Method for Using Electrochemical Bioreactor Module with Recovery of Cofactor

Abstract

Provided herein are composition and process for using an electrochemical device for the reduction of the oxidized state of phosphorylated or non-phosphorylated nicotinamide adenine dinucleotide to the reduced state in which unwanted products of the electrochemical reduction are recovered as the oxidized state of the phosphorylated or non-phosphorylated nicotinamide adenine dinucleotide and returned to the electrochemical device for reduction.

1 . A system for electrochemically generating NAD(P)H 2  reducing equivalents comprising:
 an electrochemical cell comprising an anode contained in an anode chamber and a cathode contained in a cathode chamber;   a first process stream containing NAD(P), passing through the cathode chamber and continuously in contact with the cathode from which electrons are transferred to the NAD(P) to produce a second process stream containing reduced species 1,4-NAD(P)H 2 , 1,2-NAD(P)H 2 , 1,6-NAD(P)H 2 , and [NAD(P)] 2 , while optionally producing hydrogen;   a substrate of an oxidoreductase or P450 enzyme, which, when contacted with the second process stream in the presence of the oxidoreductase or P450 enzyme, is transformed to a product while concomitantly consuming the 1,4-NAD(P)H 2  in the second process stream and producing a first recovered NAD(P) and a third process stream; and   at least one of a Mung Bean Phenol Oxidase enzyme and illumination at a wavelength of about 254 nm or exceeding about 320 nm, which, when contacted with the third process stream, convert at least one of the 1,2-NAD(P)H 2 , 1,6-NAD(P)H 2 , and [NAD(P)] 2  therein to a second and optionally a third recovered NAD(P).   
     
     
         2 . The system of  claim 1 , comprising the Mung Bean Phenol Oxidase enzyme for converting the 1,2-NAD(P)H 2 , 1,6-NAD(P)H 2 , and/or [NAD(P)] 2  into the second recovered NAD(P) and/or the third recovered NAD(P). 
     
     
         3 . The system of  claim 1 , comprising the illumination for converting the [NAD(P)] 2  into NAD(P). 
     
     
         4 . The system of  claim 1 , comprising the Mung Bean Phenol Oxidase enzyme and the illumination. 
     
     
         5 . The system of  claim 4 , wherein the third process stream is contacted with the Mung Bean Phenol Oxidase enzyme resulting in conversion of the 1,2-NAD(P)H 2  and 1,6-NAD(P)H 2  into the second recovered NAD(P) and a fourth process stream, wherein the fourth process stream is contacted with the illumination to convert the [NAD(P)] 2  therein to the third recovered NAD(P). 
     
     
         6 . The system of  claim 1 , further comprising, in one or more of the process streams, an electron transfer mediator (ETM) capable of transferring electrons to NAD(P). 
     
     
         7 . The system of  claim 1 , further comprising catalase for decomposing hydrogen peroxide produced by the Mung Bean Phenol Oxidase enzyme and/or the illumination. 
     
     
         8 . The system of  claim 2 , wherein the Mung Bean Phenol Oxidase enzyme is recombinantly expressed from microorganisms such as  Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, Pichia pastoris , and  Bacillus megaterium.    
     
     
         9 . The system of  claim 2 , wherein the Mung Bean Phenol Oxidase enzyme is a mutant form having a lower oxidation activity for 1,4-NAD(P)H 2  than the wild type Mung Bean Phenol Oxidase enzyme, and/or a higher oxidation activity for 1,2-NAD(P)H 2 , 1,6-NAD(P)H 2  and/or [NAD(P)] 2  than the wild type Mung Bean Phenol Oxidase enzyme, wherein preferably the mutant form is recombinantly expressed from microorganisms such as  Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, Pichia pastoris , and  Bacillus megaterium.    
     
     
         10 . A method for electrochemically generating NAD(P)H 2  reducing equivalents comprising:
 a. providing an electrochemical cell comprising an anode contained in an anode chamber and a cathode contained in a cathode chamber;   b. passing through the cathode chamber a first process stream which contains NAD(P) and is continuously in contact with the cathode from which electrons are transferred to the NAD(P) to produce a second process stream containing reduced species 1,4-NAD(P)H 2 , 1,2-NAD(P)H 2 , 1,6-NAD(P)H 2 , and [NAD(P)] 2 , while optionally producing hydrogen;   c. contacting the second process stream with a substrate of an oxidoreductase or P450 enzyme such that the substrate, in the presence of the oxidoreductase or P450 enzyme, is transformed to a product while concomitantly consuming the 1,4-NAD(P)H 2  in the second process stream and producing a first recovered NAD(P) and a third process stream; and   d. contacting the third process stream with at least one of a Mung Bean Phenol Oxidase enzyme and illumination at a wavelength of about 254 nm or exceeding about 320 nm, thereby converting at least one of the 1,2-NAD(P)H 2 , 1,6-NAD(P)H 2 , and [NAD(P)] 2  therein to a second recovered NAD(P) and optionally a third recovered NAD(P).   
     
     
         11 . The method of  claim 10 , further comprising contacting the third process stream with the Mung Bean Phenol Oxidase enzyme for converting the 1,2-NAD(P)H 2 , 1,6-NAD(P)H 2 , and/or [NAD(P)] 2  into the second recovered NAD(P) and/or the third recovered NAD(P). 
     
     
         12 . The method of  claim 10 , further comprising contacting the third process stream with the illumination for converting the [NAD(P)] 2  into NAD(P). 
     
     
         13 . The method of  claim 10 , further comprising contacting the third process stream with the Mung Bean Phenol Oxidase enzyme resulting in conversion of the 1,2-NAD(P)H 2  and 1,6-NAD(P)H 2  therein into the second recovered NAD(P) and a fourth process stream, and further comprising contacting the fourth process stream with the illumination to convert the [NAD(P)] 2  therein to the third recovered NAD(P). 
     
     
         14 . The method of  claim 10 , further comprising providing, in one or more of the process streams, an electron transfer mediator (ETM) capable of transferring electrons to NAD(P). 
     
     
         15 . The method of  claim 10 , further comprising providing catalase for decomposing hydrogen peroxide produced by the Mung Bean Phenol Oxidase enzyme, and/or the illumination. 
     
     
         16 . The method of  claim 10 , wherein the Mung Bean Phenol Oxidase enzyme is recombinantly expressed from microorganisms such as  Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, Pichia pastoris , and  Bacillus megaterium.    
     
     
         17 . The method of  claim 10 , wherein the Mung Bean Phenol Oxidase enzyme is a mutant form having a lower oxidation activity for 1,4-NAD(P)H 2  than the wild type Mung Bean Phenol Oxidase enzyme, and/or a higher oxidation activity for 1,2-NAD(P)H 2 , 1,6-NAD(P)H 2  and/or [NAD(P)] 2  than the wild type Mung Bean Phenol Oxidase enzyme, wherein preferably the mutant form is recombinantly expressed from microorganisms such as  Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, Pichia pastoris , and  Bacillus megaterium.    
     
     
         18 . The method of  claim 10 , further comprising returning the third process stream to the cathode chamber.