Nadph-regeneration system based on monomeric isocitrate dehydrogenase and use thereof

Abstract

The present invention relates to an NADPH-regeneration system based on monomeric isocitrate dehydrogenase (IDH) and a use thereof. Specifically, the present invention relates to a recombinant vector including a polynucleotide encoding an isocitrate dehydrogenase recombinant protein derived from Corynebacterium glutamicum (CgIDH) and an isocitrate dehydrogenase recombinant protein derived from Azotobacter vinelandii (AvIDH), a method for producing the recombinant protein, and an NADPH-regeneration system using the recombinant protein produced by the method. In the present invention, the enzyme in a monomeric form that may be efficiently used in the NADPH-regeneration system in the transformant into which the recombinant vector was introduced, was found, and the NADPH-regeneration system using the enzyme in a monomeric form has a very high utility value as biological parts and biocatalyst materials that provides NADPH to the NADPH-dependent enzyme.

1 . A recombinant expression vector for NADPH regeneration, comprising a polynucleotide encoding monomeric isocitrate dehydrogenase (IDH) from  Corynebacterium glutamicum  or  Azotobacter vinelandii.    
     
     
         2 . The recombinant expression vector of  claim 1 , further comprising a polynucleotide encoding an NADPH-dependent enzyme. 
     
     
         3 . The recombinant expression vector of  claim 1 , wherein the isocitrate dehydrogenase from  Corynebacterium glutamicum  consists of an amino acid sequence of SEQ ID NO: 1, and the isocitrate dehydrogenase from  Azotobacter vinelandii  consists of an amino acid sequence of SEQ ID NO: 2. 
     
     
         4 . The recombinant expression vector of  claim 2 , wherein the NADPH-dependent enzyme is any one or two or more selected from the group consisting of dehydrogenase, reductase, oxidoreductase, transhydrogenase, peroxidase, oxygenase, monooxygenase, flavodoxin, and dehalogenase. 
     
     
         5 . The recombinant expression vector of  claim 4 , wherein the NADPH-dependent enzyme is recombined and fused to the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2, or is linked by addition of a chemical linker. 
     
     
         6 . The recombinant expression vector of  claim 5 , wherein the chemical linker is selected from the group consisting of PEGylated bis(sulfosuccinimidyl) suberate (BS(PEG)5), PEGylated bis(sulfosuccinimidyl) suberate (BS(PEG)9), bis(sulfosuccinimidyl) glutarate-d0 (BS2G-d0), bis(sulfosuccinimidyl) 2,2,4,4-glutarate-d4 (BS2G-d4), disuccinimidyl dibutyric urea (DSBU), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), disuccinimidyl glutarate (DSG), dithiobis(succinimidyl) propionate (DSP), disuccinimidyl suberate (DSS), disuccinimidyl sulfoxide (DSSO), disuccinimidyl tartarate (DST), dimethyl-3,3-dithiobis propionimidate (DTBP), ethylene glycol bis(succinimidyl) succinate (EGS), tris-(succinimidyl) aminotriacetate (TSAT), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). 
     
     
         7 . A method for producing a recombinant protein, the method comprising:
 producing a recombinant expression vector for NADPH regeneration of comprising a polynucleotide encoding monomeric isocitrate dehydrogenase (IDH) from  Corynebacterium glutamicum  or  Azotobacter vinelandii;      transforming the recombinant expression vector to produce a transformant;   culturing the transformant to overexpress isocitrate dehydrogenase from  Corynebacterium glutamicum  or isocitrate dehydrogenase from  Azotobacter vinelandii;  and   recovering an overexpressed recombinant protein.   
     
     
         8 . The method of  claim 7 , wherein the culturing is performed at 28 to 32° C. 
     
     
         9 . The method of  claim 7 , wherein the transformant is  Escherichia coli.    
     
     
         10 . A composition for substrate hydroxylation, comprising:
 a recombinant protein produced by:
 producing a recombinant expression vector for NADPH regeneration of comprising a polynucleotide encoding monomeric isocitrate dehydrogenase (IDH) from  Corynebacterium glutamicum  or  Azotobacter vinelandii;    
 transforming the recombinant expression vector to produce a transformant; 
 culturing the transformant to overexpress isocitrate dehydrogenase from  Corynebacterium glutamicum  or isocitrate dehydrogenase from  Azotobacter vinelandii;  and 
 recovering the recombinant protein; and 
   a cytochrome P450 protein.   
     
     
         11 . The composition of  claim 10 , wherein the substrate is selected from the group consisting of omeprazole, omeprazole sulfide, ethoxycoumarin, and nitrophenol.