U.S. patent application number 17/189143 was filed with the patent office on 2022-08-25 for nadph-regeneration system based on monomeric isocitrate dehydrogenase and use thereof.
The applicant listed for this patent is INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY. Invention is credited to Sang-Oh AHN, Dae Eun CHEONG, Hye-Ji CHOI, Geun Joong KIM, Hun-Dong LEE, Su-Kyoung YOO, Chul-Ho YUN.
Application Number | 20220267742 17/189143 |
Document ID | / |
Family ID | |
Filed Date | 2022-08-25 |
United States Patent
Application |
20220267742 |
Kind Code |
A1 |
KIM; Geun Joong ; et
al. |
August 25, 2022 |
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.
Inventors: |
KIM; Geun Joong; (Gwangju,
KR) ; LEE; Hun-Dong; (Gwangju, KR) ; YOO;
Su-Kyoung; (Gwangju, KR) ; CHEONG; Dae Eun;
(Gwangju, KR) ; YUN; Chul-Ho; (Sejong-si, KR)
; CHOI; Hye-Ji; (Jeollanam-do, KR) ; AHN;
Sang-Oh; (Gwangju, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY |
Gwangju |
|
KR |
|
|
Appl. No.: |
17/189143 |
Filed: |
March 1, 2021 |
International
Class: |
C12N 9/04 20060101
C12N009/04; C12P 21/02 20060101 C12P021/02; C12P 17/16 20060101
C12P017/16 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 22, 2021 |
KR |
10-2021-0023232 |
Claims
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.degree. 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.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] The present application claims priority to Korean Patent
Application No. 10-2021-0023232 filed on Feb. 22, 2021. The entire
contents of the above-listed application is hereby incorporated by
reference for all purposes.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been filed electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Feb. 25, 2021, is named Sequence_Listing_PLS21306.txt and is
20,480 bytes in size.
TECHNICAL FIELD
[0003] The following disclosure relates to an NADPH-regeneration
system based on monomeric isocitrate dehydrogenase (IDH) and a use
thereof, and in particular, to an NADPH-regeneration system based
on isocitrate dehydrogenase in a monomeric form, for the
regeneration of NADPH, which is a cofactor used in an enzymatic
reaction of an NADPH-dependent enzyme, and an application method
thereof.
BACKGROUND
[0004] NADPH-dependent enzymes, for example, cytochrome P450, are a
group of enzymes found in most species that function as
monooxygenases.
[0005] Cytochrome P450 enzymes can mediate an oxidation reaction of
a wide range of substrates, and thus, can be applied to various
biosynthetic and/or degradative pathways. Cytochrome P450 enzymes
not only produce high value-added biological compounds, thereby
having great potential in a field of drug metabolism, but also has
a very high utility value as an enzyme in industrial processes. In
addition, cytochrome P450 enzymes perform an important oxidation
reaction in metabolic processes of drugs or hormones, etc. in vivo,
and are responsible for metabolism of greater than or equal to 75%
of drugs administered to humans. Furthermore, cytochrome P450
enzymes are able to control functionality of various substrates by
participating in hydroxylation reactions of the various substrates,
and thus, may be widely used in the discovery of optimal
metabolites or high value-added processes.
[0006] Since cytochrome P450s, especially in eukaryotes, exist
typically as a membrane protein, expression and purification in
foreign hosts are difficult. In addition, since most cytochrome
p450s need to receive electrons from reductase, they need
nicotinamide adenine dinucleotide phosphate reduced form (NADPH) as
an electron transfer material. However, NADPH, which is a cofactor
used in the enzymatic reaction, is highly unstable and very
expensive, and there are thus may restrictions on the industrial
use of cytochrome P450s.
[0007] Meanwhile, cytochrome P450 BM3, which is a cytochrome P450
from Bacillus megaterium, unlike other cytochrome P450s, has the
advantage that an oxygenase domain involved in enzyme activity and
a reductase domain that provides a reducing power required for
enzyme activity through oxidation of the cofactor are not only
expressed in a form of a monocistronic protein, but also expressed
in cytoplasm, such that expression and purification are relatively
easy. In addition, although cytochrome P450 BM3 is a multidomain
protein of about 119 kDa in size, it is characterized in that a
soluble overexpression in Escherichia coli is possible. Thus, in
order to utilize cytochrome P450 BM3 as an enzyme for the
industrial processes, a lot of studies for improving substrate
specificity and enzyme activity, etc., are being actively
conducted.
[0008] However, since NADPH, which is the cofactor of cytochrome
P450, is unstable and very expensive, as described above, there is
still a limitation in industrial use. In order to solve the above
problems, various NADPH-regeneration systems have been developed.
Specifically, the NADPH-regeneration system may use
electrochemical, optical, and enzymatic methods, of which an
enzymatic method is mainly used.
[0009] Examples of enzymes used in an enzyme-mediated
NADPH-regeneration system include enzymes that produce NADPH in
metabolic pathways in-vivo such as alcohol dehydrogenase (ADH),
formate dehydrogenase (FDH), glucose dehydrogenase (GDH), and
glucose 6-phosphate dehydrogenase (G6PDH).
[0010] However, all of the enzymes currently used in the
NADPH-regeneration system are in a multimeric form, and have
difficulties with soluble expression in E. coli. In particular,
structural characteristics of the multimeric form have a negative
influence on the enzymatic reaction, soluble expression, and upon
expression of fusion protein of the NADPH-dependent enzyme such as
cytochrome P450 and an enzyme used in the NADPH-regeneration
system, and thus, act as a serious limiting factor in the case of
producing a high value-added substance through an enzymatic process
or a whole-cell reaction.
[0011] Thus, there is a need in the art for an NADPH-regeneration
system capable of stably supplying NADPH to the enzymatic reaction
in which the NADPH-dependent enzyme acts as a catalyst. Thus, in
order to solve the problem of an enzyme used in the
NADPH-regeneration system of the prior art, there is an urgent need
to find an enzyme that may be used in the NADPH-regeneration system
in a monomeric form, is capable of the soluble overexpression in E.
coli, and is capable of a fusion expression with the
NADPH-dependent enzyme.
RELATED ART DOCUMENT
Patent Document
[0012] Korean Patent Publication No. 2022137
[0013] Korean Patent Publication No. 1152878
Non-Patent Document
[0014] Xiaodong Wang et al., Chem., 2 (5) pp. 621-654 (2017)
SUMMARY
[0015] In order to solve the problem of expensive cofactor and
cofactor regeneration, which determines productivity or price of a
product, in a process of developing a biocatalytic process with
useful activity dependent on a cofactor using enzymes or
whole-cells, an embodiment of the present invention is directed to
providing an isocitrate dehydrogenase recombinant protein from
Corynebacterium glutamicum and an isocitrate dehydrogenase
recombinant protein from Azotobacter vinelandii that are solubly
expressed in E. coli in a monomeric form and may be used in an
NADPH-regeneration system, and a novel NADPH-regeneration system
using the same.
[0016] In one general aspect, there is provided a recombinant
expression vector for NADPH regeneration, including a
polynucleotide encoding monomeric isocitrate dehydrogenase from
Corynebacterium glutamicum or Azotobacter vinelandii. The
recombinant expression vector may further include a polynucleotide
encoding an NADPH-dependent enzyme.
[0017] The isocitrate dehydrogenase from Corynebacterium glutamicum
may consist of an amino acid sequence of SEQ ID NO: 1 and the
isocitrate dehydrogenase from Azotobacter vinelandii may consist of
an amino acid sequence of SEQ ID NO: 2.
[0018] The NADPH-dependent enzyme may be any one or two or more
selected from the group consisting of dehydrogenase, reductase,
oxidoreductase, transhydrogenase, peroxidase, oxygenase,
monooxygenase, flavodoxin, and dehalogenase. The NADPH-dependent
enzyme may be recombined and fused to the amino acid sequence of
SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2, or may be
linked by addition of a chemical linker.
[0019] The chemical linker may be 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).
[0020] The present invention provides a transformant transformed
with the recombinant expression vector for NADPH regeneration. The
transformant may be Escherichia coli.
[0021] In another general aspect, there is provided a method for
producing a recombinant protein including: producing the
recombinant expression vector for NADPH regeneration as described
above; 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
overexpressed recombinant protein. The culturing may be performed
at 28 to 32.degree. C.
[0022] The present invention provides a recombinant protein
produced by the method for producing a recombinant protein. The
recombinant protein may be solubly expressed in a monomeric form,
and may be used together with the NADPH-dependent enzyme. The
NADPH-dependent enzyme may be any one or two or more selected from
the group consisting of dehydrogenase, reductase, oxidoreductase,
transhydrogenase, peroxidase, oxygenase, monooxygenase, flavodoxin,
and dehalogenase. The NADPH-dependent enzyme may be recombined and
fused to the amino acid sequence of SEQ ID NO: 1 or the amino acid
sequence of SEQ ID NO: 2, or may be linked by addition of a
chemical linker.
[0023] The chemical linker may be selected from the group
consisting of BS(PEG)5, BS(PEG)9, BS2G-d0, BS2G-d4, DSBU, DFDNB,
DMP, DMS, DSG, DSP, DSS, DSSO, DST, DTBP, EGS, TSAT, and EDC.
[0024] The present invention provides a composition for NADPH
regeneration, comprising the recombinant protein.
[0025] The present invention provides a method for regenerating
NADPH including: regenerating NADPH by adding the recombinant
protein to an NADPH-dependent enzyme reaction system.
[0026] The present invention provides a kit for NADPH regeneration,
including the recombinant protein.
[0027] In another general aspect, there is provided a composition
for substrate hydroxylation, comprising the recombinant protein;
and a cytochrome P450 protein. The substrate may be selected from
the group consisting of omeprazole, omeprazole sulfide,
ethoxycoumarin, and nitrophenol.
[0028] The present invention provides an in vitro toxicity test
method including: treating the composition for substrate
hydroxylation.
[0029] The present invention provides a method for converting a
target substrate by co-expressing or fusion expressing the
recombinant isocitrate dehydrogenase protein and the
NADPH-dependent enzyme.
[0030] Other features and aspects will be apparent from the
following detailed description, the drawings, and the claims.
BRIEF DESCRIPTION OF THE FIGURES
[0031] FIG. 1 is a schematic diagram illustrating an
NADPH-regeneration system based on isocitrate dehydrogenase in a
monomeric form according to the present invention;
[0032] FIGS. 2A and 2B illustrate schematic diagrams of the
recombinant vector for expressing a recombinant protein of
isocitrate dehydrogenase from Corynebacterium glutamicum
(hereinafter may be referred to as CgIDH) and a recombinant protein
of isocitrate dehydrogenase from Azotobacter vinelandii
(hereinafter may be referred to as AvIDH);
[0033] FIGS. 3A and 3B are each a SDS-PAGE analysis result
depending on an expression temperature of CgIDH and AvIDH;
[0034] FIGS. 4A and 4B are each a SDS-PAGE analysis result that can
confirm a pure separation and purification result of CgIDH and
AvIDH;
[0035] FIG. 5 is a size-exclusion chromatography analysis result of
CgIDH and AvIDH;
[0036] FIGS. 6A and 6B are an analysis result of the kinetic
constants (Km and Vmax) of CgIDH and AvIDH;
[0037] FIG. 7 is a measurement result of an enzyme activity of
CgIDH and AvIDH;
[0038] FIGS. 8A and 8B are graphs of results obtained by performing
a coupling reaction between CgIDH or AvIDH and cytochrome P450 BM3,
which is the NADPH-dependent enzyme, and performing an analysis
with a spectrophotometer;
[0039] FIGS. 9A and 9B are graphs showing the degree of increase of
NAPDH when determining an optimal enzyme concentration ratio for a
coupling reaction between CgIDH or AvIDH and cytochrome P450
BM3;
[0040] FIGS. 10A and 10B are a series of HPLC analysis results of a
hydroxylation reaction of a substrate by the coupling reaction
between CgIDH or AvIDH and cytochrome P450 BM3;
[0041] FIG. 11 is storage stability evaluation results of CgIDH and
AvIDH;
[0042] FIG. 12 is an evaluation result of an enzyme activity
inhibitory effect by .alpha.-ketoglutarate, which is a reaction
product of CgIDH and AvIDH recombinant proteins.
DETAILED DESCRIPTION
[0043] Hereinafter, an NADPH-regeneration system based on monomeric
isocitrate dehydrogenase of the present invention and a use thereof
will be described in detail with reference to the accompanying
table or drawings.
[0044] If the drawings are described, they are provided as examples
so that the spirit of the present invention can be sufficiently
transferred to those skilled in the art. Therefore, the present
invention is not limited to the accompanying drawings, but may be
modified in many different forms. In addition, the accompanying
drawings described below will be exaggerated in order to illustrate
the spirit and scope of the present invention.
[0045] Terms such as "first", "second", etc. may be used to
describe various components, but these components are not to be
construed as being limited to these terms. The terms are used only
to distinguish one component from another component. For example, a
first component may be referred to as a second component and the
second component may also be similarly referred to as the first
component, without departing from the scope of the present
invention.
[0046] Technical terms and scientific terms used in the present
specification have the general meaning understood by those skilled
in the art to which the present invention pertains unless otherwise
defined, and a description for the known function and configuration
unnecessarily obscuring the gist of the present invention will be
omitted in the following description and the accompanying drawings.
Terms generally used and defined by a dictionary should be
interpreted as having the same meanings as meanings within a
context of the related art and should not be interpreted as having
ideal or excessively formal meanings unless being clearly defined
otherwise in the present specification.
[0047] In addition, singular forms used in the specification of the
present invention are intended to include the plural forms as well
unless otherwise indicated in context.
[0048] Further, units used in the specification of the present
invention without special mention are by weight, and as an example,
the unit of % or ratio means % by weight or a ratio by weight,
respectively.
[0049] Furthermore, in the specification of the present invention,
the expression "comprise" is an "open" description having the
meaning equivalent to expressions such as "include," "contain,"
"have," or "feature", and does not exclude elements, materials, or
processes that are not further listed. In addition, the expression
"substantially composed of . . . " means that other elements,
materials, or processes not listed with the specified element,
material or process may be present in an amount that does not have
an unacceptably significant effect on at least one basic and novel
technical idea of the invention. Further, the expression "composed
of" means that only the described elements, materials, or processes
are present.
[0050] In the specification of the present invention, "NADPH
regeneration" is meant to include not only a process of recycling
NADPH together with an NADPH-dependent enzyme by converting
NADP.sup.+ produced by the NADPH-dependent enzyme into NADPH, but
also a reaction of producing NADPH from free NADP.sup.+, as can be
seen in FIG. 1, unless otherwise specified.
[0051] In the specification of the present invention, the term
"vector", "expression vector", or "recombinant expression vector"
is a linear or circular DNA molecule that encodes an operably
linked polynucleotide, comprising elements and additional fragments
that provide for gene transcription and translation. Additional
fragments include a promoter, a transcription termination sequence,
etc. A vector, an expression vector, or a recombinant expression
vector includes one or more origins replication, one or more
selection markers, etc. A vector, an expression vector, or a
recombinant expression vector is generally derived from plasmid or
viral DNA, or contains elements of both of them.
[0052] In the specification of the present invention, the term
"recombination protein" refers to a conventional expression protein
that expresses a gene from a cell of another species using a
heterologous host, but includes a protein to which another protein
is linked or a different amino acid sequence is added to an amino
or carboxyl terminus of a target protein sequence, if necessary. In
the present invention, an affinity tag may be further included at
the carboxyl terminus of a recombinant protein of isocitrate
dehydrogenase from Corynebacterium glutamicum (CgIDH) and a
recombinant protein of isocitrate dehydrogenase from Azotobacter
vinelandii (AvIDH) for ease of purification. A histidine tag
(his-tag) may be used as the affinity tag, but is not limited
thereto.
[0053] A recombinant expression vector for NADPH regeneration
according to the present invention includes a polynucleotide
encoding an isocitrate dehydrogenase recombinant protein (CgIDH or
AvIDH) monomer from Corynebacterium glutamicum or Azotobacter
vinelandii. Here, the recombinant expression vector may preferably
be one such that the recombinant protein monomer is solubly
overexpressed, but is not limited thereto.
[0054] The isocitrate dehydrogenase from Corynebacterium
glutamicum, as a specific example, may consist of an amino acid
sequence of SEQ ID NO: 1 and the isocitrate dehydrogenase from
Azotobacter vinelandii may consist of an amino acid sequence of SEQ
ID NO: 2.
[0055] The polynucleotide is not particularly limited as long as it
is a nucleic acid sequence capable of encoding CgIDH or AvIDH. For
example, the polynucleotide encoding CgIDH may consist of, but is
not limited to, a nucleic acid sequence of SEQ ID NO: 7, and the
polynucleotide encoding AvIDH may consist of a nucleic acid
sequence of SEQ ID NO: 8. The polynucleotide may be operably linked
to the promoter to ensure that the expression of the protein is
well achieved when introduced into a recombinant expression
vector.
[0056] Here, the term "operably linked" refers to a state in which
a nucleic acid expression control sequence and a nucleic acid
sequence encoding a target protein or RNA are functionally linked
to perform a general function. For example, the nucleic acid
sequence encoding the protein or RNA is operably linked with the
promoter, which can affect the expression of the coding sequence.
An operable linkage with the expression vector may be produced by
gene recombination techniques well known in the art, and
site-specific DNA cleavage and linkage may be used by appropriately
introducing enzymes or homologous gene recombination techniques,
etc. generally known in the art.
[0057] The promoter may be derived from, but is not limited to, a
subject (host) to which a recombinant vector of the present
invention is to be introduced, and examples of thereof include a T7
promoter.
[0058] In the transformant into which the recombinant vector was
introduced, CgIDH and/or AvIDH are/is expressed from the
polynucleotide encoding CgIDH and/or the polynucleotide encoding
AvIDH.
[0059] The recombinant vector of the present invention may be, but
is not limited to, a plasmid vector, a cosmid vector, a
bacteriophage vector, a viral vector, etc. as template. A preferred
example thereof may include a promoter, an operator, an initiation
codon, a stop codon, an expression control element such as a
polyadenylation signal and an enhancer, etc., and may be prepared
in various ways according to the purpose. The recombinant vector
may include an antibiotic resistance marker for selection of
transformants into which the vector was introduced, which may be
inherent in the vector or may be introduced from the outside.
[0060] In the recombinant expression vector for NADPH regeneration
according to an embodiment of the present invention, the
recombinant expression vector may further include a polynucleotide
encoding the NADPH-dependent enzyme. A specific example of the
NADPH-dependent enzyme may be, but is not limited to, any one or
two or more selected from the group consisting of dehydrogenase,
reductase, oxidoreductase, transhydrogenase, peroxidase, oxygenase,
monooxygenase, flavodoxin, and dehalogenase, preferably
dehydrogenase.
[0061] In the recombinant expression vector for NADPH regeneration
according to an embodiment of the present invention, the
NADPH-dependent enzyme may be recombined and fused to the amino
acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID
NO: 2, or may be linked by addition of a chemical linker.
[0062] In the recombinant expression vector for NADPH regeneration
according to an embodiment of the present invention, the chemical
linker may be selected from the group consisting of BS(PEG)5,
BS(PEG)9, BS2G-d0, BS2G-d4, DSBU, DFDNB, DMP, DMS, DSG, DSP, DSS,
DSSO, DST, DTBP, EGS, TSAT, and EDC.
[0063] The present invention provides a transformant transformed
with the recombinant expression vector for NADPH regeneration. The
type of the transformant is not limited as long as the recombinant
expression vector of the present invention can be introduced to
express CgIDH and/or AvIDH. Examples of the transformant may be
selected from strains included in genus Escherichia, genus
Salmonella, genus Shigella, genus Enterobacter, genus Proteus,
genus Pseudomonas, genus Moraxella, genus Helicobacter, genus
Stenotropomonas, genus Bdellovibrio, genus Legionella, genus
Neisseria, and genus Erwinia. Specific examples of the transformant
may be E. coli, and more specifically, E. coli BL21 (DE3).
[0064] When the transformant is produced, a transformation method
may be performed by conventional methods in the art, for example,
but is not limited to, a natural introduction method, a heat shock
method, an electric shock method, etc.
[0065] The present invention provides a method for producing a
recombinant protein including culturing the transformant, and more
specifically, the method for producing the recombinant protein
includes producing the recombinant expression vector for NADPH
regeneration as described above; transforming the recombinant
expression vector to produce a transformant; culturing the
transformant to overexpress isocitrate dehydrogenase originated
from Corynebacterium glutamicum or isocitrate dehydrogenase derived
from Azotobacter vinelandii; and recovering the overexpressed
recombinant protein.
[0066] Culture conditions are not particularly limited when the
transformant is cultured, but may be used by introducing known
culture conditions. As a specific example, the culture may be
performed at 28 to 32.degree. C., preferably 29 to 31.degree. C.,
and more preferably 30.degree. C., and the soluble expression of
90% or more of the target protein may be implemented in the culture
temperature range described above. A medium for culturing
microorganisms may also be appropriately introduced and used into a
known medium, and as a specific example, a Luria-Bertani (LB)
medium may be used, but is not limited thereto.
[0067] When the transformant expresses the recombinant protein by
the introduction of the recombinant expression vector, the culture
medium may further contain an appropriate antibiotic for selection
of a transformed microorganism, and may further contain a substance
for promoting expression of the recombinant protein, for example,
but is not limited to, isopropyl .beta.-D-1-thiogalactopyranoside
(IPTG), etc., if necessary.
[0068] The method for producing the recombinant protein may be
obtained by separating and purifying the recombinant protein from
the culture of the transformant, wherein the culture may be the
transformant or a culture medium thereof, and the culture medium
may be a medium containing the transformant or a medium obtained by
separating the transformant.
[0069] In addition, for easy separation and purification of the
recombinant protein, the transformant may be destroyed, and as
specific methods of destruction, methods, such as, but is not
limited to, physical destruction through ultrasonic decomposition
or chemical destruction through a non-ionic detergent (surfactant),
etc., may be used.
[0070] In addition, the method for preparing the recombinant
protein may further include isolating and purifying CgIDH and/or
AvIDH, and the isolating and purifying may be performed by
introducing conventional separation and purification processes in
the art, which is performed to utilize the expressed protein for
desired purpose or use. Through such separation and purification
processes, a high yield of recombinant protein may be obtained.
[0071] The present invention provides a method for regenerating
NADPH, including regenerating NADPH by adding the recombinant
protein to an NADPH-dependent enzyme reaction system, an
NADPH-regeneration system, and a method for regenerating NADPH,
which is the cofactor of the NADPH-dependent enzyme, such as
cytochrome P450.
[0072] Specific examples of the NADPH-dependent enzyme may be, but
are not limited to, any one or two or more selected from the group
consisting of dehydrogenase, reductase, oxidoreductase,
transhydrogenase, peroxidase, oxygenase, monooxygenase, flavodoxin,
and dehalogenase, preferably dehydrogenase.
[0073] The NADPH-dependent enzyme may be recombined and fused to
CgIDH and/or AvIDH, or may be linked by a chemical linker. The
chemical linker may be selected from, but is not limited to, the
group consisting of BS(PEG)5, BS(PEG)9, BS2G-d0, BS2G-d4, DSBU,
DFDNB, DMP, DMS, DSG, DSP, DSS, DSSO, DST, DTBP, EGS, TSAT, and
EDC.
[0074] The present invention provides a kit for NADPH regeneration,
including the recombinant protein.
[0075] In addition, the present invention provides a composition
for substrate hydroxylation comprising the recombinant protein; and
a NADPH dependent cytochrome P450 protein. Here, the recombinant
protein may refer to, but is not limited to, a NADPH
regeneration-related construct (system) including the same.
Specific examples of the substrate may be selected from, but is not
limited to, the group consisting of omeprazole, omeprazole sulfide,
ethoxycoumarin, and nitrophenol.
[0076] The composition for substrate hydroxylation not only may
include an NADPH-regeneration system using CgIDH and/or AvIDH to
provide expensive NADPH used for cytochrome P450, but may also be
used as a biological catalyst for a hydroxylation reaction of a
wide range of substrates including cytochrome P450 protein by
genetic fusion.
[0077] The present invention provides an in vitro toxicity test
method including treating the composition for substrate
hydroxylation. Here, the type of toxicity test is not particularly
limited, but may include a drug toxicity test, a liver toxicity
test, etc.
[0078] The present invention provides a method of converting a
target substrate by co-expressing or fusion expressing the
recombinant isocitrate deyhydrogenase protein and the
NADPH-dependent enzyme.
[0079] Hereinafter, the content of the present invention will be
described in more detail through examples. The examples are only
for describing the present invention in more detail, and the scope
of the present invention is not limited thereto.
Strain, Reagent, Material, and Experimental Protocol
[0080] In the present invention, reagents, materials, and protocols
used for polymerase chain reaction (PCR), DNA cloning,
transformation, etc. are as follows, which will be apparent to
those skilled in the art.
[0081] Corynebacterium glutamicum ATCC13032 was obtained from the
American Type Culture Collection (ATCC, USA), an international
depository organization, and used.
[0082] Azotobacter vinelandii KACC10899 was obtained from the
Korean Agricultural Culture Collection (KACC, Korea), and used.
[0083] E. coli XL1-Blue was purchased from Yeastern Biotech.
(Taiwan), and used.
[0084] E. coli BL21 (DE3) was purchased from Yeastern Biotech.
(Taiwan), and used.
[0085] pET24a plasmid was purchased from New England Labs (UK), and
used.
[0086] A PureLink.TM. Genomic DNA Kit was purchased from Thermo
Fisher Scientific Korea (Korea), and used.
[0087] Primers for gene amplification were synthesized by BIONICS
(Korea), and used.
[0088] Speed-Pfu DNA Polymerase was purchased from NanoHelix Co.,
Ltd. (Korea), and used.
[0089] An In-Fusion.RTM.HD cloning kit was purchased from Takara
Korea Biomedical Inc., and used.
[0090] Other NdeI and XhoI restriction enzymes were purchased from
Takara Korea Biomedical Inc. (Korea), and used.
[0091] The other reagents were purchased from Sigma-Aldrich (USA),
etc., and used.
Transformant
[0092] E. coli XL1-Blue was used as the transformant for plasmid
transformation and genetic manipulation.
[0093] E. coli BL21 (DE3) was used as the transformant for protein
expression.
Histidine Tag (His-Tag)
[0094] Since fusion of a histidine tag to an amino terminus
(N-terminus) of a recombinant protein may affect the expression and
the structure of a whole protein in some cases, primers were
synthesized so that the histidine tag is fused to a carboxyl
terminus (C-terminus).
Example 1
Construction of Recombinant Vector for Overexpression and
Production of Recombinant Protein
[0095] Plasmid construction was performed based on standard DNA
manipulation techniques. First, a PureLink.TM. Genomic DNA Kit was
used to extract each genomic DNA from Corynebacterium glutamicum
ATCC13032 and Azotobacter vinelandii KACC10899. Thereafter, PCR was
performed using the extracted genomic DNA as templates.
[0096] Here, for the PCR reaction, primers composed of each
sequence in Table 1 below were each synthesized and used.
TABLE-US-00001 TABLE 1 SEQ Restriction ID No. Primer name Sequence
(5'.fwdarw.3') enzyme 3 CgIDH infusion 5'-GAA GGA GAT ATA CAT ATG
GCT AAG ATC ATC TGG ACC NdeI F CG-3' 4 CgIDH infusion 5'-GTG GTG
GTG GTG CTC GAG CTT CTT CAG TGC GTC XhoI R AAC GAT CTC-3' 5 AvIDH
infusion 5'-GAA GGA GAT ATA CAT ATG TCC ACA CCG AAG ATT ATC NdeI F
TAT ACG C-3' 6 AvIDH infusion 5'-GTG GTG GTG GTG CTC GAG TGC AAG
AGG TGC CAG XhoI R AGC C-3'
[0097] Using primers of SEQ ID NOs: 3 and 4 with the template of
genomic DNA extracted from Corynebacterium glutamicum ATCC13032,
polynucleotide 1 encoding an isocitrate dehydrogenase protein from
Corynebacterium glutamicum (CgIDH) was amplified by PCR. Using
primers of SEQ ID NOs: 5 and 6 with the template for genomic DNA
extracted from Azotobacter vinelandii KACC10899, polynucleotide 2
encoding an isocitrate dehydrogenase protein from Azotobacter
vinelandii (AvIDH) was amplified in the same manner. During the PCR
amplification, a Speed-Pfu DNA polymerase, which is a polymerase
having a low mutation frequency, was used.
[0098] DNA fragments obtained through PCR were cloned into pET24a
plasmids treated with NdeI and XhoI restriction enzymes,
respectively, using an In-Fusion.RTM. HD cloning kit. The results
are shown in FIGS. 2A and 2B.
[0099] Each of FIGS. 2A and 2B illustrates schematic diagrams of
the recombinant vector for transcribing and translating genes
(cgIDH and avIDH) encoding amino acid sequences of SEQ ID NO: 1 and
SEQ ID NO: 2, under the control of a T7 promoter.
Example 2
Establishment of Expression Conditions and Purification of
Recombinant Protein
Establishment of Expression Conditions of Recombinant Protein
[0100] In order to provide high expression conditions for
purification of the recombinant protein, the expression conditions
were established while confirming an expression pattern of the IDH
protein.
[0101] The recombinant expression vectors, pET24a-CgIDH and
pET24a-AvIDH constructed in Example 1 above were transformed into
E. coli XL1-Blue, respectively, using a method that is apparent to
those skilled in the art, plated on an LB solid medium containing
50 ug/mL kanamycin, and then cultured overnight at 37.degree. C.
Then, E. coli XL1-Blues into which the recombinant expression
vectors were introduced, were each inoculated into an LB liquid
medium containing 50 ug/mL of kanamycin, and the recombinant
expression vectors were purely isolated from the harvested bacteria
by centrifuging the cultured bacteria at 220 rpm at 37.degree.
C.
[0102] The recombinant expression vectors isolated through the
above process were each transformed into E. coli BL21 (DE3), plated
on the LB solid medium containing 50 ug/mL of kanamycin, and then
cultured overnight at 37.degree. C. Thereafter, a single clone
grown in the medium was inoculated into the LB liquid medium
containing 50 ug/mL of kanamycin, and then absorbance (OD.sub.600)
was measured at 600 nm while being pre-cultured at 220 rpm at
37.degree. C.
[0103] When the absorbance of a culture solution reached 2.0 to
2.5, after passage to the LB liquid medium having the same
composition, the culturing was performed until the absorbance
reached 0.6. Thereafter, 100 mM IPTG was added so that a final
concentration was 0.2 mM, and the medium was incubated for further
2.5 hours at 220 rpm at 37.degree. C. and 30.degree. C. After
completion of the culture cells were harvested by centrifugation
and then adjusted to be 2.0 of OD.sub.600 for complete washing with
DDW.
[0104] The cells were resuspended in 200 uL of 1.times.
phosphate-buffered saline (PBS, pH 7.4), and then disrupted by
sonication. Immediately after crushing, the whole protein fractions
were taken and centrifuged at 16,000.times.g at 4.degree. C. for 30
minutes to remove insoluble aggregates, and aliquoted soluble
fractions.
[0105] To the samples taken in each step, 5.times. sample loading
buffer (0.225 M Tris-HCl pH 6.8, 50% glycerol, 5% SDS, 0.005 M
bromophenol blue, and 0.25 M dithiothreitol (DTT)) was added in a
ratio of 5:1, and the resulting samples was heated at 95.degree. C.
for 15 minutes to induce denaturation of the whole proteins. Then,
after slowly cooling each sample, the prepared sample was loaded on
a 10% acrylamide gel and fixed at 150V, followed by
electrophoresis. After completion of the electrophoresis, the
acrylamide gel was stained with a Coomassie Brilliant Blue
solution, and expression patterns of CgIDH and AvIDH depending on
culture temperature were compared and shown in FIGS. 3A and 3B.
[0106] Each of FIGS. 3A and 3B is the SDS-PAGE analysis result for
confirming the expression patterns after expressing CgIDH and AvIDH
in E. coli BL21 (DE3) under differently set induction temperate at
30.degree. C. and 37.degree. C., respectively. As a result, when
CgIDH was expressed at 30.degree. C., a soluble overexpressed band
was confirmed at a position of about 80 kDa, and when AvIDH was
expressed at 30.degree. C., a soluble overexpressed band was
confirmed at a position of about 70 kDa. It was confirmed from
these results that soluble expression did not occur when the
expression temperature of both of the recombinant proteins was set
to 37.degree. C., but 90% or more soluble expression was achieved
when the expression temperature was set to 30.degree. C.
Purification of Recombinant Protein
[0107] For the purification of the protein expressed as described
above, after increasing culture volume to 100 mL, the culturing was
performed in the same procedure as the above culture method.
[0108] Each of the cells harvested by the procedure as described
above was resuspended by adding 40 mL of 60 mM potassium phosphate
buffer (pH 7.7) containing 300 mM sodium chloride. The resuspended
cells were destroyed by sonication, and centrifuged at
16,000.times.g at 4.degree. C. for 60 minutes to separate
supernatant from which the insoluble aggregates were removed.
Thereafter, 40 mL of soluble protein solutions were each loaded
onto a 5 mL Histrap column (GE Healthcare Life Science, USA). After
completion of loading of each of soluble protein fractions, they
were sufficiently washed with the same buffer, and the recombinant
protein was eluted by gradient to a concentration of 250 mM
imidazole.
[0109] FIGS. 4A and 4B is a result of SDS-PAGE that can confirm a
purification result of CgIDH and AvIDH using affinity
chromatography. It was confirmed from FIGS. 4A and 4B that CgIDH
was eluted at a concentration of about 40 to 60 mM imidazole, and
AvIDH was eluted at a concentration of about 90 to 110 mM
imidazole. In particular, it was confirmed that both of the eluted
CgIDH and AvIDH were purified with a high purity of 95% or
more.
[0110] Additionally, as a result of quantifying the protein of an
eluted fraction by a protein quantification test (Bradford assay),
it was confirmed that both recombinant proteins exhibited a
purification yield of 1 g/L, indicating a high yield even in
laboratory-level purification in which culture conditions were not
optimized.
Example 3
Quaternary Structure of Purified Recombinant Protein
[0111] In order to confirm whether a multimer (quaternary
structure) of the recombinant protein purified by the process as
described above is formed, size exclusion chromatography was
performed.
[0112] First, after CgIDH and AvIDH purified in Example 2 were each
loaded onto a Superdex.TM. 200 10/300 GL column (GE Healthcare Life
Science, USA), size exclusion chromatography was performed using
1.times.PBS (pH 7.4). Cytochrome P450 BM3 (blue, 119 kDa) was used
as a control group, and conalbumin (purple, 75 kDa) and ovalbumin
(orange, 45 kDa) were used as standard size markers. The results of
performing size exclusion chromatography are shown in FIG. 5.
[0113] It was confirmed from FIG. 5 that CgIDH and AvIDH were
eluted at the peak position consistent with the SDS-PAGE analysis
result, which means that both recombinant proteins have a monomeric
form.
Example 4
Kinetic Constant Analysis of Purified Recombinant Protein
[0114] The analysis of kinetic properties related to an enzyme
activity of the recombinant protein, that is, a kinetic constant,
was performed by the following procedure.
[0115] The analysis of kinetic activity for each of the recombinant
proteins purified in Example 2 was performed based on the method
described in Chen, R. & Yang, H. Biochemistry and Biophysics,
383 (2):238-245 (2000); and Watanabe, S. Microbiology 151
(4):1083-1094 (2005).
[0116] Here, a K.sub.m (uM) value representing the affinity of the
enzyme to the substrate as a reaction constant used in enzyme
kinetics, a K.sub.cat(S-1) value, which means the metabolic
turnover number of the enzyme, and a K.sub.cat/K.sub.m (S-1, M-1)
value representing a reaction efficiency of the enzyme, were
determined. The parameters of the wild-types CgIDH and AvIDH were
used as control groups, and the results are shown in FIGS. 6A and
6B.
[0117] It was confirmed from FIGS. 6A and 6B that both CgIDH and
AvIDH according to the present invention showed a slight decrease
in K.sub.cat compared with the wild-types, but the enzyme activity
for providing NADPH required for the reaction of the
NADPH-dependent enzyme such as cytochrome P450 was sufficiently
high.
[0118] Thus, the activity of the recombinant protein according to
the present invention can be seen to be the same level as that of
the wild-types, which supports the reliability of the result of the
quaternary structure analysis of Example 3. That is, the above
result can be interpreted as an indirect or considerable result
indicating that there was no change in the quaternary structure
during the producing process of the recombinant protein.
Example 5
Specific Activity Analysis of Recombinant Protein
[0119] In order to confirm the degree of NADPH regeneration by the
activity of the IDH recombinant protein, CgIDH and AvIDH were each
added to a reaction solution containing 100 mM potassium phosphate
buffer (pH 7.4), 0.8 mM manganese sulfate (MnSO.sub.4), 0.8 mM
DL-isocitric acid, and 0.5 mM NADP+ so that the final
concentrations were 5 nM, and the degree of increase of NADPH
depending on the enzyme activity was measured with a
spectrophotometer. When fluorescence was measured using a
spectrophotometer, an excitation wavelength was set to 350 nm and
an emission wavelength was set to 450 nm, and the measurement
results are shown in FIG. 7.
[0120] It was confirmed from FIG. 7 that NADPH increased over time
in each reaction solution containing each protein of CgIDH
indicated by a circle and AvIDH indicated by a square, which
suggests that both CgIDH and AvIDH generate NADPH by an enzymatic
reaction that converts isocitric acid to .alpha.-ketoglutarate.
[0121] In addition, when the enzyme activity of the same amount of
the recombinant protein is compared, a CgIDH enzyme reaction slope
was found to be higher than that of an AvIDH enzyme reaction slope,
which means that CgIDH has higher enzyme activity. These results
are also consistent with those in FIGS. 6A and 6B of Example 4.
[0122] From the above results, it was confirmed that the specific
activities of CgIDH and AvIDH correspond to 57 U/mg and 28 U/mg,
respectively.
Example 6
Analysis of Coupling Reaction between NADPH-Regeneration System
Based on Isocitrate Dehydrogenase and Cytochrome P450 BM3
[0123] In order to confirm whether NADPH, which is the cofactor of
the NADPH-dependent enzyme, can be continuously supplied, through
the NADPH-regeneration system using the IDH recombinant protein,
CgIDH and cytochrome P450 BM3 were added to a reaction solution
containing 100 mM potassium phosphate buffer (pH 7.4), 0.8 mM
manganese sulfate, 40 mM DL-isocitric acid, 0.5 mM NADP, and 2 mM
omeprazole to prepare a reaction product, and the reaction product
was prepared using AvIDH and cytochrome P450 BM3 in the same
manner. Thereafter, coupling reactions between CgIDH and AvIDH, and
cytochrome P450 BM3 were performed at 37.degree. C. for each
reaction to prepare the product.
NADPH Self-Fluorescence Analysis to Confirm Cofactor
Regeneration
[0124] In the above process, 2 nM CgIDH and 500 nM cytochrome P450
BM3 were each added to the reaction solution at the final
concentrations, and 2 nM AvIDH and 500 nM cytochrome P450 BM3 were
each added to the reaction solution at the final concentration in
the same manner. Thereafter, after each reactant was reacted at
37.degree. C., whether or not NADPH was increased was measured
using the spectrophotometer. When fluorescence was measured using
the spectrophotometer, an excitation wavelength was set to 350 nm
and an emission wavelength was set to 450 nm, and the measurement
results are shown in FIGS. 8A and 8B.
[0125] Here, the case in which the IDH recombinant protein was not
added to the reaction solution was used as a negative control group
(circle in FIG. 8A), and two cases in which cytochrome P450 BM3 was
not added to the reaction solution were used as positive control
groups (square and triangle in FIG. 8A).
[0126] FIG. 8B illustrates fluorescence measurement results for two
coupling reactions (CgIDH and cytochrome P450 BM3 (square); AvIDH
and cytochrome P450 BM3 (triangle)) together with the negative
control group (circle), wherein a fluorescence intensity scale on a
vertical axis (y-axis) is set from 0 to 500 a.u. and is
enlarged.
[0127] It was confirmed from FIGS. 8A and 8B that only in the case
of the reaction solution in which both of the coupled proteins were
present, the fluorescence intensity slightly increased due to NADPH
generated by the IDH recombinant protein at the beginning of the
reaction, but the fluorescence intensity remained at a certain
level after a certain period of time, which suggests that NADPH
produced by the IDH recombinant protein is consumed by cytochrome
P450 BM3, which is the NADPH-dependent enzyme and NADP produced at
the same time is again regenerated into NADPH by the IDH
recombinant protein, thereby efficiently regenerating the cofactor,
NADPH. A schematic representation of a reaction involving IDH
recombinant protein, NADPH-dependent enzyme, NADP+, and NADPH in
terms of regeneration of NADPH, is shown in Scheme 1 below:
NADP.sup.30 +IDH recombinant protein.fwdarw.NADPH+NADPH-dependent
enzyme.fwdarw.NADP.sup.30 Scheme 1
Determination of Enzyme Concentration Ratio
[0128] In the above reaction, the amount of cytochrome P450 BM3 was
high compared with the IDH recombinant protein, so NADPH was
present in a small concentration in the reaction solution. Thus, in
order to establish a more appropriate enzyme concentration ratio,
500 nM cytochrome P450 BM3 was added to a reaction solution
containing 100 mM potassium phosphate buffer (pH 7.4), 0.8 mM
manganese sulfate, 40 mM DL-isocitric acid, 0.5 mM NADP, and 2 mM
omeprazole, and CgIDH and AvIDH were each added at 2 nM, 5 nM, and
10 nM (concentration ratio of 1:250, 1:100, 1:50, respectively) to
prepare a reaction product. Thereafter, each reactant was subjected
to a coupling reaction at 37.degree. C., and then the degree of
increase of NADPH was measured in the same manner. The results are
shown in FIGS. 9A and 9B.
[0129] FIG. 9A illustrates the result of the coupling reaction
between CgIDH and cytochrome P450 BM3, wherein the concentration
ratio of the two enzymes was set to 1:250 (square), 1:100
(triangle) and 1:50 (inverted triangle). In addition, FIG. 9B
illustrates the result of the coupling reaction between AvIDH and
cytochrome P450 BM3, wherein the concentration ratio of the two
enzymes was set to 1:250 (square), 1:100 (triangle) and 1:50
(inverted triangle). In the same manner as in FIGS. 8A and 8B, the
case in which the IDH recombinant protein was not added to the
reaction solution was used as the negative control group (circle in
FIGS. 9A and 9B).
[0130] As can be seen from FIGS. 9A and 9B, an interval in which
NADPH was maintained at a certain level was different depending on
the concentration of CgIDH and AvIDH, and subsequent analysis was
performed by setting the ratio of an enzyme concentration (for
CgIDH, 1:50 and for AvIDH, 1:100) at which the fluorescence
intensity was maintained at 2000 a.u. as the ratio of an
appropriate enzyme concentration.
HPLC Analysis of Hydroxylation Product of Substrate
[0131] In the above reaction, in order to confirm that the activity
of cytochrome P450 BM3 is maintained by substantial NADPH
regeneration, and the hydroxylation reaction of omeprazole, which
is one of the substrates, occurs, to 1 mL of a reaction solution
containing 100 mM potassium phosphate buffer (pH 7.4), 0.8 mM
manganese sulfate, 40 mM DL-isocitric acid, 0.5 mM NADP, and 2 mM
omeprazole, CgIDH and cytochrome P450 BM3 were added so that the
final concentrations were 10 nM and 500 nM, respectively, and AvIDH
and cytochrome P450 BM3 were added so that the final concentrations
were 5 nM and 500 nM, respectively, to prepare a reaction
product.
[0132] The reaction mixture was recovered by 90 uL each time, mixed
with 90 uL of methanol to stop the reaction, and then HPLC analysis
was performed. Specific HPLC performance conditions are as follows:
[0133] HPLC was performed using an Alliance HPLC system (Waters).
[0134] A SunFire 18C column was used as a column [0135] The amount
of sample loading was set to 20 uL. [0136] As a HPLC mobile phase,
30% acetonitrile was flowed at a flow rate of 1 mL/min, and an
eluate was measured with ultraviolet (UV) light at 302 nm. [0137]
Analysis was performed using an Empower 3 (Waters) program.
[0138] Instead of using the NADPH-regeneration system as a control
group, NADPH itself and a substrate were added to perform the
hydroxylation reaction of the substrate through cytochrome P450 BM3
activity, and HPLC analysis was performed in the same manner. The
results are shown in FIGS. 10A and 10B.
[0139] It was confirmed from FIGS. 10A and 10B that the product of
the coupling reaction, that is, 5'-hydroxyomeprazole, which is a
hydroxylated form of the substrate omeprazole, was eluted from all
three reaction solutions (a coupling reaction between CgIDH and
cytochrome P450 BM3, a coupling reaction between AvIDH and
cytochrome P450 BM3, control) when five minutes has elapsed, and
the shape of each peak was similar.
[0140] In addition, the elution amount of 5'-hydroxyomeprazole
produced by the coupling reaction (5 min) with the
NADPH-regeneration system of the present invention was measured to
be 126% in the case of the coupling reaction between CgIDH and P450
BM3, and 114% in the case of the coupling reaction between AvIDH
and P450 BM3, based on the elution amount of 5'-hydroxyomeprazole
produced in the control group.
[0141] The above-mentioned elution amount suggests that the
NADPH-regeneration system of the present invention may more
effectively supply NADPH to the enzymatic reaction of the
NADPH-dependent enzyme.
Example 7
Storage Stability of Recombinant Protein
[0142] In order to evaluate the stability of the recombinant
protein produced in the present invention, after a certain period
of time, each of the recombinant proteins stored at 4.degree. C.
was added to a reaction solution containing 100 mM potassium
phosphate buffer (pH 7.4), 0.8 mM manganese sulfate, 40 mM
DL-isocitric acid, and 0.5 mM NADP+so that the final concentration
was 5 nM, the reaction was proceeded as described in Example 5, and
the degree of increase of NADPH was measured. The measurement
results are shown in FIG. 11.
[0143] It was confirmed from FIG. 11 that after storage at
4.degree. C. for 30 days, the enzyme activity of the CgIDH and
AvIDH recombinant proteins measured under the same conditions did
not decrease.
[0144] In particular, it was confirmed that the activity increased
by 10 to 20% compared with before storage, which suggests that the
activity is maintained in the range of 90 to 110% of the initial
activity, considering that a deviation occurs depending on reaction
conditions and protein quantification.
[0145] That is, it was confirmed that the NADPH-regeneration system
of the present invention may be stably utilized as an independent
component for an NADPH-dependent enzymatic reaction, and may be
applied as key parts by maintaining favorable stability for mass
production and long-term storage in an NADPH-regeneration process
for NADPH-dependent enzyme activity including cytochrome P450
BM3.
Example 8
Enzyme Activity Inhibitory Effect Depending on Product of
Recombinant IDH Protein
[0146] In order to confirm an enzyme activity inhibitory effect by
.alpha.-ketoglutarate, which is an enzyme reaction product of the
recombinant protein produced in the present invention, a reaction
product, in which 5 nM of CgIDH and 5 nM AvIDH were each added to a
reaction solution containing 100 mM potassium phosphate buffer (pH
7.4), 0.8 mM manganese sulfate, 40 mM DL-isocitric acid, and 0.5 mM
NADP, was prepared. Thereafter, .alpha.-ketoglutarate was added for
each concentration to proceed the reaction as described in Example
5, and the degree of increase of NADPH was measured. The
measurement results are shown in FIG. 12. Here, an experimental
group to which .alpha.-ketoglutarate was not added was used as a
control group.
[0147] It was confirmed from FIG. 12 that for CgIDH, the enzyme
activity was reduced by 30% by the added 10 mM
.alpha.-ketoglutarate, and for AvIDH, the enzyme activity was
reduced by 30% by the added 20 mM .alpha.-ketoglutarate.
[0148] The above-mentioned reduction means that when the
NADPH-regeneration system is operated in a batch process, an enzyme
activity inhibition phenomenon may occur due to the enzyme reaction
product, which suggests that it is preferable to perform a process
of removing the enzyme reaction product by introducing a continuous
process, a hollow tube membrane reactor, etc. in order to solve the
above phenomenon. However, it was also confirmed that when the pH
of the reaction process is corrected by adding a pH adjuster, etc.,
the inhibitory effect can be improved by 50% or more (results not
attached).
Example 9
Analysis of Coupling Reaction between NADPH-Regeneration System and
Cytochrome P450 BM3 Depending on Substrate Change
[0149] In Example 6, the substrate omeprazole was changed to
omeprazole sulfide, ethoxycoumarin, and nitrophenol, respectively,
and then NADPH-regeneration ability was analyzed under the same
conditions.
[0150] As a result, the same substrate conversion result was
confirmed even when the substrates other than omeprazole were used,
which suggests that the NADPH-regeneration system using the IDH
recombinant protein, regardless of the type of substrate, has the
NADPH-regeneration ability to maintain NADPH-dependent enzyme
activity. That is, it can be said that it is suitable as robust key
parts of the NADPH-regeneration system.
Example 10
Analysis of Coupling Reaction Depending on Changes in
NADPH-Regeneration System and NADPH-Dependent Enzyme
[0151] In Example 6, after changing cytochrome P450, which is the
NADPH-dependent enzyme, to mannitol 2-dehydrogenase,
methylmalonate-semialdehyde dehydrogenase, glutamate dehydrogenase,
and phenylalanine dehydrogenase, respectively, the
NADPH-regeneration ability was analyzed under the same
conditions.
[0152] As a result, the same results were confirmed for other types
of NADPH-dependent enzymes other than cytochrome P450 BM3, which
suggests that the NADPH-regeneration system using the IDH
recombinant protein, regardless of the type of NADPH-dependent
enzyme, has the NADPH regeneration ability to maintain
NADPH-dependent enzyme activity. That is, it can be said that it is
suitable as robust key parts of the NADPH-regeneration system.
Example 11
Whole Cell Reaction Analysis Through Co-Expression of Recombinant
IDH Protein and Cytochrome P450
[0153] Transformants transformed with a plasmid in which the IDH
recombinant protein according to the present invention and
cytochrome P450 BM3 are co-expressed, were cultured as in the above
Example, and 0.8 mM manganese sulfate, 2 mM omeprazole, a
sufficient amount of potassium phosphate buffer (pH 7.4),
DL-isocitric acid, and 0.5 mM NADP were added to a reaction
solution with harvested cells after cultivation. Thereafter, HPLC
analysis was performed in the same manner as in the above
Example.
[0154] As a result, it was confirmed that 5'-hydroxyomeprazole was
detected in reaction solution, which suggests that substrate
conversion is possible even through a whole-cell reaction in which
the NADPH-regeneration system using the IDH recombinant protein and
the NADPH-dependent enzymes are simultaneously expressed.
Example 12
Whole Cell Reaction Analysis Through Fused Protein Expression of
Recombinant IDH Protein With Cytochrome P450
[0155] Transformants transformed with a plasmid engineered so that
the IDH recombinant protein according to the present invention and
cytochrome P450 BM3 are expressed as a fusion protein in cells,
were cultured as in the above Example. 0.8 mM manganese sulfate, 2
mM omeprazole, a sufficient amount of potassium phosphate buffer
(pH 7.4), DL-isocitric acid, and 0.5 mM NADP were added to a
reaction solution with harvested cells after cultivation.
Thereafter, HPLC analysis was performed in the same manner as in
the above Example.
[0156] As a result, it was confirmed that 5'-hydroxyomeprazole was
produced by whole cell catalyst, which suggests that substrate
conversion is possible even through the whole-cell reaction in
which the NADPH-regeneration system using the IDH recombinant
protein and the NADPH-dependent enzymes are expressed as a fusion
protein.
[0157] The above results show that the difficulty of efficient
regeneration of NADPH through the expression of fusion protein,
which is one of the fundamental disadvantages of the conventional
cytochrome P450 BM3 and the NADPH-regeneration system (e.g., G6PDH,
etc), has been overcome, through the fusion protein expression of
the NADPH-regeneration system using the IDH recombinant protein and
the NADPH-dependent enzyme. In the future, the continuous reaction
system with NADPH-regeneration part f, which fused the
NADPH-dependent enzyme with IDH recombinant protein as a single
protein, can be widely used in the development of new processes
with excellent productivity.
[0158] The present invention relates to a recombinant vector
including a polynucleotide encoding an isocitrate dehydrogenase
recombinant protein from Corynebacterium glutamicum and an
isocitrate dehydrogenase recombinant protein from Azotobacter
vinelandii, 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.
[0159] Special portions of contents of the present invention have
been described in detail herein above, and it will be obvious to
those skilled in the art that this detailed description is only an
exemplary embodiment and the scope of the present invention is not
limited by this detailed description. Therefore, the substantial
scope of the present invention will be defined by the claims and
equivalents thereof.
Sequence CWU 1
1
81746PRTArtificial SequenceCgIDH protein 1Met Ala Lys Ile Ile Trp
Thr Arg Thr Asp Glu Ala Pro Leu Leu Ala1 5 10 15Thr Tyr Ser Leu Lys
Pro Val Val Glu Ala Phe Ala Ala Thr Ala Gly 20 25 30Ile Glu Val Glu
Thr Arg Asp Ile Ser Leu Ala Gly Arg Ile Leu Ala 35 40 45Gln Phe Pro
Glu Arg Leu Thr Glu Asp Gln Lys Val Gly Asn Ala Leu 50 55 60Ala Glu
Leu Gly Glu Leu Ala Lys Thr Pro Glu Ala Asn Ile Ile Lys65 70 75
80Leu Pro Asn Ile Ser Ala Ser Val Pro Gln Leu Lys Ala Ala Ile Lys
85 90 95Glu Leu Gln Asp Gln Gly Tyr Asp Ile Pro Glu Leu Pro Asp Asn
Ala 100 105 110Thr Thr Asp Glu Glu Lys Asp Ile Leu Ala Arg Tyr Asn
Ala Val Lys 115 120 125Gly Ser Ala Val Asn Pro Val Leu Arg Glu Gly
Asn Ser Asp Arg Arg 130 135 140Ala Pro Ile Ala Val Lys Asn Phe Val
Lys Lys Phe Pro His Arg Met145 150 155 160Gly Glu Trp Ser Ala Asp
Ser Lys Thr Asn Val Ala Thr Met Asp Ala 165 170 175Asn Asp Phe Arg
His Asn Glu Lys Ser Ile Ile Leu Asp Ala Ala Asp 180 185 190Glu Val
Gln Ile Lys His Ile Ala Ala Asp Gly Thr Glu Thr Ile Leu 195 200
205Lys Asp Ser Leu Lys Leu Leu Glu Gly Glu Val Leu Asp Gly Thr Val
210 215 220Leu Ser Ala Lys Ala Leu Asp Ala Phe Leu Leu Glu Gln Val
Ala Arg225 230 235 240Ala Lys Ala Glu Gly Ile Leu Phe Ser Ala His
Leu Lys Ala Thr Met 245 250 255Met Lys Val Ser Asp Pro Ile Ile Phe
Gly His Val Val Arg Ala Tyr 260 265 270Phe Ala Asp Val Phe Ala Gln
Tyr Gly Glu Gln Leu Leu Ala Ala Gly 275 280 285Leu Asn Gly Glu Asn
Gly Leu Ala Ala Ile Leu Ser Gly Leu Glu Ser 290 295 300Leu Asp Asn
Gly Glu Glu Ile Lys Ala Ala Phe Glu Lys Gly Leu Glu305 310 315
320Asp Gly Pro Asp Leu Ala Met Val Asn Ser Ala Arg Gly Ile Thr Asn
325 330 335Leu His Val Pro Ser Asp Val Ile Val Asp Ala Ser Met Pro
Ala Met 340 345 350Ile Arg Thr Ser Gly His Met Trp Asn Lys Asp Asp
Gln Glu Gln Asp 355 360 365Thr Leu Ala Ile Ile Pro Asp Ser Ser Tyr
Ala Gly Val Tyr Gln Thr 370 375 380Val Ile Glu Asp Cys Arg Lys Asn
Gly Ala Phe Asp Pro Thr Thr Met385 390 395 400Gly Thr Val Pro Asn
Val Gly Leu Met Ala Gln Lys Ala Glu Glu Tyr 405 410 415Gly Ser His
Asp Lys Thr Phe Arg Ile Glu Ala Asp Gly Val Val Gln 420 425 430Val
Val Ser Ser Asn Gly Asp Val Leu Ile Glu His Asp Val Glu Ala 435 440
445Asn Asp Ile Trp Arg Ala Cys Gln Val Lys Asp Ala Pro Ile Gln Asp
450 455 460Trp Val Lys Leu Ala Val Thr Arg Ser Arg Leu Ser Gly Met
Pro Ala465 470 475 480Val Phe Trp Leu Asp Pro Glu Arg Ala His Asp
Arg Asn Leu Ala Ser 485 490 495Leu Val Glu Lys Tyr Leu Ala Asp His
Asp Thr Glu Gly Leu Asp Ile 500 505 510Gln Ile Leu Ser Pro Val Glu
Ala Thr Gln Leu Ser Ile Asp Arg Ile 515 520 525Arg Arg Gly Glu Asp
Thr Ile Ser Val Thr Gly Asn Val Leu Arg Asp 530 535 540Tyr Asn Thr
Asp Leu Phe Pro Ile Leu Glu Leu Gly Thr Ser Ala Lys545 550 555
560Met Leu Ser Val Val Pro Leu Met Ala Gly Gly Gly Leu Phe Glu Thr
565 570 575Gly Ala Gly Gly Ser Ala Pro Lys His Val Gln Gln Val Gln
Glu Glu 580 585 590Asn His Leu Arg Trp Asp Ser Leu Gly Glu Phe Leu
Ala Leu Ala Glu 595 600 605Ser Phe Arg His Glu Leu Asn Asn Asn Gly
Asn Thr Lys Ala Gly Val 610 615 620Leu Ala Asp Ala Leu Asp Lys Ala
Thr Glu Lys Leu Leu Asn Glu Glu625 630 635 640Lys Ser Pro Ser Arg
Lys Val Gly Glu Ile Asp Asn Arg Gly Ser His 645 650 655Phe Trp Leu
Thr Lys Phe Trp Ala Asp Glu Leu Ala Ala Gln Thr Glu 660 665 670Asp
Ala Asp Leu Ala Ala Thr Phe Ala Pro Val Ala Glu Ala Leu Asn 675 680
685Thr Gly Ala Ala Asp Ile Asp Ala Ala Leu Leu Ala Val Gln Gly Gly
690 695 700Ala Thr Asp Leu Gly Gly Tyr Tyr Ser Pro Asn Glu Glu Lys
Leu Thr705 710 715 720Asn Ile Met Arg Pro Val Ala Gln Phe Asn Glu
Ile Val Asp Ala Leu 725 730 735Lys Lys Leu Glu His His His His His
His 740 7452749PRTArtificial SequenceAvIDH protein 2Met Ser Thr Pro
Lys Ile Ile Tyr Thr Leu Thr Asp Glu Ala Pro Ala1 5 10 15Leu Ala Thr
Tyr Ser Leu Leu Pro Ile Ile Lys Ala Phe Thr Gly Ser 20 25 30Ser Gly
Ile Ala Val Glu Thr Arg Asp Ile Ser Leu Ala Gly Arg Leu 35 40 45Ile
Ala Thr Phe Pro Glu Tyr Leu Thr Asp Thr Gln Lys Ile Ser Asp 50 55
60Asp Leu Ala Glu Leu Gly Lys Leu Ala Thr Thr Pro Asp Ala Asn Ile65
70 75 80Ile Lys Leu Pro Asn Ile Ser Ala Ser Val Pro Gln Leu Lys Ala
Ala 85 90 95Ile Lys Glu Leu Gln Gln Gln Gly Tyr Lys Leu Pro Asp Tyr
Pro Glu 100 105 110Glu Pro Lys Thr Asp Thr Glu Lys Asp Val Lys Ala
Arg Tyr Asp Lys 115 120 125Ile Lys Gly Ser Ala Val Asn Pro Val Leu
Arg Glu Gly Asn Ser Asp 130 135 140Arg Arg Ala Pro Leu Ser Val Lys
Asn Tyr Ala Arg Lys His Pro His145 150 155 160Lys Met Gly Ala Trp
Ser Ala Asp Ser Lys Ser His Val Ala His Met 165 170 175Asp Asn Gly
Asp Phe Tyr Gly Ser Glu Lys Ala Ala Leu Ile Gly Ala 180 185 190Pro
Gly Ser Val Lys Ile Glu Leu Ile Ala Lys Asp Gly Ser Ser Thr 195 200
205Val Leu Lys Ala Lys Thr Ser Val Gln Ala Gly Glu Ile Ile Asp Ser
210 215 220Ser Val Met Ser Lys Asn Ala Leu Arg Asn Phe Ile Ala Ala
Glu Ile225 230 235 240Glu Asp Ala Lys Lys Gln Gly Val Leu Leu Ser
Val His Leu Lys Ala 245 250 255Thr Met Met Lys Val Ser Asp Pro Ile
Met Phe Gly Gln Ile Val Ser 260 265 270Glu Phe Tyr Lys Asp Ala Leu
Thr Lys His Ala Glu Val Leu Lys Gln 275 280 285Ile Gly Phe Asp Val
Asn Asn Gly Ile Gly Asp Leu Tyr Ala Arg Ile 290 295 300Lys Thr Leu
Pro Glu Ala Lys Gln Lys Glu Ile Glu Ala Asp Ile Gln305 310 315
320Ala Val Tyr Ala Gln Arg Pro Gln Leu Ala Met Val Asn Ser Asp Lys
325 330 335Gly Ile Thr Asn Leu His Val Pro Ser Asp Val Ile Val Asp
Ala Ser 340 345 350Met Pro Ala Met Ile Arg Asp Ser Gly Lys Met Trp
Gly Pro Asp Gly 355 360 365Lys Leu His Asp Thr Lys Ala Val Ile Pro
Asp Arg Cys Tyr Ala Gly 370 375 380Val Tyr Gln Val Val Ile Glu Asp
Cys Lys Gln His Gly Ala Phe Asp385 390 395 400Pro Thr Thr Met Gly
Ser Val Pro Asn Val Gly Leu Met Ala Gln Lys 405 410 415Ala Glu Glu
Tyr Gly Ser His Asp Lys Thr Phe Gln Ile Pro Ala Asp 420 425 430Gly
Val Val Arg Val Thr Asp Glu Ser Gly Lys Leu Leu Leu Glu Gln 435 440
445Ser Val Glu Ala Gly Asp Ile Trp Arg Met Cys Gln Ala Lys Asp Ala
450 455 460Pro Ile Gln Asp Trp Val Lys Leu Ala Val Asn Arg Ala Arg
Ala Thr465 470 475 480Asn Thr Pro Ala Val Phe Trp Leu Asp Pro Ala
Arg Ala His Asp Ala 485 490 495Gln Val Ile Ala Lys Val Glu Arg Tyr
Leu Lys Asp Tyr Asp Thr Ser 500 505 510Gly Leu Asp Ile Arg Ile Leu
Ser Pro Val Glu Ala Thr Arg Phe Ser 515 520 525Leu Ala Arg Ile Arg
Glu Gly Lys Asp Thr Ile Ser Val Thr Gly Asn 530 535 540Val Leu Arg
Asp Tyr Leu Thr Asp Leu Phe Pro Ile Met Glu Leu Gly545 550 555
560Thr Ser Ala Lys Met Leu Ser Ile Val Pro Leu Met Ser Gly Gly Gly
565 570 575Leu Phe Glu Thr Gly Ala Gly Gly Ser Ala Pro Lys His Val
Gln Gln 580 585 590Phe Leu Glu Glu Gly Tyr Leu Arg Trp Asp Ser Leu
Gly Glu Phe Leu 595 600 605Ala Leu Ala Ala Ser Leu Glu His Leu Gly
Asn Ala Tyr Lys Asn Pro 610 615 620Lys Ala Leu Val Leu Ala Ser Thr
Leu Asp Gln Ala Thr Gly Lys Ile625 630 635 640Leu Asp Asn Asn Lys
Ser Pro Ala Arg Lys Val Gly Glu Ile Asp Asn 645 650 655Arg Gly Ser
His Phe Tyr Leu Ala Leu Tyr Trp Ala Gln Ala Leu Ala 660 665 670Ala
Gln Thr Glu Asp Lys Glu Leu Gln Ala Gln Phe Thr Gly Ile Ala 675 680
685Lys Ala Leu Thr Asp Asn Glu Thr Lys Ile Val Gly Glu Leu Ala Ala
690 695 700Ala Gln Gly Lys Pro Val Asp Ile Ala Gly Tyr Tyr His Pro
Asn Thr705 710 715 720Asp Leu Thr Ser Lys Ala Ile Arg Pro Ser Ala
Thr Phe Asn Ala Ala 725 730 735Leu Ala Pro Leu Ala Leu Glu His His
His His His His 740 745338DNAArtificial SequenceCgIDH infusion F
3gaaggagata tacatatggc taagatcatc tggacccg 38442DNAArtificial
SequenceCgIDH infusion R 4gtggtggtgg tgctcgagct tcttcagtgc
gtcaacgatc tc 42543DNAArtificial SequenceAvIDH infusion F
5gaaggagata tacatatgtc cacaccgaag attatctata cgc 43637DNAArtificial
SequenceAvIDH infusion R 6gtggtggtgg tgctcgagtg caagaggtgc cagagcc
3772241DNAArtificial SequenceCgIDH polynucleotide 7atggctaaga
tcatctggac ccgcaccgac gaagcaccgc tgctcgcgac ctactcgctg 60aagccggtcg
tcgaggcatt tgctgctacc gcgggcattg aggtcgagac ccgggacatt
120tcactcgctg gacgcatcct cgcccagttc ccagagcgcc tcaccgaaga
tcagaaggta 180ggcaacgcac tcgcagaact cggcgagctt gctaagactc
ctgaagcaaa catcattaag 240cttccaaaca tctccgcttc tgttccacag
ctcaaggctg ctattaagga actgcaggac 300cagggctacg acatcccaga
actgcctgat aacgccacca ccgacgagga aaaagacatc 360ctcgcacgct
acaacgctgt taagggttcc gctgtgaacc cagtgctgcg tgaaggcaac
420tctgaccgcc gcgcaccaat cgctgtcaag aactttgtta agaagttccc
acaccgcatg 480ggcgagtggt ctgcagattc caagaccaac gttgcaacca
tggatgcaaa cgacttccgc 540cacaacgaga agtccatcat cctcgacgct
gctgatgaag ttcagatcaa gcacatcgca 600gctgacggca ccgagaccat
cctcaaggac agcctcaagc ttcttgaagg cgaagttcta 660gacggaaccg
ttctgtccgc aaaggcactg gacgcattcc ttctcgagca ggtcgctcgc
720gcaaaggcag aaggtatcct cttctccgca cacctgaagg ccaccatgat
gaaggtctcc 780gacccaatca tcttcggcca cgttgtgcgc gcttacttcg
cagacgtttt cgcacagtac 840ggtgagcagc tgctcgcagc tggcctcaac
ggcgaaaacg gcctcgctgc aatcctctcc 900ggcttggagt ccctggacaa
cggcgaagaa atcaaggctg cattcgagaa gggcttggaa 960gacggcccag
acctggccat ggttaactcc gctcgcggca tcaccaacct gcatgtccct
1020tccgatgtca tcgtggacgc ttccatgcca gcaatgattc gtacctccgg
ccacatgtgg 1080aacaaagacg accaggagca ggacaccctg gcaatcatcc
cagactcctc ctacgctggc 1140gtctaccaga ccgttatcga agactgccgc
aagaacggcg cattcgatcc aaccaccatg 1200ggtaccgtcc ctaacgttgg
tctgatggct cagaaggctg aagagtacgg ctcccatgac 1260aagaccttcc
gcatcgaagc agacggtgtg gttcaggttg tttcctccaa cggcgacgtt
1320ctcatcgagc acgacgttga ggcaaatgac atctggcgtg catgccaggt
caaggatgcc 1380ccaatccagg attgggtaaa gcttgctgtc acccgctccc
gtctctccgg aatgcctgca 1440gtgttctggt tggatccaga gcgcgcacac
gaccgcaacc tggcttccct cgttgagaag 1500tacctggctg accacgacac
cgagggcctg gacatccaga tcctctcccc tgttgaggca 1560acccagctct
ccatcgaccg catccgccgt ggcgaggaca ccatctctgt caccggtaac
1620gttctgcgtg actacaacac cgacctcttc ccaatcctgg agctgggcac
ctctgcaaag 1680atgctgtctg tcgttccttt gatggctggc ggcggactgt
tcgagaccgg tgctggtgga 1740tctgctccta agcacgtcca gcaggttcag
gaagaaaacc acctgcgttg ggattccctc 1800ggtgagttcc tcgcactggc
tgagtccttc cgccacgagc tcaacaacaa cggcaacacc 1860aaggccggcg
ttctggctga cgctctggac aaggcaactg agaagctgct gaacgaagag
1920aagtccccat cccgcaaggt tggcgagatc gacaaccgtg gctcccactt
ctggctgacc 1980aagttctggg ctgacgagct cgctgctcag accgaggacg
cagatctggc tgctaccttc 2040gcaccagtcg cagaagcact gaacacaggc
gctgcagaca tcgatgctgc actgctcgca 2100gttcagggtg gagcaactga
ccttggtggc tactactccc ctaacgagga gaagctcacc 2160aacatcatgc
gcccagtcgc acagttcaac gagatcgttg acgcactgaa gaagctcgag
2220caccaccacc accaccactg a 224182250DNAArtificial SequenceAvIDH
polynucleotide 8atgtccacac cgaagattat ctatacgctc actgatgaag
cacccgcact ggcgacttac 60tctctgcttc ccatcatcaa agcgttcacc ggatcttcag
gtatcgccgt tgaaacccgc 120gatatctccc ttgcaggccg cctcatcgca
accttccccg aatacctgac cgatacccag 180aaaatctccg acgatttggc
cgaactggga aaactggcca ccacgccgga cgccaacatc 240atcaagctgc
cgaacatcag cgcctccgtc ccgcaactca aggccgccat caaggaactg
300cagcagcagg gctacaagct cccggactac cctgaagagc ccaagaccga
caccgagaag 360gacgtcaagg cccgctacga caagatcaag ggcagcgccg
tgaaccccgt cctgcgcgaa 420ggcaactccg accgccgcgc gccactgtcc
gtcaagaact acgccagaaa gcaccctcac 480aagatgggcg cctggagtgc
ggactccaag tcccatgtcg cccacatgga caacggtgat 540ttctacggca
gcgagaaggc cgctctgatt ggcgcccccg gcagtgtgaa aatcgagctg
600atcgccaaag acggcagcag cactgttctg aaggcaaaga cctctgttca
ggctggcgag 660atcatcgact cttcggtaat gagcaagaac gccttgcgca
acttcatcgc cgctgaaatc 720gaggatgcga agaagcaggg agtactgctg
tccgtgcacc tgaaggcgac catgatgaag 780gtgtccgacc ccatcatgtt
cggccagatc gtctccgagt tctacaagga cgccctcacc 840aagcacgcag
aggtgctgaa gcagatcggc ttcgacgtca acaatggcat cggtgatctc
900tacgcccgga tcaagactct tcccgaagca aagcagaagg aaatcgaggc
cgacatccag 960gcggtttacg cccagcgccc gcaattggcg atggtgaact
ccgacaaggg catcaccaac 1020ctgcatgtgc cgagcgacgt catcgtcgac
gcctcgatgc cggcgatgat ccgcgactcc 1080ggcaagatgt ggggccccga
cggcaagctg catgacacca aggcggtcat ccccgaccgt 1140tgctatgccg
gcgtgtacca ggtggtcatc gaggactgca agcagcacgg cgccttcgac
1200cccaccacca tgggcagcgt gcccaacgtc ggtttgatgg ctcagaaagc
cgaggaatac 1260ggctcccacg acaagacctt ccagattcct gcagacggcg
tggtccgtgt gaccgatgaa 1320agcggcaagc tcttgctgga gcaaagcgtg
gaggccggcg acatttggcg catgtgccag 1380gcgaaagacg ccccgatcca
ggactgggtc aagctggccg tcaaccgcgc ccgcgccacc 1440aataccccgg
cggtgttctg gctggacccg gcgcgtgccc atgatgccca ggttattgcc
1500aaggtcgagc gttacctgaa ggactacgat accagcggtc tcgacatccg
catcttgtcg 1560ccggtcgagg caacccgctt ctcgctggcc cgcatccgcg
aaggcaagga caccatttcc 1620gtcaccggca acgtcctgcg cgactacctg
accgacctgt tcccgatcat ggaactgggt 1680accagcgcca aaatgttgtc
gatcgtcccg ctgatgagcg gcggcggtct gttcgaaacc 1740ggcgcgggcg
gctcggctcc caagcatgtc cagcagttcc tcgaggaagg ttacctgcgt
1800tgggattcgc tcggcgagtt cctcgctctt gccgcatccc tggagcactt
gggcaacgcc 1860tacaagaacc cgaaagcgct tgtcctggcc agcaccctgg
accaggctac cggcaagatt 1920ctcgataaca acaaatcgcc ggcacgtaag
gttggcgaga tcgataaccg cggtagccac 1980ttctacttgg cactctactg
ggcccaggca ttggcagcgc aaaccgagga caaggaactg 2040caagcccagt
tcaccggcat tgccaaggct ctgaccgaca acgagaccaa gatcgtcggc
2100gagttggctg cagcccaagg caagcctgtg gatatcgctg gctactacca
tccgaatacc 2160gacctgacca gcaaggccat ccgcccgagc gctactttca
acgcggctct ggcacctctt 2220gcactcgagc accaccacca ccaccactga 2250
* * * * *