Protein complex capable of catalyzing asymmetric oxidation reaction and method for producing same

Abstract

Provided are: a protein complex capable of selectively and asymmetrically oxidizing an enantiomer of a secondary alcohol without adding a coenzyme and having an asymmetric oxidation activity in a water-soluble solvent system in the presence of oxygen; a method for producing the same; and a method for coating the protein complex with a high molecular weight compound. The method for producing the protein complex includes: (1) enclosing a crude water-soluble protein in a gel, air-oxidizing the gel, and eluting the protein complex into an aqueous solution; and (2) applying gravity to concentrate and precipitate the protein complex, redissolving the precipitate in an aqueous glycine sodium hydroxide solution of about 0.5 mM and allowing the same to homogeneously coexist with a high molecular weight compound, and re-precipitating the solution and dehydrating and drying the same to yield a protein complex coated with a high molecular weight compound.

1 - 8 . (canceled) 
     
     
         9 . An iron-containing protein complex derived from an animal or plant, which has reactivity with hydrogen peroxide, even in the absence of NAD(H) and/or NADP(H). 
     
     
         10 . The protein complex according to  claim 9 , which has a function attributable to a cell organelle having asymmetric oxidation activity and/or catalase activity. 
     
     
         11 . The protein complex according to  claim 10 , wherein the cell organelle is a mitochondrion, and the function is derived from iron-oxygen-dependent electron transfer-based oxidation activity of PQQ-dehydrogenase and cytochrome c oxidase present as membrane protein. 
     
     
         12 . The protein complex according to  claim 10 , wherein the cell organelle is a chloroplast or endoplasmic reticulum, and the function is derived from tissue in which catalase activity is localized that exhibits reactivity with hydrogen peroxide. 
     
     
         13 . The protein complex according to  claim 10 , wherein the cell organelle is present as a membrane protein, and contains two or more enzymes of any of PQQ-dehydrogenase, cytochrome c oxidase, peroxidase and catalase. 
     
     
         14 . The protein complex according to  claim 9 , which has at least one peak in the region of 1085±50 cm −1  in an FT-IR spectrum thereof. 
     
     
         15 . The protein complex according to  claim 14 , wherein the region is a peak derived from iron oxide (S—O or S═O) that is formed due to air oxidation of amino acid cysteine in the protein. 
     
     
         16 . The protein complex according to  claim 9 , comprising a component that contains the following amino acid sequence as an amino acid sequence from the N-terminal thereof to the 33rd amino acid:
 S*SISYST*YATNTVAQYL*DW*AYFGDLNHRE   
       and has reactivity with hydrogen peroxide even in the absence of NAD(H) and/or NADP(H). 
     
     
         17 . The protein complex according to  claim 16 , wherein an iron-binding protein supported by the sequence is derived from a membrane protein and/or enzyme group composed of the enzymes PQQ-dehydrogenase, cytochrome c oxidase, peroxidase and catalase. 
     
     
         18 . The protein complex according to  claim 17 , wherein the enzyme group contains two types of enzymes capable of catalyzing not only an R-isomer-selective asymmetric oxidation reaction of a racemic alcohol, but also an S-isomer-selective asymmetric oxidation reaction thereof, and both enantiomers can be used separately corresponding to the purpose. 
     
     
         19 . The protein complex according to  claim 18 , wherein a protein complex derived from ovalbumin can be used in the S-isomer-selective asymmetric oxidation reaction. 
     
     
         20 . The protein complex according to  claim 9 , wherein the protein complex is coated with a polymer compound. 
     
     
         21 . The protein complex according to  claim 9 , wherein the protein complex is subjected to chemical modification treatment. 
     
     
         22 - 29 . (canceled) 
     
     
         30 . The protein complex according to  claim 9 , wherein the reactivity with hydrogen peroxide is reactivity of a level at which bubbling occurs that can be visually confirmed with the naked eye based on the generation of oxygen when 15 g of the protein complex (particle diameter: 50 μm) is placed in a test tube having a diameter of 18 mm and 1.0 mL of hydrogen peroxide is added thereto. 
     
     
         31 . The protein complex according to  claim 9 , which has substrate reactivity of 10 units/g (per hour) or more as specific activity calculated according to average rate for a reaction time of 15 hours in the case of reacting 20 mg of the protein complex (particle diameter: 50 μm) under conditions of a room temperature of 40° C. using 0.8 mM racemic 6-methoxy-1-(2-naphthyl) ethanol (Rac-1) as substrate in an aqueous medium (4 mL). 
     
     
         32 . The protein complex according to  claim 9 , wherein enantioselectivity imparted to a naproxen precursor (s-1) for a reaction time of 15 hours is 95% ee or more in the case of reacting 20 mg of the protein complex (particle diameter: 50 μm) under conditions of a room temperature of 40° C. using 0.8 mM racemic 6-methoxy-1-(2-naphthyl) ethanol (Rac-1) as substrate in an aqueous medium (4 mL). 
     
     
         33 - 36 . (canceled) 
     
     
         37 . The protein complex according to  claim 20 , wherein the polymer compound has a molecular weight of 4000 or more. 
     
     
         38 . The protein complex according to  claim 37 , wherein the polymer compound is polyethylene glycol. 
     
     
         39 . The protein complex according to  claim 20 , wherein the polymer compound is a mixture of polymer compounds having different molecular weights, and
 the mixture is composed of a polymer compound having a molecular weight of 4000 or more and a polymer compound having a molecular weight of 1000 or less, and contains one-third or less of the low molecular weight polymer compound.   
     
     
         40 . The protein complex according to  claim 39 , wherein the polymer compound is polyethylene glycol. 
     
     
         41 - 44 . (canceled)