U.S. patent application number 17/182039 was filed with the patent office on 2021-06-10 for method for using electrochemical bioreactor module with recovery of cofactor.
The applicant listed for this patent is BIOCHEMINSIGHTS, INC.. Invention is credited to William B. Armiger, David R. Dodds, Mattheos Koffas, Clifford S. Morrison.
Application Number | 20210171995 17/182039 |
Document ID | / |
Family ID | 1000005417671 |
Filed Date | 2021-06-10 |
United States Patent
Application |
20210171995 |
Kind Code |
A1 |
Morrison; Clifford S. ; et
al. |
June 10, 2021 |
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.
Inventors: |
Morrison; Clifford S.;
(Riverside, CA) ; Armiger; William B.; (Malvern,
PA) ; Dodds; David R.; (Manlius, NY) ; Koffas;
Mattheos; (Niskayuna, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BIOCHEMINSIGHTS, INC. |
Malvern |
PA |
US |
|
|
Family ID: |
1000005417671 |
Appl. No.: |
17/182039 |
Filed: |
February 22, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16084579 |
Sep 13, 2018 |
10927395 |
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PCT/US2017/022241 |
Mar 14, 2017 |
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17182039 |
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62308175 |
Mar 14, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/0006 20130101;
C12R 2001/84 20210501; C12N 1/205 20210501; C12R 2001/865 20210501;
C12R 2001/15 20210501; H01M 8/16 20130101; C12P 19/32 20130101;
C07H 19/207 20130101; C12N 9/0071 20130101; C07H 21/00 20130101;
C12Y 101/01 20130101; C12P 19/36 20130101; C12N 9/0008 20130101;
C07H 1/00 20130101; C12Y 103/01 20130101; C12Y 113/11001 20130101;
C12N 9/0036 20130101; C12N 9/0059 20130101; C12Y 114/11 20130101;
C12Y 110/03001 20130101; C12N 1/185 20210501; A61K 31/7084
20130101; C12R 2001/19 20210501; C12R 2001/11 20210501; C12N 9/0069
20130101; C12N 1/165 20210501; C12R 2001/125 20210501; Y02E 60/50
20130101 |
International
Class: |
C12P 19/36 20060101
C12P019/36; C07H 1/00 20060101 C07H001/00; C12N 9/02 20060101
C12N009/02; C07H 21/00 20060101 C07H021/00; A61K 31/7084 20060101
A61K031/7084; C07H 19/207 20060101 C07H019/207; C12N 9/04 20060101
C12N009/04; C12P 19/32 20060101 C12P019/32; C12R 1/11 20060101
C12R001/11; C12R 1/125 20060101 C12R001/125; C12R 1/15 20060101
C12R001/15; C12R 1/19 20060101 C12R001/19; C12R 1/84 20060101
C12R001/84; C12R 1/865 20060101 C12R001/865 |
Claims
1. A system for electrochemically generating NAD(P)H.sub.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.sub.2,
1,2-NAD(P)H.sub.2, 1,6-NAD(P)H.sub.2, and [NAD(P)].sub.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.sub.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.sub.2, 1,6-NAD(P)H.sub.2, and [NAD(P)].sub.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.sub.2, 1,6-NAD(P)H.sub.2,
and/or [NAD(P)].sub.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)].sub.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.sub.2 and 1,6-NAD(P)H.sub.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)].sub.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.sub.2 than the wild type Mung Bean Phenol Oxidase
enzyme, and/or a higher oxidation activity for 1,2-NAD(P)H.sub.2,
1,6-NAD(P)H.sub.2 and/or [NAD(P)].sub.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.sub.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.sub.2, 1,2-NAD(P)H.sub.2,
1,6-NAD(P)H.sub.2, and [NAD(P)].sub.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.sub.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.sub.2,
1,6-NAD(P)H.sub.2, and [NAD(P)].sub.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.sub.2, 1,6-NAD(P)H.sub.2, and/or
[NAD(P)].sub.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)].sub.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.sub.2 and 1,6-NAD(P)H.sub.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)].sub.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.sub.2 than the wild type Mung Bean Phenol Oxidase
enzyme, and/or a higher oxidation activity for 1,2-NAD(P)H.sub.2,
1,6-NAD(P)H.sub.2 and/or [NAD(P)].sub.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.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional patent application of U.S.
application Ser. No. 16/084,579, filed Sep. 13, 2018, now U.S. Pat.
No. 10,927,395, which is a U.S. National Phase application under 35
U.S.C. 371 of International Application No. PCT/US2017/022241,
filed Mar. 14, 2017, which claims priority to and the benefit of
U.S. Provisional Application No. 62/308,175 filed Mar. 14, 2016,
the disclosure of each of which is incorporated herein by reference
in their entireties.
TECHNICAL FIELD
[0002] The present disclosure relates generally to the use of
biologically mediated reactions that alter the oxidation state of
compounds, and specifically the oxidation state of carbon atoms in
a given chemical compound.
[0003] More specifically, the present disclosure relates to an
improved method of utilizing the Electrochemical Bioreactor Module
(EBM) in which undesired forms of nicotinamide adenine dinucleotide
cofactor which are produced during the electrochemical reduction of
the oxidized form of the nicotinamide adenine dinucleotide cofactor
are recovered for recycling through the EBM.
BACKGROUND
[0004] The use of enzymes which perform a reduction and are capable
of reducing a provided substrate to a desired reduced product, has
been widely reported. (Dodds et al, J. Am. Chem. Soc., (1988)
110(2), 577-583; Dodds et al, Proceedings Chiral Europe'95. London,
(1995), 55-62; A. Liese et al, Appl Microbiol Biotechnol (2007)
76:237-248; Liese, A.; 2nd ed. Enzyme Catalysis in Organic
Synthesis, (2002), 3, 1419-1459; Kula, M. R.; Kragl, U.
Stereoselect. Biocatal. (2000), 839-866; Chartrain, M.; Greasham,
R.; Moore, J.; Reider, P.; Robinson, D.; Buckland, B. J. Mol.
Catal. B: Enzym. (2001), 11, 503-512; Patel, R. N. Enzyme Microb.
Technol. (2002), 31, 804-826; Patel, R. N. Adv. Appl. Microbiol.
(2000), 47, 33-78; Patel, R. N.; Hanson, R. L.; Banerjee, A.;
Szarka, J. Am. Oil Chem. Soc. (1997), 74, 1345-1360; Hummel, W.
Adv. Biochem. Eng. Biotechnol. (1997), 58, 145-184; Whitesides et
al, Appl. Biochem. and Biotech. (1987) 14, 147-197; Whitesides et
al, Biotechnology and Genetic Engineering Reviews, Vol. 6.
September 1988).
[0005] To accomplish such a reduction of a provided substrate,
electrons must be provided to the reaction. In biological systems,
both in vivo and in vitro, these electrons, generally called
"reducing equivalents", are provided by small molecules generally
termed "cofactors". The most common cofactor is nicotinamide
adenine dinucleotide, NAD. A phosphorylated form of the
cofactoralso exists, and both forms provide reducing equivalents to
the enzymes that catalyze reactions requiring reducing
equivalents.
[0006] The nicotinamide adenine dinucleotide cofactors exist in an
oxidized state and in a reduced state, and these are respectively
abbreviated as NAD(P)+ and NAD(P)H in the open literature. However,
these abbreviations will be avoided here.
[0007] Most generally, the enzymes accepting reducing equivalents
from the reduced state of the nicotinamide adenine dinucleotide
cofactors are termed "oxidoreductases" and are classified by the
Nomenclature Committee of the International Union of Biochemistry
and Molecular Biology as EC 1.n.n.n. Of particular interest are the
enzymes generally termed "dehydrogenases" or "ketoreductases" in
the classes EC 1.1.n.n and EC 1.2.n.n, as well as those termed
"mono-oxygenases" in the classes EC1.13.n.n and EC.1.14.n.n. This
last group of enzymes are also termed "P450" enzymes or "CYP"
enzymes.
[0008] When performing reactions catalyzed by such enzymes using
micro-organisms growing on a carbon source such as a carbohydrate,
the reducing equivalents are generated by oxidizing a portion of
the carbohydrate to CO.sub.2, that is, some of the carbohydrate
provided to the micro-organism is sacrificially oxidized in order
to provide electrons for the micro-organism to use in various
metabolic processes. While the resulting electrons are desirable
and useful to the microorganism, the carbon atoms sacrificed by
oxidation to CO.sub.2 are lost. It is also possible to provide
carbon sources other than carbohydrates that are also sacrificed to
produce the desired reducing equivalents. It will be clear that
while such an in vivo process may perform the desired reaction
catalyzed by the oxidoreductase enzyme, recovering the desired
product from the milieu of the in vivo system, e.g. a fermentation
broth, can be difficult. It is thus advantageous to utilize an in
vitro system for performing the desired reaction.
[0009] It is possible to isolate the needed oxidoreductase enzyme
and use it to catalyze reactions in vitro. Significantly improved
chemical processes could be achieved by an in vitro system which
allows the use of the plethora of oxidoreductase enzymes in
processes resembling standard catalytic chemical processes. Such
systems avoid the issues of recovering the desired product of the
reaction from fermentation broths and can provide further
advantages by allowing the enzyme to be used under
non-physiological conditions, and the use of a variety of methods
to immobilize or otherwise contain the enzyme, and allow it to be
used for extended periods of time. Such immobilization or other
containment methods also allow simple and efficient recovery of the
desired product of the reaction from the enzyme system. A further
advantage of using isolated enzymes in a cell-free system is that
multiple enzymes can be used, allowing a series of reactions to be
performed.
[0010] Micro-organisms containing useful oxidoreductases are widely
known in nature and can be found quickly by simple screening (A.
Zaks et al, Tetrahedron (2004) 60,789-797). If a particular
oxidoreductase enzyme is required, the enzyme can be readily cloned
and over-expressed in a standard industrially useful host such as
S. cerevisiae or E. coli, and isolated by standard methods, and
used in an in vitro system.
[0011] However, the reducing equivalents must still be provided to
such in vitro systems. Analogous to in vivo systems, a sacrificial
substrate can be provided, which is oxidized. This generates the
necessary reduced state of the nicotinamide adenine dinucleotide
cofactors for providing reducing equivalents for the desired
reaction.
[0012] It will be clear that the need of a sacrificial substrate
removes at least some of the advantages of an in vivo system, since
a second product, the product of the oxidation of the sacrificial
substrate, must now be separated from the desired product. While
formate may be used sacrificial substrate, generating CO.sub.2 as
the result, this requires the use of an additional enzyme, formate
dehydrogenase, which may not be convenient.
[0013] Thus it is most desirable to provide the electrons required
for the reaction, that is, the reducing equivalents required by the
oxidoreductase enzyme, in a manner that does not require
sacrificial substrates or additional enzymes.
[0014] If electrons could be provided from an external source, that
is, an electrical current, then the need to provide a sacrificial
substrate to provide electrons would be eliminated, and
oxidoreductase enzymes could be used as conventional catalysts,
performing reactions without the need for living cells or
associated enzyme systems or processes.
[0015] This situation has been recognized by others, and a number
of attempts to deliver electrons to biological systems by
electrochemical methods have been published.
[0016] It has been reported by Park and Zeikus in J. Bacteriol.
181:2403-2410, 1999 that the compound called Neutral Red would
undergo reversible chemical oxidoreductions with the nicotinamide
adenine dinucleotide cofactor, that is, Neutral Red in its reduced
form (NR.sub.red) has a sufficiently low redox value that it will
transfer electrons to, and thus reduce, the nicotinamide adenine
dinucleotide cofactor from its oxidized state to its reduced state.
In this process, the Neutral Red becomes oxidized to the species
NR.sub.ox which is then available to accept an electron from the
cathode and thus return to the reduced form NR.sub.red, which is in
turn available to again reduce the oxidized state of the
nicotinamide adenine dinucleotide cofactor.
[0017] U.S. Pat. No. 7,250,288 B2 to Zeikus et al. discusses the
need for improving electrode efficiencies in electrochemical
bioreactor system and proposes improvements such as linking the
nicotinamide adenine dinucleotide cofactor, Neutral Red, and the
enzyme fumarate reductase to the electrode in order to improve
electron transfer characteristics. While the above may improve
electron transfer characteristics, it may also be advantageous to
improve upon the electrochemical bioreactor system design and its
use in other ways such as those described below.
[0018] The requirements for providing reducing equivalents by
electrochemical methods are disclosed in PCT publication No.
WO2014039767 A1, "Electrochemical Bioreactor Module and Methods of
Using the Same". The use of isolated enzymes in conjunction with
the electrochemical reduction of the oxidized form of nicotinamide
adenine dinucleotide cofactor to its reduced form, and
considerations to be made, are disclosed in PCT publication No.
WO2016070168 A1, "Improved Electrochemical Bioreactor Module and
Use Thereof".
[0019] When the oxidized state of the nicotinamide adenine
dinucleotide cofactor is electrochemically reduced, several
different reduced species can result. These species differ only in
the position on the nicotinamide ring at which the reduction has
formally occurred. This can be at the 2-position, the 4-position or
the 6-position on the nicotinamide ring, and these species are
termed the 1,2-dihydro-NAD, 1,4-dihydro-NAD and 1,6-dihydro-NAD
isomers respectively. (Chakraverty et al, Biochem. Biophys. Res
Comm., (1964), 15(3), 262-268; Chakreverty et al, Jour. Biol. Chem
(1969), 244(15), 4208-4217). These are shown below in Illustration
A. The carbon atoms of the nicotinamide ring are numbered in the
oxidized form, with the nitrogen atom as position 1. The desired
reduced product is the 1,4-dihydro-NAD isomer, commonly called
(3-NADH, and this is the biologically active form of the co-factor
required for most dehydrogenase, ketoreductase and P450 enzymes.
The other isomers, 1,2-dihydro-NAD and 1,6-dihydro-NAD do not have
known useful biological activity with dehydrogenase, ketoreductase
or P450 enzymes.
[0020] A fourth species, a dimer of the NAD cofactor, can also form
during the electrochemical reduction of the oxidized state of
nicotinamide adenine dinucleotide. This is termed the 4,4'-dimer
and is also shown in Illustration A. Like the 1,2-dihydro-NAD and
1,6-dihydro-NAD forms of the cofactor, the dimer has no known
useful biological activity with respect to oxidoreductase enzymes,
and cannot deliver reducing equivalents to them (Burnett et al,
Biochem. (1968), 10(7), 3328-3333; Biellmann et al, Tetrahedron
Letters (1978) 7, 683-686; Kirkor et al, Eur. J. Biochem. (2000),
267, 5014-5022).
Illustration A
##STR00001##
[0022] In a system where an initial amount of the oxidized state of
the nicotinamide adenine dinucleotide cofactor is electrochemically
reduced for the purpose of providing reducing equivalents to an
oxidoreductase enzyme, the desired, biologically active
1,4-dihydro-NAD isomer will be formed and will provide reducing
equivalents to the oxidoreductase enzyme (or enzymes) in the
system, and be oxidized back to the oxidized nicotinamide adenine
dinucleotide cofactor, ready for another cycle of electrochemical
reduction. However, the two non-biologically active isomers, the
1,2-dihydro-NAD and the 1,6-dihydro-NAD, as well as the 4,4'-dimer
initially produced by the electrochemical reduction of the oxidized
nicotinamide adenine dinucleotide cofactor will not be consumed and
will simply remain in the system. The oxidized nicotinamide adenine
dinucleotide cofactor resulting from the productive delivery of
reducing equivalents to the oxidoreductase enzyme will be again
reduced to give a mixture of the three isomers, plus the dimer, as
in the previous reduction process. In this cyclic manner, the
amount of oxidized nicotinamide adenine dinucleotide cofactor
available for electrochemical reduction decreases continually, and
after a short period of time, essentially none is left. This
problem was recognized four decades ago (Whitesides et al, Appl.
Biochem. and Biotech. (1987) 14, 147-197; Whitesides et al,
Biotechnology and Genetic Engineering Reviews, Vol. 6. September
1988).
[0023] Thus, it is useful to remove the undesired 1,2-dihydro-NAD
and the 1,6-dihydro-NAD isomers as well as the 4,4'-dimer should
they be produced during the electrochemical reduction of oxidized
nicotinamide adenine dinucleotide cofactor to the desired
1,4-dihydro-NAD species, (i.e. .beta.-NADH). Further, it is useful
to recover the 1,2-dihydro-NAD and the 1,6-dihydro-NAD isomers as
either as the desired 1,4-dihydro-NAD isomer, or as the initial
oxidized state of the nicotinamide adenine dinucleotide
cofactor.
[0024] Provided herein are methods and systems for achieving the
above objectives, Other objectives, features, and advantages of the
present disclosure will be apparent on review of the specification
and claims.
SUMMARY
[0025] The present disclosure, in one aspect, provides a system for
electrochemically generating NAD(P)H.sub.2 reducing equivalents,
the system comprising: [0026] (a) an electrochemical cell
comprising an anode contained in an anode chamber and a cathode
contained in a cathode chamber; [0027] (b) 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.sub.2, 1,2-NAD(P)H.sub.2,
1,6-NAD(P)H.sub.2, and [NAD(P)].sub.2, while optionally producing
hydrogen; [0028] (c) 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.sub.2 in the
second process stream and producing a first recovered NAD(P) and a
third process stream; and [0029] (d) at least one of a renalase
enzyme, 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.sub.2, 1,6-NAD(P)H.sub.2, and [NAD(P)].sub.2
therein to a second and optionally a third recovered NAD(P).
[0030] In some embodiments, the system includes the renalase enzyme
for converting the 1,2-NAD(P)H.sub.2 and 1,6-NAD(P)H.sub.2 into the
second recovered NAD(P).
[0031] In some embodiments, the system includes the Mung Bean
Phenol Oxidase enzyme for converting the 1,2-NAD(P)H.sub.2,
1,6-NAD(P)H.sub.2, and/or [NAD(P)].sub.2 into the second recovered
NAD(P) and/or the third recovered NAD(P).
[0032] In some embodiments, the system includes the illumination
for converting the [NAD(P)].sub.2 into NAD(P).
[0033] In some embodiments, the system includes the renalase enzyme
and the Mung Bean Phenol Oxidase enzyme. For example, the third
process stream can be contacted with the renalase enzyme resulting
in conversion of the 1,2-NAD(P)H.sub.2 and 1,6-NAD(P)H.sub.2 into
the second recovered NAD(P) and a fourth process stream, wherein
the fourth process stream can be contacted with the Mung Bean
Phenol Oxidase enzyme to convert the [NAD(P)].sub.2 therein to the
third recovered NAD(P).
[0034] In some embodiments, the system includes the renalase enzyme
and the illumination. For example, the third process stream can be
contacted with the renalase enzyme resulting in conversion of the
1,2-NAD(P)H.sub.2 and 1,6-NAD(P)H.sub.2 into the second recovered
NAD(P) and a fourth process stream, wherein the fourth process
stream can be contacted with the illumination to convert the
[NAD(P)].sub.2 therein to the third recovered NAD(P).
[0035] In some embodiments, the system includes the Mung Bean
Phenol Oxidase enzyme and the illumination. For example, the third
process stream can be contacted with the Mung Bean Phenol Oxidase
enzyme resulting in conversion of the 1,2-NAD(P)H.sub.2 and
1,6-NAD(P)H.sub.2 into the second recovered NAD(P) and a fourth
process stream, wherein the fourth process stream can be contacted
with the illumination to convert the [NAD(P)].sub.2 therein to the
third recovered NAD(P).
[0036] In some embodiments, the system can include, in one or more
of the process streams, an electron transfer mediator (ETM) capable
of transferring electrons to NAD(P). The system can also include
catalase for decomposing hydrogen peroxide produced by the renalase
enzyme, the Mung Bean Phenol Oxidase enzyme, and/or the
illumination.
[0037] Another aspect relates to a method for electrochemically
generating NAD(P)H.sub.2 reducing equivalents, the method
comprising: [0038] (a) providing an electrochemical cell comprising
an anode contained in an anode chamber and a cathode contained in a
cathode chamber; [0039] (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.sub.2, 1,2-NAD(P)H.sub.2, 1,6-NAD(P)H.sub.2,
and [NAD(P)].sub.2, while optionally producing hydrogen; [0040] (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.sub.2 in the
second process stream and producing a first recovered NAD(P) and a
third process stream; and [0041] (d) contacting the third process
stream with at least one of a renalase enzyme, 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.sub.2, 1,6-NAD(P)H.sub.2, and [NAD(P)].sub.2 therein to
a second recovered NAD(P) and optionally a third recovered
NAD(P).
[0042] The method in some embodiments can further include
contacting the third process stream with the renalase enzyme for
converting the 1,2-NAD(P)H.sub.2 and 1,6-NAD(P)H.sub.2 into the
second recovered NAD(P).
[0043] In some embodiments, the method can further include
contacting the third process stream with the Mung Bean Phenol
Oxidase enzyme for converting the 1,2-NAD(P)H.sub.2,
1,6-NAD(P)H.sub.2, and/or [NAD(P)].sub.2 into the second recovered
NAD(P) and/or the third recovered NAD(P).
[0044] In some embodiments, the method can further include
contacting the third process stream with the illumination for
converting the [NAD(P)].sub.2 into NAD(P).
[0045] In some embodiments, the method can further include
contacting the third process stream with the renalase enzyme
resulting in conversion of the 1,2-NAD(P)H.sub.2 and
1,6-NAD(P)H.sub.2 therein into the second recovered NAD(P) and a
fourth process stream, and further comprising contacting the fourth
process stream with the Mung Bean Phenol Oxidase enzyme to convert
the [NAD(P)].sub.2 therein to the third recovered NAD(P).
[0046] In some embodiments, the method can further include
contacting the third process stream with the renalase enzyme
resulting in conversion of the 1,2-NAD(P)H.sub.2 and
1,6-NAD(P)H.sub.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)].sub.2
therein to the third recovered NAD(P).
[0047] In some embodiments, the method can further include
contacting the third process stream with the Mung Bean Phenol
Oxidase enzyme resulting in conversion of the 1,2-NAD(P)H.sub.2 and
1,6-NAD(P)H.sub.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)].sub.2
therein to the third recovered NAD(P).
[0048] In some embodiments, the method can further include
providing, in one or more of the process streams, an electron
transfer mediator (ETM) capable of transferring electrons to
NAD(P).
[0049] In some embodiments, the method can further include
providing catalase for decomposing hydrogen peroxide produced by
the renalase enzyme, the Mung Bean Phenol Oxidase enzyme, and/or
the illumination.
[0050] In some embodiments, the method can further include
returning the third process stream to the cathode chamber.
[0051] In any of the systems and methods disclosed herein, the
renalase enzyme and/or Mung Bean Phenol Oxidase enzyme may be
recombinantly expressed from microorganisms such as Escherichia
coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces
cerevisiae, Pichia pastoris, and Bacillus megaterium.
[0052] The renalase enzyme in certain embodiments can be a mutant
form (naturally occurring or genetically engineered) having a lower
oxidation activity for 1,4-NAD(P)H.sub.2 than the wild type
renalase enzyme, and/or a higher oxidation activity for
1,2-NAD(P)H.sub.2 and 1,6-NAD(P)H.sub.2 than the wild type renalase
enzyme. In some examples, the mutant form can be recombinantly
expressed from microorganisms such as Escherichia coli, Bacillus
subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae,
Pichia pastoris, and Bacillus megaterium.
[0053] The Mung Bean Phenol Oxidase enzyme can be a mutant form
(naturally occurring or genetically engineered) having a lower
oxidation activity for 1,4-NAD(P)H.sub.2 than the wild type Mung
Bean Phenol Oxidase enzyme, and/or a higher oxidation activity for
1,2-NAD(P)H.sub.2, 1,6-NAD(P)H.sub.2 and/or [NAD(P)].sub.2 than the
wild type Mung Bean Phenol Oxidase enzyme. In some embodiments, the
mutant form is recombinantly expressed from microorganisms such as
Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum,
Saccharomyces cerevisiae, Pichia pastoris, and Bacillus
megaterium.
[0054] Also provided herein is a process for providing
electrochemically generated reducing power via the reduction of an
oxidized form of phosphorylated or non-phosphorylated forms of
nicotinamide adenine dinucleotide cofactor to a redox enzyme system
for the purpose of catalyzing a transformation of a substrate
molecule.
[0055] The process can include steps for preventing the loss of
cofactor material to reduced forms that are not used by the redox
enzyme system for the purpose of catalyzing a transformation of a
substrate molecule.
[0056] The process can include the electrochemical reduction of the
oxidized state of nicotinamide adenine dinucleotide cofactor to
give a mixture of the desired 1,4-dihydro-NAD species with the
undesired 1,2-dihydro-NAD and 1,6-dihydro-NAD species, and the
undesired 4,4'-dimer. The desired 1,4-dihydro-NAD species is
converted back to the oxidized form of the nicotinamide adenine
dinucleotide cofactor in the course of providing reducing
equivalents to the oxidoreductase enzyme which is present to
catalyze the transformation of the substrate molecule. The
remaining mixture of the 1,2-dihydro-NAD and 1,6-dihydro-NAD
species, the 4,4'-dimer, together with the oxidized state of the
nicotinamide adenine dinucleotide cofactor is acted upon by the
renalase enzyme. This enzyme oxidizes the 1,2-dihydro-NAD and
1,6-dihydro-NAD species back to the oxidized state of the
nicotinamide adenine dinucleotide cofactor in the presence of
oxygen, producing hydrogen peroxide in the process. The Mung Bean
enzyme oxidizes the 4,4'-dimer back to two molecules of the
oxidized state of the nicotinamide adenine dinucleotide cofactor,
also by using oxygen and generating hydrogen peroxide. Thus all
species originally produced by the electrochemical reduction of the
oxidized form of the nicotinamide adenine dinucleotide cofactor,
both desired and undesired, are returned to the original oxidized
state of the nicotinamide adenine dinucleotide cofactor and no
cofactor material is lost. The 4,4'-dimer is photo-active, and can
be returned to the oxidized state of the nicotinamide adenine
dinucleotide cofactor species by irradiation with the appropriate
wavelength; this may be performed in place of, or in conjunction
with, the Mung Bean oxidase.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] FIG. 1 illustrates, in a schematic, the utilization of
renalase, Mung Bean Phenol Oxidase, and optionally illumination to
recover and recycle cofactor material from the mixture of the
desired 1,4-dihydro-NAD species plus the undesired 1,2-dihydro-NAD
and 1,6-dihydro-NAD species and the undesired 4,4'-dimer which are
formed via the electrochemical reduction of the oxidized state of
nicotinamide adenine dinucleotide cofactor. In FIG. 1, the
1,2-dihydro-NAD, 1,4-dihydro-NAD and the 1,6-dihydro-NAD species of
the nictotinamide adenine dinucleotide cofactor are named
1,2-NAD(P)H.sub.2, 1,4-NAD(P)H.sub.2 and 1,6-NAD(P)H.sub.2
respectively, while the 4,4'-dimer is named [NAD(P)].sub.2 and the
oxidized form is named NAD(P).
DETAILED DESCRIPTION
[0058] The current literature most commonly uses the descriptor
"NAD(P)+" to indicate the oxidized state of the phosphorylated and
non-phosphorylated forms of the nicotinamide adenine dinucleotide,
and "NAD(P)H" to indicate the reduced state. The reduced state of
the phosphorylated and non-phosphorylated forms of the nicotinamide
adenine dinucleotide are produced by adding two electrons and two
protons to the oxidized state. This common nomenclature is
misleading, as plain reading of the common descriptors "NAD(P)+"
and "NAD(P)H" show the oxidized state of the phosphorylated and
non-phosphorylated forms of the nicotinamide adenine dinucleotide
to be a positively charged species "NAD(P)+", while the reduced
state of the phosphorylated and non-phosphorylated forms of the
nicotinamide adenine dinucleotide, "NAD(P)H", is shown to have only
one hydrogen added relative to the oxidized state. Neither of these
representations is true. The oxidized state of the phosphorylated
and non-phosphorylated forms of the nicotinamide adenine
dinucleotide is a neutral molecule in which the nitrogen of the
nicotinamide ring bears a formal positive charge with is balanced
by a negative charge one of the deprotonated phosphate linkages,
thus forming an internal salt or zwitterion. The reduced state of
the phosphorylated and non-phosphorylated forms of the nicotinamide
adenine dinucleotide has formally accepted a hydrogen molecule,
H.sub.2, relative to the oxidized state, and this achieved by two
one-electron transfers balanced by two protons. This is shown
formally by the following balanced chemical reaction:
NAD(P)+2H.sup.++2e.sup.-.fwdarw.NAD(P)H.sub.2
[0059] The published molecular weights of the oxidized and reduced
states of the non-phosphorylated form of the nicotinamide adenine
dinucleotide indicate the reality of this reaction clearly, the
molecular weights being 663.43 Da and 665.44 Da respectively and
the difference being the molecular weight of a single hydrogen
molecule, H2.
[0060] In the present disclosure, the descriptor "NAD(P)" indicates
the oxidized state of the phosphorylated and non-phosphorylated
forms of the nicotinamide adenine dinucleotide, while the
descriptor "NAD(P)H.sub.2" indicates the reduced state of the
phosphorylated and non-phosphorylated forms of the nicotinamide
adenine dinucleotide. Further, the descriptors "1,2-NAD(P)H.sub.2",
"1,4-NAD(P)H.sub.2" and "1,6-NAD(P)H.sub.2" are used to indicate
the three stereoisomers of the nicotinamide ring that can occur
upon reduction of NAD(P), in which a hydrogen molecule has been
formally added across the 1,2-, 1,4- and 1,6-positions of the
nicotinamide ring. It will be clear to those skilled in the art
that 1,4-NAD(P)H.sub.2 is commonly called .beta.-NADH, and is the
stereoisomer of the reduced state of the phosphorylated and
non-phosphorylated forms of nicotinamide adenine dinucleotide which
is active with oxidoreductase and P450 enzymes.
[0061] In the present disclosure, the 4,4'-dimer that is also known
to form as a consequence of a one-electron transfer to NAD(P) is
indicated by the descriptor "[NAD(P)].sub.2".
[0062] The present disclosure, in some embodiments, is directed to
an improved version and improved use of the "Electrochemical
Bioreactor Module" (EBM) previously described in PCT Patent
Application Publication Nos. WO2014039767 A1 and WO2016070168 A1,
which are incorporated herein by reference in its entirety.
[0063] An EBM can include one or more of the following components:
[0064] a. an anode contained in an anode chamber and a cathode
contained in a cathode chamber; [0065] b. deionized water in the
anode chamber in contact with the anode; [0066] c. a proton
permeable membrane that separates the anode and cathode chambers;
[0067] d. a liquid phase in the cathode chamber continuously in
contact with the cathode, said liquid phase optionally comprising
an electron transfer mediator (ETM) capable of transferring
reducing equivalents to a redox enzyme system, said redox enzyme
system comprising a redox enzyme and a cofactor; [0068] e. a
process stream containing a substrate to be transformed via
catalysis by the redox enzyme system into a desired product; [0069]
f. a membrane located between the cathode and the process stream,
said membrane capable of preventing the optional ETM and the redox
enzyme system from significantly entering into the process stream;
and [0070] g. an external power source providing a voltage between
the anode and the cathode.
[0071] In certain embodiments, an improved EBM or process using the
EBM of the present disclosure can include the enzyme renalase, the
Mung Bean Phenol Oxidase, and illumination to recover the undesired
forms of cofactor that result from electrochemical reduction of
NAD(P).
[0072] The enzyme renalase (Moran et al, J. Am. Chem. Soc. (2013),
135, 13980-13987; Moran, G. R., Biochimica et Biophysica Acta 1864
(2016) 177-186), is capable of oxidizing the 1,2-dihydro-NAD and
1,6-dihydro-NAD species back to the oxidized state of the
nicotinamide adenine dinucleotide cofactor.
[0073] Another enzyme isolated from Mung Bean and currently termed
"Mung Bean Phenol Oxidase" (Fricks et al, Arch. Biochem. Biophys,
(1973)169, 837-841). This enzyme oxidizes the 4,4'-dimer,
[NAD(P)].sub.2, that can be formed under electrochemically reducing
conditions back to the oxidized state of nicotinamide adenine
dinucleotide. This enzyme has also been reported as having the same
activity as renalase and being able to oxidize the
1,2-NAD(P)H.sub.2 and 1,6-NAD(P)H.sub.2 species back to the
oxidized state of nicotinamide adenine dinucleotide as well (Kono
et al, Bull. Agr. Chem. Soc. Japan, (1958) 22(6) 404-410).
[0074] It has also recently been shown that illumination of
electrochemically prepared 4,4'-dimer at about 254 nm or at
wavelengths exceeding 320 nm leads to regeneration of the oxidized
state of nicotinamide adenine dinucleotide. (Czochralska et al,
(1980) Arch. Biochem. Biophys. 199, 497; Czochralska et al, (1990)
Photochem. Photobiol. 51, 401-410).
[0075] Thus it is useful to incorporate the renalase and Mung Bean
Phenol Oxidase enzymes, and the illumination of the [NAD(P)].sub.2
dimer of nicotinamide adenine dinucleotide in a process for the
recovery of cofactor material, and for the prevention of the loss
of cofactor material to forms which are not used by oxidoreductase
enzymes such as dehydrogenases, ketoreductases or P450 enzymes.
[0076] In one embodiment, the present disclosure provides a process
that comprises: [0077] a) a process stream containing NAD(P) which
is passed through the cathode chamber of an electrochemical cell
such that the desired, biologically active 1,4-NAD(P)H.sub.2 is
produced, together with the undesired 1,2-NAD(P)H.sub.2,
1,6-NAD(P)H.sub.2 and [NAD(P)].sub.2 species; [0078] b) providing a
substrate molecule that is to be transformed via the catalytic
reaction of an oxidoreductase enzyme, such as a dehydrogenase or
ketoreductase, or a P450 enzyme, to produce a desired product
molecule; [0079] c) contacting the process stream containing both
the desired and undesired cofactor species with an oxidoreductase,
such as a dehydrogenase or ketoreductase, or a P450 enzyme, capable
of using the reducing equivalents presented by the
1,4-NAD(P)H.sub.2 to perform a desired reaction in which a
substrate molecule is transformed to a desired product molecule,
while the 1,4-NAD(P)H.sub.2 is concomitantly transformed back to
NAD(P); [0080] d) recovering the cofactor material from the
undesired, leaving the undesired 1,2-NAD(P)H.sub.2 and
1,6-NAD(P)H.sub.2 cofactor species present in the process stream by
contacting the process stream with the renalase enzyme in the
presence of oxygen for a sufficient time that the renalase enzyme
can catalyze the oxidation of the undesired 1,2-NAD(P)H.sub.2 and
1,6-NAD(P)H.sub.2 species that may be present in the process stream
to the oxidized form of the cofactor, NAD(P); [0081] e) recovering
the cofactor material from the undesired and [NAD(P)].sub.2 species
present in the process stream by contacting the process stream with
Mung Bean Phenol Oxidase in the presence of oxygen for a sufficient
time that the Mung Bean Phenol Oxidase enzyme can catalyze the
oxidation and any [NAD(P)].sub.2 present in the process stream to
the oxidized form of the cofactor NAD(P); [0082] f) returning the
process stream now containing all of the original nicotinamide
adenine dinucleotide as the oxidized form NAD(P) to the cathode
chamber of the electrochemical cell for reduction.
[0083] In one embodiment, a process stream containing NAD(P) is
passed through the cathode chamber of the electrochemical cell such
that 1,2-NAD(P)H.sub.2, 1,4-NAD(P)H.sub.2, 1,6-NAD(P)H.sub.2 and
the 4,4'-dimer, [NAD(P)].sub.2 are produced. The process stream
containing the mixture of NAD(P)H.sub.2 species is then contacted
with an oxidoreductase enzyme (e.g. a dehydrogenase, ketoreductase,
or P450 enzyme) capable of using the reducing equivalents from the
1,4-NAD(P)H.sub.2 to perform a desired reaction in which a
substrate molecule is transformed to a desired product molecule,
while the 1,4-NAD(P)H.sub.2 is concomitantly transformed back to
NAD(P). The enzyme utilizing the 1,4-NAD(P)H.sub.2 may be present
in the process stream, immobilized on beads or other suitable
material and held in a packed column, or contained by a membrane as
described in PCT Patent Application Publication No. WO2016070168
A1, which is incorporated herein by reference in its entirety.
[0084] The process stream, after contacting the oxidoreductase
enzyme and after the 1,4-NAD(P)H.sub.2 has been transformed back to
NAD(P), is then contacted with the renalase enzyme. In the presence
of oxygen, the renalase enzyme oxidizes the undesired
1,2-NAD(P)H.sub.2 and 1,6-NAD(P)H.sub.2 species that may be present
back to NAD(P) with the concomitant production of hydrogen
peroxide. An enzyme such as catalase, or a suitable inorganic
catalyst (e.g. MnO.sub.2), may be present to decompose the hydrogen
peroxide and thus prevent deleterious effects by the hydrogen
peroxide.
[0085] The renalase enzyme may be immobilized on beads or a
suitable support, immobilized on, or contained by, a membrane or
kept from freely circulating in the process stream by other
standard methods known to those skilled in the art of enzymatic
reactions. As the renalase enzyme has some activity towards the
desirable 1,4-NAD(P)H.sub.2 species and will oxidize this back to
NAD(P), it is better if the renalase enzyme is not freely
circulating in the process stream, and that the process stream is
contacted with the renalase after the 1,4-NAD(P)H.sub.2 present in
the process stream has been consumed by the oxidoreductase enzyme,
and has been used to perform a useful reaction on a substrate.
[0086] The process stream is then brought into contact with the
Mung Bean Phenol oxidase in the presence of oxygen and any
undesired 4,4'-dimer, [NAD(P)].sub.2, present is thus oxidized back
to NAD(P) with the concomitant production of hydrogen peroxide. The
Mung Bean Phenol Oxidase may be immobilized on beads or a suitable
support, immobilized on, or contained by, a membrane or kept from
freely circulating in the process stream by other standard methods
known to those skilled in the art of enzymatic reactions. As the
Mung Bean Phenol Oxidase enzyme has some activity towards the
desirable 1,4-NAD(P)H.sub.2 species and will oxidize this back to
NAD(P), it is better if the Mung Bean Phenol Oxidase enzyme is not
freely circulating in the process stream, and that the process
stream is contacted with the Mung Bean Phenol Oxidase after the
desirable 1,4-NAD(P)H.sub.2 present in the process stream has been
consumed by the oxidoreductase enzyme.
[0087] In another embodiment, the process stream, after contacting
the oxidoreductase enzyme, may be contacted first with the Mung
Bean Phenol Oxidase enzyme for oxidation of the undesired
[NAD(P)].sub.2 back to NAD(P), and then subsequently with the
renalase enzyme for oxidation of the undesired 1,2-NAD(P)H.sub.2
and 1,6-NAD(P)H.sub.2 species back to NAD(P).
[0088] In another embodiment, the process stream, after contacting
the oxidoreductase enzyme may be contacted with the renalase enzyme
and subsequently irradiated with light at 254 nm or at wavelengths
exceeding 320 nm for decomposing the undesired [NAD(P)].sub.2 dimer
back to NAD(P).
[0089] In another embodiment, the process stream, after contacting
the oxidoreductase enzyme, may be contacted not only with the Mung
Bean Phenol Oxidase enzyme for oxidation of the undesired
[NAD(P)].sub.2 dimer back to NAD(P), and also for oxidation of the
undesired 1,2-NAD(P)H.sub.2 and 1,6-NAD(P)H.sub.2 species to NAD(P)
as the Mung Bean Phenol Oxidase enzyme is known to have activity
for the oxidation of the undesired 1,2-NAD(P)H.sub.2 and
1,6-NAD(P)H.sub.2 species back to NAD(P).
[0090] It will be clear to those skilled in the art that the order
of the steps in the process for recovering cofactor material as
NAD(P) from the undesired 1,2-NAD(P)H.sub.2, 1,6-NAD(P)H.sub.2, and
[NAD(P)].sub.2 species by the above described steps of contacting
the process stream with renalase, Mung Bean Phenol Oxidase or
illuminating it with light at 254 nm or at wavelengths exceeding
320 nm may be done in any order or combination.
[0091] It will be clear to those skilled in the art that the Mung
Bean Phenol Oxidase and the renalase enzymes may be engineered by
known methods to allow greater stability and therefore lifetime in
the process, or to allow greater activity, or to allow increased
ease and efficiency of immobilization, or to allow the more
efficient expression and isolation of these enzymes.
[0092] It will be equally clear to those skilled in the art of
enzymatic reactions that the Mung Bean Phenol Oxidase enzyme may be
engineered to allow increased activity for the oxidation of the
undesired 1,2-NAD(P)H.sub.2 and 1,6-NAD(P)H.sub.2, species to
NAD(P), in addition to its activity for oxidizing the undesired
[NAD(P)].sub.2 dimer to NAD(P).
[0093] In certain embodiments, the renalase enzyme and the Mung
Bean Phenol Oxidase enzyme may be engineered to have insignificant
(e.g., lower than wide-type) activity on the desirable
1,4-NAD(P)H.sub.2 species, but useful (e.g., higher than wide-type)
activity on the undesired 1,2-NAD(P)H.sub.2, 1,6-NAD(P)H.sub.2, and
[NAD(P)].sub.2 species resulting from the electrochemical reduction
of NAD(P). These enzymes may then be circulated with the process
stream, or the enzymes may be immobilized or otherwise contained
and contacted with the process stream in any order that is
convenient, including prior to contacting the process stream with
the oxidoreductase or P450 enzyme which is acting upon the provided
substrate.
EXAMPLES
Example I
[0094] This example describes an exemplary process in which the
undesired 1,2-NAD(P)H.sub.2 and 1,6-NAD(P)H.sub.2 isomers are
re-oxidized to NAD(P) by renalase, and the undesired [NAD(P)].sub.2
dimer is oxidatively cleaved to NAD(P) by Mung Bean Phenol
Oxidase.
[0095] To demonstrate the process described in the present
disclosure, Mung Bean Phenol Oxidase is extracted from Mung Bean
seedlings as described by Burnett and Underwood (Biochem. 7(10),
3328-3333, 1968). The gene human renalase isoform 1 is cloned onto
a plasmid for expression in E. coli, including codon optimization.
The sequence of the renalase is revealed by Pandini et al. (Protein
Expression and Purification 72 (2010) 244-253). The enzyme is
expressed as an inclusion body, the inclusion bodies collected and
refolded as described by Padini et al (Protein Expression and
Purification 72 (2010) 244-253). The renalase enzyme (MW=35 KDa) is
held in a container in which an inlet and an outlet are provided,
with a permeable membrane between the inlet and the outlet that is
of a sufficiently low molecular weight cutoff to prevent the
renalase from passing through it, but also having a sufficiently
high molecular weight cutoff that all species of the nicotinamide
adenine dinucleotide present in the process stream can pass through
it. A membrane with a molecular weight cutoff of 10 KDa is used. In
a similar manner, the Mung Bean Phenol Oxidase enzyme is held in a
separate container with an inlet and an outlet and that also
includes a membrane between the inlet and the outlet that is
permeable, having a sufficiently low molecular weight cutoff that
the Mung Bean Phenol Oxidase cannot pass through it, but a
sufficiently high molecular weight cutoff that all species of the
nicotinamide adenine dinucleotide present in the process stream can
pass through it. A membrane with a molecular weight cutoff of 10
KDa is used. To allow the use of an oxidoreductase enzyme that
demonstrates the presence of useful amounts of the desired
1,4-NAD(P)H.sub.2 cofactor, the membrane system described in
published PCT Patent Application WO2016070168 A1 published 6 May
2016, and which is also described in U.S. Pat. Nos. 4,705,704 and
5,077,217 for the purpose of containing an enzyme. The enzyme Horse
Liver Alcohol Dehydrogenase is used, with cyclohexanone or
benzaldehyde used as the substrate for the alcohol dehydrogenase
enzyme. A process stream containing 1 to 2 mM of NAD(P) is prepared
and buffered to a pH suitable for all three enzymes; a pH of 7.5 is
used. The process stream containing the NAD(P) is pumped through
the cathode chamber of the electrochemical cell, which is energized
at approximately 2 volts, and the NAD(P) cofactor is reduced to
give the desired 1,4-NAD(P)H.sub.2 form of the cofactor, and also
the undesired 1,2-NAD(P)H.sub.2, 1,6-NAD(P)H.sub.2 and 4,4'-dimer,
[NAD(P)].sub.2 forms of the cofactor. Exiting the cathode chamber,
the NAD(P) process stream goes through the membrane system
containing the alcohol dehydrogenase, where the desired
1,4-NAD(P)H.sub.2 form of the cofactor provides reducing
equivalents to the alcohol dehydrogenase, the cyclohexanone or
benzaldehyde substrate is reduced to cyclohexanol or benzyl alcohol
product respectively, thus consuming the 1,4-NAD(P)H.sub.2 form of
the cofactor and producing the oxidized form NAD(P). The process
stream then proceeds to the inlet of the container holding the
renalase enzyme. The process stream mixes with the renalase enzyme
and the renalase enzyme oxidizes the undesired 1,2-NAD(P)H.sub.2
and 1,6-NAD(P)H.sub.2 forms of the cofactor to NAD(P). The process
stream then passes through the membrane but the renalase enzyme is
held in its container by the membrane. After passing through the
membrane the process stream proceeds to the outlet of the container
holding the renalase enzyme. From here the process stream passes to
the inlet of the container holding the Mung Bean Phenol Oxidase
enzyme. The process stream mixes with the Mung Bean Phenol Oxidase
enzyme and the enzyme oxidizes the undesired 4,4'dimer,
[NAD(P)].sub.2 form of the cofactor to NAD(P). The process stream
then passes through the membrane but the Mung Bean Phenol Oxidase
enzyme is held in its container by the membrane. The process stream
exits through the outlet of the container holding the Mung Bean
Phenol Oxidase enzyme, and now contains only NAD(P) as the other
forms of the cofactor have all been oxidized by the alcohol
dehydrogenase, the renalase and the Mung Bean Phenol Oxidase
enzymes. The process stream is returned to the pump, and sent into
the cathode chamber to repeat the cycle.
Example II
[0096] This example describes an exemplary process in which the
undesired 1,2-NAD(P)H.sub.2 and 1,6-NAD(P)H.sub.2 isomers are
re-oxidized to NAD(P) by renalase, and the undesired [NAD(P)].sub.2
dimer is oxidatively cleaved to NAD(P) by illumination with UV
light at 254 nm.
[0097] The process described in Example I is used, excepting the
container holding the Mung Bean Phenol Oxidase is replaced by a
quartz tube through which the process stream can flow unimpeded by
any membrane. Outside the quartz tube an ultraviolet lamp is
present, and illuminates the tube with light at 254 nm which passes
through the quartz and photolytically cleaves the 4,4-dimer of the
cofactor, [NAD(P)].sub.2, to NAD(P). Upon exiting the quartz tube,
the process stream is returned to the pump, and sent into the
cathode chamber to repeat the cycle.
EQUIVALENTS
[0098] The present disclosure provides among other things novel
methods and devices for providing reducing equivalents to
biological systems. While specific embodiments of the subject
disclosure have been discussed, the above specification is
illustrative and not restrictive. Many variations of the disclosure
will become apparent to those skilled in the art upon review of
this specification. The full scope of the disclosure should be
determined by reference to the claims, along with their full scope
of equivalents, and the specification, along with such
variations.
INCORPORATION BY REFERENCE
[0099] All publications, patents and patent applications cited
above are incorporated by reference herein in their entirety for
all purposes to the same extent as if each individual publication
or patent application were specifically indicated to be so
incorporated by reference.
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