-sulfur cluster structure (ferredoxin, rubredoxin, etc.).
  • the aforementioned proteins (IRP, IRE, IRR) involved in HasA expression are also redoxins of the HasA-redoxin complex. As included.
  • Example 8 catalase activity of HasA-redoxin complex
  • Example 8 Measurement of Catalase Activity; Measurement by Visualization of “Bubbling”
  • PC precipitate 15 mg
  • microbial expression HasApf 15 mg obtained in Example 7
  • 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.
  • oxygen was generated about twice in the solution surface layer portion, and bubbling stopped after 5 minutes.
  • 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.
  • 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.
  • the identity of the “bubble” generated in the experiment is oxygen, and the involvement of the enzyme of the catalase activity that converts hydrogen peroxide into water and oxygen is confirmed. It was. Therefore, the ME precipitate (15 mg) consumed about 10,000 ppm of catalase activity with respect to 5% hydrogen peroxide solution, the PC precipitate (15 mg) consumed about 8000 ppm with catalase activity, the microorganism-expressed HasApf (15 mg). ) Showed about 4000 ppm consumption of catalase activity.
  • HasApf 15 mg
  • the catalase activity of the ME precipitate (50 ⁇ g) capable of changing 1 ⁇ mol of the substrate (H 2 O 2 : MW34) per 5 minutes is the same as that of the PC precipitate (63.5 ⁇ g). It was found to be 3 times, 2.3 times the microbial expression HasApf (118 ⁇ g). 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.
  • 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 for about 50 hours in nano-oxygen bubble water; 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 mixed with distilled water (4 mL) / DMSO ( ⁇ 1.0% (v / v)) in the substrate Rac-2 (20,000 ppm, 30 ⁇ L). 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 ⁇ L) was added and reacted.
  • CMME can synthesize S-2 (> 99% ee / ⁇ 50%) by stereoselective R-form oxidation
  • AGME can synthesize R-2 (> 99% ee by stereoselective S-form oxidation. / -50%) could be synthesized.
  • 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.
  • 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).
  • CMME is PEG4000 coating
  • AGME is PEG1000 coating.
  • Example 11 Enzyme-immobilized support-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%.
  • Toyonite 1000 mg
  • 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.
  • E. coli expression-fluorescent HasA amount prepared from Example 2 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.
  • the fluorescent bacterium HasApf approximately 0.4 mg / 4 mL-ion-exchanged water expressed in E. coli.
  • 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 ⁇ L) 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 results 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.
  • the reaction time of the produced ketone spectrum 350 nm
  • 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.
  • Hemophor HasApf / heme is an oxidation reaction mechanism (Fe II + O 2 ⁇ Fe III —O—O ⁇ ⁇ Fe IV ⁇ O (oxidizing rac-1) ⁇ Fe II + H) in the same manner as cytochrome-P450. 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.
  • 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
  • HasApf is further adsorbed and immobilized on immobilized carrier toyonite
  • 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.
  • 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.
  • Strong chemical resistance Since it is a kaolinite ceramic carrier, it is excellent in chemical resistance such as acid, alkali and organic solvent.
  • Toyonite types Toyonite has carriers with 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 Toyonite200-carrier having a hydroxyl group as an organic functional group
  • Type 2 Toyonite200A-carrier having an amino group as an organic functional group
  • Type 3 Toyonite200P-carrier having a phenylamino group as an organic functional group
  • Type 4 Toyonite200M-
  • HasA solution (1.8 mg / mL) 5-fold amount was adsorbed and immobilized on enzyme-immobilized carrier-Toyoden (1,000 mg) manufactured by Toyo Denka Kogyo.
  • HasA solution An Escherichia coli expression-fluorescent fungus HasA solution (concentration: 1.8 mg / mL) is prepared according to the method of Example 2 (Method of microbial expression of HasA gene).
  • Immobilization Toyonite (1000 mg) is dropped into 5 mL of the HasA solution obtained in step (1) above and stirred for 20 to 40 hours. Stirring uses a stirring blade or a shaker.
  • 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).
  • 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.
  • HasApf was suitably adsorbed and immobilized on a toyonite-immobilized carrier and can be used as an immobilizing agent as in the case where toyonite is lipase.
  • 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.
  • 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 Immobilized 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
  • immobilized HasApf carrier (30 mg) / ion-exchanged water (4 mL) in an environment of 40 ° C., followed by extraction with hexane (4 mL) after 50 h or 72 h of reaction time.
  • the results of confirming the optical purity (% ee) and the conversion rate (%) to the resulting ketone are shown.
  • CMME / AGME system which is 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.
  • the immobilized HasApf after hexane extraction is further equivalent to the substrate rac-1 (0.8 mM, 1.2 mM, 2.0 mM; 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.
  • the results when the substrate concentration is changed from 0.8 mM to 1.2 mM or 2.0 mM are summarized as follows.
  • Substrate concentration of 1.2 mM is the optimal concentration as PP-HasA, while 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.
  • 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 is a 2 mM / 4 mL-water system and can contribute to the separation of the substrate racemic alcohol about 1.5 times.
  • 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 thereof, has been studied as a system catalyzed in an oxygen-dependent manner by heme oxygenase (HO) or (MhuD: Mycobacterium tuberculosis enzyme). On the other hand, a functional system that catalyzes an asymmetric oxidation reaction is the first case discovered this time.
  • HO heme oxygenase
  • MhuD Mycobacterium tuberculosis enzyme
  • 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
  • sodium borohydride NaBH4: 50 mg
  • 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.
  • 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.
  • PP-HasApf is capable of cyclic deracemization with NaBH 4 and can improve the yield of the target optically active alcohol 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 a HasApf-immobilized carrier.
  • FIG. 18 SDS-PAGE results after purification of the EsA-expressed HasA gene.
  • 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
  • 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 field of fine chemicals 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.

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Abstract

L'invention concerne un complexe HasA-rédoxine (un HasA de type à activité de fer héminique) qui peut oxyder de manière asymétrique uniquement un des énantiomères d'un alcool secondaire sans nécessité d'ajouter une coenzyme, et présente une activité d'oxydation asymétrique dans un solvant aqueux en présence d'oxygène; elle concerne aussi une forme immobilisée du complexe HasA-rédoxine. L'invention concerne des procédés de production du complexe HasA-rédoxine et de la forme immobilisée du complexe HasA-rédoxine, respectivement; et un procédé de production concernant l'enrobage du complexe protéique avec un composé polymérique. Elle concerne également des procédés de production d'un complexe HasA-rédoxine (un HasA de type à activité de fer héminique) présentant une réaction d'oxydation asymétrique, et d'une forme immobilisée du complexe, respectivement. Les procédés de l'invention se caractérisent en ce qu'ils comprennent: une première étape comprenant un traitement d'encapsulation dans un gel d'une protéine brute d'origine végétale soluble dans l'eau, un traitement d'oxydation du gel avec l'air, et un traitement d'élution en solution aqueuse d'un complexe HasA-rédoxine à partir du gel; et une seconde étape comprenant un traitement d'application d'une gravité à la solution aqueuse du complexe HasA-rédoxine pour concentrer et précipiter celui-ci, un traitement de redissolution dans une solution aqueuse des précipités du complexe HasA-rédoxine pour permettre à ce dernier et à un composé polymère de co-exister de manière homogène, et un traitement de déshydratation/séchage de la solution aqueuse coexistante pour produire un complexe HasA-rédoxine enrobé d'un composé polymérique. Les procédés de l'invention se caractérisent également en ce que HasA peut s'exprimer à l'aide d'un transformant (un micro-organisme) contenant HasA et issu de Pseudomonas fluorescens, puis HasA est agité et oxydé avec l'air dans une solution aqueuse oxygéné pour produire ainsi un complexe HasA-rédoxine (un HasA de type à activité de fer héminique).
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CN110283839A (zh) * 2019-07-08 2019-09-27 天津大学青岛海洋技术研究院 生物电催化系统促进p450催化甾体羟基化反应的方法

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WO2014073673A1 (fr) * 2012-11-09 2014-05-15 サンヨー食品株式会社 Complexe protéique capable de catalyser une réaction d'oxydation asymétrique et procédé de production de celui-ci

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WO2014073673A1 (fr) * 2012-11-09 2014-05-15 サンヨー食品株式会社 Complexe protéique capable de catalyser une réaction d'oxydation asymétrique et procédé de production de celui-ci

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110283839A (zh) * 2019-07-08 2019-09-27 天津大学青岛海洋技术研究院 生物电催化系统促进p450催化甾体羟基化反应的方法
CN110283839B (zh) * 2019-07-08 2023-06-27 天津大学青岛海洋技术研究院 生物电催化系统促进p450催化甾体羟基化反应的方法

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