U.S. patent application number 15/543909 was filed with the patent office on 2018-01-11 for method of continuously producing glutathione using photosynthetic membrane vesicles.
This patent application is currently assigned to Sogang University Research Foundation. The applicant listed for this patent is SOGANG UNIVERSITY RESEARCH FOUNDATION. Invention is credited to Hyeon Jun KIM, Jeong Kug LEE, Eun Kyoung OH.
Application Number | 20180010163 15/543909 |
Document ID | / |
Family ID | 56406093 |
Filed Date | 2018-01-11 |
United States Patent
Application |
20180010163 |
Kind Code |
A1 |
LEE; Jeong Kug ; et
al. |
January 11, 2018 |
METHOD OF CONTINUOUSLY PRODUCING GLUTATHIONE USING PHOTOSYNTHETIC
MEMBRANE VESICLES
Abstract
The present invention relates to a method of producing
glutathione, wherein photosynthetic membrane vesicles and enzymes
catalyzing glutathione synthesis are combined and glutamate,
cysteine and glycine are used as reaction substrates. As enzymes
catalyzing glutathione synthesis, .gamma.-glutamylcysteine
synthetase and glutathione synthetase may be used together, or
bifunctional glutathione synthetase may be used alone. According to
the conventional methods, there is a problem in that expensive
adenosine triphosphate should be continuously supplied when
glutathione is produced. However, according to the present
invention, since photosynthetic membrane vesicles are used as a
source to regenerate adenosine triphosphate, it is possible to
continuously produce glutathione without additionally adding
adenosine triphosphate, thereby reducing production costs of
glutathione.
Inventors: |
LEE; Jeong Kug; (Seoul,
KR) ; KIM; Hyeon Jun; (Seoul, KR) ; OH; Eun
Kyoung; (Seoul, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SOGANG UNIVERSITY RESEARCH FOUNDATION |
Seoul |
|
KR |
|
|
Assignee: |
Sogang University Research
Foundation
Seoul
KR
|
Family ID: |
56406093 |
Appl. No.: |
15/543909 |
Filed: |
January 15, 2016 |
PCT Filed: |
January 15, 2016 |
PCT NO: |
PCT/KR2016/000433 |
371 Date: |
July 14, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 5/0819 20130101;
C12N 9/00 20130101; C12Y 603/02002 20130101; C12N 9/93 20130101;
C12Y 603/02003 20130101; C12P 21/02 20130101 |
International
Class: |
C12P 21/02 20060101
C12P021/02; C12N 9/00 20060101 C12N009/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 16, 2015 |
KR |
10-2015-0008174 |
Claims
1. A method of producing glutathione, comprising: a) a step of
generating adenosine triphosphate (ATP) from adenosine diphosphate
(ADP) and an inorganic phosphate by irradiating photosynthetic
membrane vesicles with light; and b) a step of synthesizing
glutathione by enzymes that catalyze glutathione synthesis using
ATP generated in step a), and forming ADP and an inorganic
phosphate.
2. The method according to claim 1, further comprising c) a step of
reusing ADP and an inorganic phosphate forming in step b) to
generate ATP in photosynthetic membrane vesicles.
3. The method according to claim 1, wherein the photosynthetic
membrane vesicles are selected from the group consisting of
chromatophore membrane vesicles isolated from purple non-sulfur
bacteria and thylakoid membrane vesicles isolated from
cyanobacteria or algae.
4. The method according to claim 1, wherein the enzymes that
catalyze glutathione synthesis are one or more selected from the
group consisting of .gamma.-glutamylcysteine synthetase (GSH-I),
glutathione synthetase (GSH-II) and bifunctional glutathione
synthetase (bifunctional .gamma.-glutamylcysteine
synthetase/glutathione synthetase, GshF).
5. The method according to claim 1, further comprising a step of
adding glutamate, cysteine and glycine as substrates for producing
glutathione.
6. The method according to claim 1, wherein step b) comprises a
step of synthesizing .gamma.-glutamylcysteine from glutamate and
cysteine by .gamma.-glutamylcysteine synthetase while converting
adenosine triphosphate generated in step a) into adenosine
diphosphate and an inorganic phosphate; and a step of synthesizing
glutathione from the synthesized .gamma.-glutamylcysteine and
glycine by glutathione synthetase while converting adenosine
triphosphate generated in step a) into adenosine diphosphate and an
inorganic phosphate.
7. The method according to claim 1, wherein step b) comprises a
step of synthesizing glutathione from glutamate, cysteine and
glycine by bifunctional glutathione synthetase while converting
adenosine triphosphate generated in step a) into adenosine
diphosphate and an inorganic phosphate.
8. The method according to claim 6, wherein a relative activity
ratio of .gamma.-glutamylcysteine synthetase to glutathione
synthetase is 4:1 to 20:1.
9. The method according to claim 6, wherein a relative activity
ratio of the photosynthetic membrane vesicles to
.gamma.-glutamylcysteine synthetase to glutathione synthetase is
1:12:1 to 50:12:1.
10. The method according to claim 7, wherein a relative activity
ratio of photosynthetic membrane vesicles to bifunctional
glutathione synthetase is 10:1 to 500:1.
11. A composition for producing glutathione, comprising: i)
photosynthetic membrane vesicles; and ii) enzymes that catalyze
glutathione synthesis, wherein the enzymes are selected from the
group consisting of .gamma.-glutamylcysteine synthetase (GSH-I),
glutathione synthetase (GSH-II) and bifunctional glutathione
synthetase (bifunctional .gamma.-glutamylcysteine
synthetase/glutathione synthetase, GshF).
12. A system for continuously producing glutathione, comprising: i)
photosynthetic membrane vesicles as a means for supplying adenosine
triphosphate; ii) enzymes that catalyze glutathione synthesis,
wherein the enzymes are selected from the group consisting of
.gamma.-glutamylcysteine synthetase (GSH-I), glutathione synthetase
(GSH-II) and bifunctional glutathione synthetase (bifunctional
.gamma.-glutamylcysteine synthetase/glutathione synthetase, GshF),
as a means for catalyzing glutathione synthesis
13. A method of continuously producing glutathione, the method
comprising a step of adding glutamate, cysteine and glycine as
substrates for producing glutathione to the system according to
claim 12.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method of continuously
producing glutathione using photosynthetic membrane vesicles.
BACKGROUND ART
[0002] Glutathione (GSH) is a tripeptide composed of L-glutamate,
L-cysteine, and glycine and is synthesized in most eukaryotes and
some prokaryotes. Glutathione is biosynthesized by two step
enzymatic reactions catalyzed by .gamma.-glutamylcysteine
synthetase (GSH-I, EC 6.3.2.2) and glutathione synthetase (GSH-II,
EC 6.3.2.3) using L-glutamate, L-cysteine, and glycine as starting
materials. Recent studies have revealed a novel biosynthetic
pathway mediated by one enzyme in addition to the previously known
glutathione synthesis pathway mediated by the two enzymes mentioned
above, and it has been found that an enzyme catalyzing the novel
biosynthetic pathway is bifunctional glutathione synthetase
(bifunctional .gamma.-glutamylcysteine synthetase/glutathione
synthetase, GshF) (see Janowiak and Griffith. 2005. J. Biol. Chem.
280: 11829-11839, Vergauwen et al. 2006. J. Biol. Chem. 281:
4380-4394).
[0003] Glutathione contains a thiol group having strong reducing
power, and it acts in vivo in concert with glutathione peroxidase
(GSH peroxidase), glutathione reductase (GSH reductase), and
glutathione S-transferase (GSH S-transferase) to perform an
antioxidant function. It may be involved in immune stimulation and
detoxification of xenobiotics (see Pastore et al. 2003. Clinica
Chimica Acta 333: 19-39). In general, it is known that glutathione
synthesis genes are essential for survival, and organisms deficient
in these genes are less resistant to oxidative stress and various
harmful substances. In addition, there is a report that various
human diseases are associated with a decrease in glutathione
concentration in body (Wu et al. 2004. J. Nutr. 134: 489-492).
Accordingly, based on the antioxidant activity of glutathione,
glutathione is widely used as an additive in foods, medicines and
cosmetics for the purpose of preventing aging and promoting
health.
[0004] To date, known methods of producing glutathione include
organic synthesis methods, enzymatic methods, and fermentative
methods. The organic synthesis method refers to a method of
chemically synthesizing glutathione, and was developed in 1935 and
commercialized in 1950 (Harington and Mead. 1935. Biochem. J. 29:
1602-1611, Li et al. 2004. Appl. Microbiol. Biotechnol. 66:
233-242). Glutathione synthesized by the organic synthesis method
is optically inactive (racemic). However, since organisms
selectively use only L-glutathione, an additional step of isolating
L-glutathione is required to use glutathione synthesized by the
organic synthesis method.
[0005] As soon as the biosynthetic pathway of glutathione was
discovered in the 1950s, attempts to biologically produce
glutathione began (Bloch. 1949. J. Biol. Chem. 179: 1245-1254).
Among such methods, a method of producing glutathione by
fermentation using Escherichia coli or yeast (Saccharomyces
cerevisiae and Candida sp.) is currently the most commercially used
method. In the fermentative method, glucose or molasses is used as
a carbon source. Generally, Escherichia coli or yeast are cultured
for 20 to 30 hours under fermentation conditions with the carbon
source, and then glutathione is extracted. When the fermentation
method is used to produce glutathione, various methods may be
additionally performed to enhance glutathione production. For
example, L-cysteine may be added to medium to enhance a glutathione
production rate (Alfafara et al. 1992. Appl. Microbiol. Biotechol.
37: 141-146). In addition, efforts were made to increase
glutathione production by overexpressing two genes (GSH-I, GSH-II)
responsible for glutathione synthesis in fermentation strains, but
no significant increase in glutathione production was observed.
[0006] Another way to biologically produce glutathione is to use
enzymes. In commonly used biological methods, Escherichia coli or
yeast is treated with a surfactant to increase the permeability to
substrates, and then L-glutamate, L-cysteine, glycine and adenosine
triphosphate are added to the surfactant-treated cells. After
subsequent incubation, L-glutathione is produced in cells. As
another example, there is a method of producing glutathione using
purified .gamma.-glutamylcysteine synthetase and glutathione
synthetase, but the method is not widely used. The enzymatic
methods are advantageous in that the reaction is selective and
yield is higher than that of the fermentative methods. However, in
the case of the methods, since the unit price of adenosine
triphosphate required for the reaction is high, the enzymatic
methods are difficult to commercialize. To overcome this cost
problem, it is possible to supply adenosine triphosphate by using
an enzyme that regenerates adenosine triphosphate or by using
glycolysis that takes place in cells, but the cost problem cannot
be completely solved by such methods. Therefore, when a system
capable of continuously supplying adenosine triphosphate at low
cost is provided, production of glutathione using enzymes is likely
be commercialized.
[0007] The above described background art has been provided to aid
in understanding of the present invention and should not be
interpreted as conventional technology known to a person having
ordinary skill in the art.
DISCLOSURE
Technical Problem
[0008] Glutathione is very useful as an additive in foods,
medicines and cosmetics. When such glutathione is synthesized using
enzymes, there is an advantage in that reaction process is simple,
but there is a problem in that the unit cost of coenzymes used in
the reaction process is expensive. Accordingly, the present
inventors have studied a method of producing glutathione at a
relatively low cost. As a result, vesicles present in the cell
membranes of photosynthetic bacteria or algae were isolated, which
contain apparatuses for performing photosynthetic light reaction.
When enzymes that catalyze glutathione synthesis were added to the
vesicles and then irradiated with light, it was verified that
glutathione was continuously synthesized without continued addition
of adenosine triphosphate or the like having a very high unit cost,
thereby completing the present invention.
[0009] Therefore, it is an objective of the present invention to
provide a method of producing glutathione using photosynthetic
membrane vesicles and .gamma.-glutamylcysteine synthetase and
glutathione synthetase.
[0010] It is another objective of the present invention to provide
a method of producing glutathione using photosynthetic membrane
vesicles and bifunctional glutathione synthetase.
[0011] It is still another objective of the present invention to
provide a composition including photosynthetic membrane vesicles
for producing glutathione.
[0012] It is yet another objective of the present invention to
provide a system for continuously producing glutathione, the system
including photosynthetic membrane vesicles as a means for supplying
adenosine triphosphate.
[0013] It is yet another objective of the present invention to
provide a method of continuously producing glutathione, the method
including a step of adding substrates for producing glutathione to
the system.
[0014] Other objects and advantages of the present invention will
become more apparent from the following detailed description of the
invention, claims and drawings.
Technical Solution
[0015] One aspect of the present invention provides a method of
producing glutathione using photosynthetic membrane vesicles,
.gamma.-glutamylcysteine synthetase (GSH-I) and glutathione
synthetase (GSH-II).
[0016] Another aspect of the present invention provides a method of
producing glutathione using photosynthetic membrane vesicles and
bifunctional glutathione synthetase (bifunctional
.gamma.-glutamylcysteine synthetase/glutathione synthetase,
GshF).
[0017] The present inventors have studied a method of continuously
producing glutathione as an additive in foods, medicines and
cosmetics. As a result, vesicles present in the cell membranes of
photosynthetic bacteria or algae were isolated (photosynthetic
membrane vesicles). When enzymes that catalyze glutathione
synthesis were added to reaction mixture containing the vesicles,
and then it was irradiated with light, glutathione was continuously
synthesized without addition of adenosine triphosphate having a
very high unit cost.
[0018] As used herein, "photosynthetic membrane vesicles" may be
isolated in the form of vesicle from photosynthetic bacteria or
algae capable of performing photosynthesis using light energy, and
refer to cell membrane-protein complexes capable of conducting a
photosynthetic reaction upon irradiation with appropriate
wavelengths of light.
[0019] Photosynthetic membranes forming the photosynthetic membrane
vesicles may include chromatophore membranes (also known as
intracytoplasmic membranes, ICMs) present in anoxygenic
photosynthetic bacteria and thylakoid membranes (TMs) present in
oxygenic photosynthetic bacteria or algae.
[0020] Chromatophore membrane vesicles are derived from
chromatophore membranes, and refer to cell membrane-protein
complexes containing reaction centers, light-harvesting complexes,
adenosine triphosphate synthase (ATP synthase) and electron
transfer chain proteins. In chromatophore membranes, adenosine
triphosphate is generated from adenosine diphosphate and an
inorganic phosphate by a proton motive force formed as a result of
cyclic electron flow. In addition, reduced nicotinamide adenine
dinucleotide (NADH) may be generated from oxidized nicotinamide
adenine dinucleotide (NAD.sup.+) through reverse electron flow
mediated by complex I and complex II present in chromatophore
membranes. The generated NADH may be converted into reduced
nicotinamide adenine dinucleotide phosphate (NADPH) by pyridine
nucleotide transhydrogenase.
[0021] Anoxygenic photosynthetic bacteria from which the
chromatophore membrane vesicles can be isolated are preferably
purple non-sulfur bacteria, more preferably purple non-sulfur
bacteria selected from the group consisting of Rhodobacter sp.,
Rhodospirillum sp., Rhodopseudomonas sp., Roseobacter sp.,
Bradyrhizobium sp., and Rubrivivax sp., without being limited
thereto.
[0022] Thylakoid membrane vesicles are derived from thylakoid
membranes, and refer to cell membrane-protein complexes containing
two types of photosynthetic systems (photosystem I/II), adenosine
triphosphate synthase and electron transfer chain proteins.
Electron transfer using water as an electron donor occurs in
thylakoid membranes, resulting in generation of reduced
nicotinamide adenine dinucleotide phosphate (NADPH) and formation
of the proton motive force. The formed proton motive force is used
to synthesize adenosine triphosphate from adenosine diphosphate and
an inorganic phosphate by adenosine triphosphate synthase.
[0023] Oxygenic photosynthetic bacteria or algae from which the
thylakoid membrane vesicles can be isolated are preferably
cyanobacteria, more preferably cyanobacteria selected from the
group consisting of Synechocystis sp., Synechococcus sp., Nostoc
sp., Anabaena sp., Gloeobacter sp., and Cyanobacterium sp., without
being limited thereto.
[0024] According to a preferred embodiment of the present
invention, the method of the present invention includes a step of
generating adenosine triphosphate (ATP) from adenosine diphosphate
(ADP) and an inorganic phosphate by irradiating the photosynthetic
membrane vesicles with light.
[0025] In the present invention, photosynthetic membrane vesicles
are used as a source of adenosine triphosphate and, in the process
of producing glutathione, enzymes that catalyze glutathione
synthesis consume adenosine triphosphate to form adenosine
diphosphate and an inorganic phosphate (FIGS. 1a and 1b). The
adenosine diphosphate and inorganic phosphate may be converted into
adenosine triphosphate by a photoreaction carried out in
photosynthetic membrane vesicles under photosynthesis conditions,
and the adenosine triphosphate may be reused by enzymes involved in
glutathione synthesis.
[0026] As used herein, the term "photosynthesis conditions" refer
to conditions for inducing a photoreaction in photosynthetic
membrane vesicles. For example, in the case of chromatophore
membrane vesicles, a photoreaction is preferably carried out under
conditions of a temperature range of 20 to 37.degree. C., a
luminous intensity range of 3 to 300 Watts/m.sup.2 and irradiation
with light having a wavelength of 350 to 1000 nm. An incandescent
lamp may be used to meet this wavelength range. In addition, in the
case of thylakoid membrane vesicles, a photoreaction is preferably
carried out under conditions of a temperature range of 20 to
37.degree. C., a light intensity range of 5 to 500
.mu.Einstein/m.sup.2s (.mu.mole photons/m.sup.2s) and irradiation
with light having a wavelength of 400 to 700 nm. A fluorescent lamp
may be used to meet this wavelength range.
[0027] Still another aspect of the present invention provides a
method of producing glutathione including: a) a step of generating
adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and
an inorganic phosphate by irradiating photosynthetic membrane
vesicles; b) a step of synthesizing glutathione by enzymes that
catalyze glutathione synthesis using ATP generated in step a), and
forming ADP and an inorganic phosphate; and c) a step of reusing
ADP and an inorganic phosphate generated in step b) to generate ATP
by photosynthetic membrane vesicles.
[0028] As used herein, "enzymes that catalyze glutathione
synthesis" refers to enzymes that consume adenosine triphosphate to
form adenosine diphosphate and an inorganic phosphate, thereby
catalyzing a reaction that produces glutathione, and preferably
include one or more selected from the group consisting of
.gamma.-glutamylcysteine synthetase (GSH-I), glutathione synthetase
(GSH-II) and bifunctional glutathione synthetase (bifunctional
.gamma.-glutamylcysteine synthetase/glutathione synthetase,
GshF).
[0029] The enzymes that catalyze glutathione synthesis use
substrates for producing glutathione, and preferably use glutamate,
cysteine and glycine as substrates. Considering that organisms
selectively produce L-glutathione and use the same, it is effective
to use L-glutamate and L-cysteine for glutamate and cysteine.
[0030] According to a preferred embodiment of the present
invention, among enzymes that catalyze glutathione synthesis,
.gamma.-glutamylcysteine synthetase catalyzes synthesis of
.gamma.-glutamylcysteine from glutamate and cysteine while
converting adenosine triphosphate into adenosine diphosphate and an
inorganic phosphate, and the glutathione synthetase catalyzes
synthesis of glutathione from .gamma.-glutamylcysteine and glycine
while converting adenosine triphosphate into adenosine diphosphate
and an inorganic phosphate.
[0031] That is, in the method, step b) may include a step of
synthesizing .gamma.-glutamylcysteine from glutamate and cysteine
by .gamma.-glutamylcysteine synthetase while converting adenosine
triphosphate generated in step a) into adenosine diphosphate and an
inorganic phosphate; and a step of synthesizing glutathione from
the synthesized .gamma.-glutamylcysteine and glycine by glutathione
synthetase while converting adenosine triphosphate generated in
step a) into adenosine diphosphate and an inorganic phosphate (see
overall reaction A of FIG. 1a).
[0032] According to a preferred embodiment of the present
invention, among the enzymes that catalyze glutathione synthesis,
bifunctional glutathione synthetase is capable of both the reaction
catalyzed by .gamma.-glutamylcysteine synthetase and the reaction
catalyzed by glutathione synthetase.
[0033] That is, in the method, step b) may include a step of
synthesizing glutathione from glutamate, cysteine and glycine by
bifunctional glutathione synthetase while converting adenosine
triphosphate generated in step a) into adenosine diphosphate and an
inorganic phosphate (see overall reaction B of FIG. 1b).
[0034] In the present invention, enzyme activity is used to express
the relative amount of photosynthetic membrane vesicles and each
enzyme. As used herein, the term "enzyme activity" (hereinafter,
activity) refers to the amount of product that may be produced per
unit time (.mu.mole product/min) by a certain amount of enzyme, and
reflects the amount of enzyme that is actually active. The term
"relative activity" refers to the ratio of corresponding enzymes or
chromatophore membrane vesicles when the actual activity ratio
between chromatophore membrane vesicles and enzymes is expressed as
a simple integer ratio. In overall reaction A, the relative
activity ratio of .gamma.-glutamylcysteine synthetase to
glutathione synthetase is preferably 1:1 to 100:1, more preferably
4:1 to 20:1, most preferably 8:1 to 16:1, without being limited
thereto. In addition, the relative activity ratio of photosynthetic
membrane vesicles to .gamma.-glutamylcysteine synthetase to
glutathione synthetase is preferably 1:12:1 to 100:12:1, more
preferably 1:12:1 to 50:12:1, most preferably 1:12:1 to 25:12:1,
without being limited thereto. In addition, the relative activity
ratio of photosynthetic membrane vesicles to bifunctional
glutathione synthetase is preferably 1:1 to 1000:1, more preferably
10:1 to 500:1, most preferably 40:1 to 200:1, without being limited
thereto. Although production of glutathione is still possible even
upon deviating from these relative activity ratios, it is more
efficient to follow the ratio in consideration of the total yield
of glutathione and production costs.
[0035] According to a preferred embodiment of the present
invention, the overall reaction A or overall reaction B is
performed under anaerobic conditions, which may maximize the
activity of photosynthetic membrane vesicles and prevent oxidation
of the glutathione product.
[0036] Yet another aspect of the present invention provides a
composition for producing glutathione, the composition including i)
photosynthetic membrane vesicles; and ii) enzymes that catalyze
glutathione synthesis, wherein the enzymes are selected from the
group consisting of .gamma.-glutamylcysteine synthetase (GSH-I),
glutathione synthetase (GSH-II) and bifunctional glutathione
synthetase (bifunctional .gamma.-glutamylcysteine
synthetase/glutathione synthetase, GshF).
[0037] The substance of the present invention may be produced in
the form of a composition, and the composition of the present
invention may be distributed in suitable containers (e.g., glass
bottles, plastic containers, etc.). Thus, the composition of the
present invention may be utilized in the production of glutathione.
A person who intends to produce glutathione may continuously
produce glutathione by irradiating the composition of the present
invention containing the photosynthetic membrane vesicles and
enzymes that catalyze glutathione synthesis with light.
[0038] Yet another aspect of the present invention provides a
system for continuously producing glutathione, the system including
i) photosynthetic membrane vesicles as a means for supplying
adenosine triphosphate; and ii) enzymes that catalyze glutathione
synthesis, wherein the enzymes are selected from the group
consisting of .gamma.-glutamylcysteine synthetase (GSH-I),
glutathione synthetase (GSH-II) and bifunctional glutathione
synthetase (bifunctional .gamma.-glutamylcysteine
synthetase/glutathione synthetase, GshF), as a means for catalyzing
glutathione synthesis.
[0039] As used herein, the term "system for continuously producing
glutathione" refers to a system in which additional input is not
necessarily required other than initial input of adenosine
triphosphate or initial input of adenosine diphosphate and an
inorganic phosphate and a system that can continuously produce
glutathione even when only glutamate, cysteine and glycine are
added, and the system includes reactors, kits, devices, equipment,
and the like.
[0040] Yet another aspect of the present invention provides a
method of continuously producing glutathione, the method including
a step of adding glutamate, cysteine and glycine as substrates for
producing glutathione to the system of the present invention.
Advantageous Effects
[0041] The features and advantages of the present invention are
summarized as follows:
[0042] (i) The present invention provides a method of producing
glutathione using photosynthetic membrane vesicles,
.gamma.-glutamylcysteine synthetase and glutathione synthetase.
[0043] (ii) In addition, the present invention provides a method of
producing glutathione using photosynthetic membrane vesicles and
bifunctional glutathione synthetase.
[0044] (iii) According to the present invention, photosynthetic
membrane vesicles are used as a means for supplying adenosine
triphosphate which is consumed when .gamma.-glutamylcysteine
synthetase, glutathione synthetase or bifunctional glutathione
synthetase catalyzes the synthesis of glutathione. That is, since
adenosine triphosphate is continuously supplied under light
conditions, it is not necessary to additionally supply adenosine
triphosphate. Thus, the cost can be reduced.
DESCRIPTION OF DRAWINGS
[0045] FIG. 1a illustrates a schematic diagram (overall reaction A)
showing a method of synthesizing glutathione using chromatophore
membrane vesicles, .gamma.-glutamylcysteine synthetase and
glutathione synthetase. FIG. 1b illustrates a schematic diagram
(overall reaction B) showing a method of synthesizing glutathione
using chromatophore membrane vesicles and bifunctional glutathione
synthetase.
[0046] FIG. 2 is an image showing the result of sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) performed on
purified proteins of the present invention. Each band shown on the
image represents purified .gamma.-glutamylcysteine synthetase,
glutathione synthetase or bifunctional glutathione synthetase.
[0047] FIG. 3 is a graph showing the activity of
.gamma.-glutamylcysteine synthetase measured using a method of
detecting adenosine diphosphate formation.
[0048] FIG. 4 is a graph showing the activity of glutathione
synthetase measured using a method of detecting adenosine
diphosphate formation.
[0049] FIG. 5 is a graph showing the amount of glutathione produced
depending on the relative activity of .gamma.-glutamylcysteine
synthetase while the relative activity of glutathione synthetase is
fixed at 1.
[0050] FIG. 6 is a graph showing the amount of glutathione produced
depending on the relative activity of chromatophore membrane
vesicles while the relative activity of .gamma.-glutamylcysteine
synthetase to the relative activity of glutathione synthetase was
fixed at 12:1.
[0051] FIG. 7 is a graph showing the activity of bifunctional
glutathione synthetase for producing glutathione in a reaction
solution containing L-glutamate, L-cysteine, glycine and adenosine
triphosphate.
[0052] FIG. 8 is a graph showing the amount of glutathione produced
depending on the relative activity of chromatophore membrane
vesicles while the relative activity of bifunctional glutathione
synthetase is fixed at 1.
MODES OF THE INVENTION
[0053] Hereinafter, the present invention is described in more
detail with reference to the following examples. These examples are
only intended to explain the present invention more specifically,
and it will be apparent to those skilled in the art that the scope
of the present invention is not limited by these examples.
EXAMPLES
Example 1: Isolation of Chromatophore Membrane Vesicles
[0054] Chromatophore membrane vesicles are isolated using the
method described in Korean Patent Application No. 10-2014-0151907.
Rhodobacter sphearoides (Rhodobacter sphearoides 2.4.1, ATCC
BAA-808, Cohen-Bazire et al. 1956. J. Cell. Comp. Physiol. 49:
25-68), a type of purple non-sulfur bacteria, was used to isolate
chromatophore membrane vesicles. The strain was cultured in
Sistrom's minimal medium (Sistrom. 1962. J. Gen. Microbiol. 28:
607-616, Table 1). The culture method is as follows. First, a test
tube containing 5 ml of the medium was inoculated with the strain,
and was subjected to shaking culture at 30.degree. C. and 250 rpm.
When culture absorbance at 660 nm was about 2.0, the medium was
subcultured in an 18-ml screw cap test tube to an initial
absorbance of 0.05, and then the screw cap test tube was filled
with a fresh medium and sealed to block the exposure to oxygen.
Culture was performed under photosynthesis conditions.
Specifically, the culture was performed for 18 hours in an
incubator, wherein culture conditions were set as follows:
temperature is maintained at 30.degree. C. and light is irradiated
by an incandescent lamp at a luminous intensity of 15
Watts/m.sup.2. After culture, 8 ml of the strain cultured in the 18
ml screw cap test tube was added to a 260 ml transparent bottle,
and the remaining volume of the transparent bottle was filled with
a fresh medium and sealed. The culture medium was subjected to
anaerobic culture, as described above, at a temperature of
30.degree. C. and at a luminous intensity of 15 Watts/m.sup.2 for
18 hours. Thereafter, the process of isolating chromatophore
membranes was carried out in an anaerobic chamber (anaerobic
chamber, model 10, Coy laboratory product). The gas composition in
the anaerobic chamber is 90% nitrogen, 5% carbon dioxide and 5%
hydrogen. The Rhodobacter sphearoides strain cultured in the 260 ml
transparent bottle was subjected to centrifugation at 7,000 g and
4.degree. C. for 10 minutes, and then a supernatant was discarded
and a cell pellet was obtained. The cell pellet was resuspended in
4 ml of a phosphate buffer (10 mM Na.sub.2HPO.sub.4, 2 mM
KH.sub.2PO.sub.4, pH 7.6), and a protease inhibitor mixture
(protease inhibitor cocktail, Roche) was added thereto according to
the manufacturer's instructions. In subsequent procedure, all
samples were kept on ice. Next, cell lysis was performed using a
sonicator (model VCX130, Sonics & Materials) under the
following conditions: ultrasonic irradiation for 2 minutes with
100% amplification and then cooling in ice water for 2 minutes, and
repeating this process three times. When irradiating ultrasonic
waves, the cell pellets were cooled with ice water to prevent
overheating. The lysed cells were subjected to centrifugation at
6,000 g and 4.degree. C. for 10 minutes to obtain a supernatant,
and then the supernatant was subjected to centrifugation at 200,000
g and 4.degree. C. for 1 hour using an ultracentrifuge (Optima
XE-90, Beckman Coulter). After centrifugation, a supernatant was
removed, and a pellet containing chromatophore membrane vesicles
was dissolved in 1 ml of a phosphate buffer to perform
sucrose-density gradient centrifugation. A sucrose-density gradient
was formed in the order of 8 ml of a 60% (w/v, dissolved in a
phosphate buffer) sucrose solution, 1 ml of a 40% sucrose solution
and 1 ml of a 20% sucrose solution from the bottom layer of an
ultracentrifuge tube with a volume of 13.5 ml. 1 ml of the pellet
containing chromatophore membrane vesicles was placed on the top
layer, and ultracentrifugation was performed at 200,000 g for 4
hours. A reddish brown layer containing chromatophore membrane
vesicles was located between a layer of the 20% sucrose solution
and a layer of the 40% sucrose solution. The reddish brown layer
was separated, and then diluted by addition of the same volume of a
phosphate buffer. In addition, kanamycin was added at a
concentration of 100 .mu.g/ml to prevent the growth of common
contaminants, and a protease inhibitor mixture was added according
to the manufacturer's instructions. The mixture was anaerobically
sealed and stored at 4.degree. C.
TABLE-US-00001 TABLE 1 Composition of Sistrom's minimal medium for
culturing Rhodobacter sphearoides Additives Final Concentration
KH.sub.2PO.sub.4 20 mM NaCl 8.5 mM (NH.sub.4).sub.2SO.sub.4 3.78 mM
L-Glutamate 0.67 mM L-Aspartic acid 0.25 mM Succinic acid 34 mM
Nitrilotriacetic acid 1.05 mM MgCl.sub.2.cndot.6H.sub.2O 1.2 mM
CaCl.sub.2.cndot.2H.sub.2O 0.23 mM FeSO.sub.4.cndot.7H.sub.2O 7
.mu.M (NH.sub.4).sub.6Mo.sub.7O.sub.24 0.16 .mu.M EDTA 4.7 .mu.M
ZnSO.sub.4.cndot.7H.sub.2O 38 .mu.M MnSO.sub.4.cndot.H.sub.2O 9.1
.mu.M CuSO.sub.4.cndot.5H.sub.2O 1.6 .mu.M
Co(NO.sub.3).sub.2.cndot.6H.sub.2O 0.85 .mu.M H.sub.3BO.sub.3 1.8
.mu.M Nicotinic acid 8.1 .mu.M Thiamine hydrochloride 1.5 .mu.M
Biotin 41 nM
Example 2: Preparation of Genes Encoding Enzymes Involved in
Glutathione Synthesis
[0055] To clone genes encoding .gamma.-glutamylcysteine synthetase
and glutathione synthetase of overall reaction A (FIG. 1a),
respectively, and a gene encoding bifunctional glutathione
synthetase of overall reaction B (FIG. 1b), polymerase chain
reaction (PCR) was performed. In the case of
.gamma.-glutamylcysteine synthetase, SEQ ID NO. 1 and SEQ ID NO. 2
were used as a forward primer and a reverse primer, respectively.
In the case of glutathione synthetase, SEQ ID NO. 3 and SEQ ID NO.
4 were used as a forward primer and a reverse primer, respectively.
In both cases, the chromosomal DNA of Escherichia coli (Escherichia
coli str. K-12 substr. MG1655) was used as a PCR template. In the
case of bifunctional glutathione synthetase, SEQ ID NO. 5 and SEQ
ID NO. 6 were used as a forward primer and a reverse primer,
respectively, and the chromosomal DNA of Streptococcus agalactiae
(Streptococcus agalactiae str. 2603V/R, ATCC BAA-611) was used as a
PCR template. The recognition sequences of a restriction enzyme,
Bsa I, and additional sequences recommended by IBA Co. were
inserted at both ends of the gene fragments amplified by PCR, and
the resulting sequences were ligated to expression vectors,
pASK-IBA7plus (in the case of .gamma.-glutamylcysteine synthetase
and bifunctional glutathione synthetase) and pASK-IBA3plus (in the
case of glutathione synthetase), provided by IBA Co. As a result,
gene constructs encoding .gamma.-glutamylcysteine synthetase and
bifunctional glutathione synthetase, respectively, in which a
strep-tag was attached at the N-terminal, and a gene construct
encoding glutathione synthetase, in which a strep-tag was attached
to the C-terminal, were obtained.
TABLE-US-00002 TABLE 2 Primers for amplifying gene encoding
.gamma.- glutamylcysteine synthetase SEQ ID NO. Direction Sequences
1 gshA-F 5'-AAAAAAGGTCTCTGCGCTTGATCCCGGACG TATCACA-3' 2 gshA-R
5'-AAAAAAGGTCTCTTATCATCAGGCGTGTTT TTCCAGCC-3'
TABLE-US-00003 TABLE 3 Primers for amplifying gene encoding
glutathione synthetase SEQ ID NO. Direction Sequences 3 gshB-F
5'-AAAAAAGGTCTCTAATGATCAAGCTCGGC ATCGT-3' 4 gshB-R
5'-AAAAAAGGTCTCTGCGCTCTGCTGCTGTA AACGTGCTT-3'
TABLE-US-00004 TABLE 4 Primers for amplifying gene encoding
bifunctional glutathione synthetase SEQ ID NO. Direction Sequences
5 gshF-F 5'-AAAAAAGGTCTCAGCGCATGATTATCGAT CGACTGTTAC-3' 6 gshF-R
5'-AAAAAAGGTCTCGTATCATTATAATTCTG GGAACAGTTTAG-3'
Example 3: Purification of Enzymes that Catalyze Glutathione
Synthesis Reaction
[0056] Escherichia coli strains BL21 (DE3) were transformed with
the expression vectors prepared in Example 2, and transformed
strains overexpressing .gamma.-glutamylcysteine synthetase,
glutathione synthetase and bifunctional glutathione synthetase,
respectively, were obtained. The same method was used for purifying
these enzymes. First, 5 ml of LB (Luria-Bertani) medium containing
50 .mu.g/ml ampicillin was added to a test tube, and the
transformed strain was inoculated in the test tube, followed by
shaking culture at 250 rpm and 37.degree. C. for 12 hours. The
cells, then, were inoculated into a 1 L flask filled with 500 ml LB
medium containing 50 .mu.g/ml ampicillin. At the time of
inoculation, an initial absorbance at 600 nm was adjusted to 0.05,
and then shaking culture was performed at 250 rpm and 37.degree. C.
until absorbance reached 0.4. Anhydrotetracycline was added to the
culture at a concentration of 0.2 .mu.g/ml and shaking culture was
further continued at 250 rpm and 30.degree. C. for about 12 hours.
After culture, cells were centrifuged at 4,000 g for 10 minutes,
and a cell pellet was obtained, followed by suspension in 10 ml of
buffer W (100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA). A
protease inhibitor mixture (Roche) was added thereto in an amount
recommended by the manufacturer, and the cell pellet was lysed
using a sonicator (Branson sonifier 250). Sonication was performed
on the suspended cell pellet for 5 minutes at the intensity of
output 3, followed by cooling in ice water for 5 minutes. This
process was repeated three times. After cell lysis, centrifugation
was performed at 6,000 g for 10 minutes to separate a supernatant
containing water-soluble enzymes, and the enzymes were purified
using strep-tag affinity chromatography. The strep-tag affinity
chromatography was performed according to the manufacturer (IBA)'s
recommended method. The purified enzymes were verified by 10% SDS
polyacrylamide gel electrophoresis (FIG. 2). The expected molecular
weights were about 58 kDa for .gamma.-glutamylcysteine synthetase
(Watanabe et al. 1986. Nucleic Acids Res. 14: 4393), about 35 kDa
for glutathione synthetase (Gushima et al. 1984. Nucleic Acids Res.
12: 9299), and about 85 kDa for bifunctional glutathione synthetase
(Janowiak and Griffith. 2005. J. Biol. Chem. 280: 11829-11839).
Example 4: Measurement of Adenosine Triphosphate Production
Activity of Chromatophore Membrane Vesicles Under Light
Irradiation
[0057] The quantification and activity measurement of chromatophore
membrane vesicles were carried out according to the method
described in Korean Patent Application No. 10-2014-0151907.
Chromatophore membrane vesicles were quantified using a
bacteriochlorophyll a (bch a) concentration. When measuring
adenosine triphosphate (ATP) production activity, chromatophore
membrane vesicles were used at a concentration of 0.25-0.5 .mu.g
bch a/ml. An appropriate amount of chromatophore membrane vesicles
was added to a buffer containing 10 mM sodium phosphate
(Na.sub.2HPO.sub.4), 2 mM potassium phosphate (KH.sub.2PO.sub.4),
10 mM magnesium chloride (MgCl.sub.2), and 0.4 mM adenosine
diphosphate, and the mixture was allowed to react under anaerobic
conditions in which a temperature was maintained at 30.degree. C.
and light having a luminous intensity of 15 Watts/m.sup.2 was
irradiated by incandescent lamp. The amount of adenosine
triphosphate produced over time was measured by an adenosine
triphosphate detection kit (Sigma-Aldrich). The activity of
chromatophore membrane vesicles was expressed as the amount of
adenosine triphosphate produced per unit time (nmole ATP/min).
Example 5: Measurement of Enzyme Activity of
.gamma.-Glutamylcysteine Synthetase and Glutathione Synthetase
[0058] To measure enzyme activity of .gamma.-glutamylcysteine
synthetase and glutathione synthetase purified in Example 3, a
method of measuring adenosine diphosphate formation (Seelig and
Meister. 1985. Methods in Enzymol. 113: 379-390) was used. A
reaction buffer containing 0.1 mM Tris (Tris-C1, pH 7.6), 10 mM
magnesium chloride (MgCl.sub.2), 0.8 mM adenosine triphosphate
(ATP), 2 mM phosphoenolpyruvate, 0.2 mM reduced nicotinamide
adenine dinucleotide (NADH), 14.3 Unit/ml pyruvate kinase, and 14.3
Unit/ml lactic dehydrogenase was used for activity measurement.
When measuring the activity of .gamma.-glutamylcysteine synthetase,
10 mM L-glutamate and 10 mM L-cysteine were added to the reaction
buffer. When measuring the activity of glutathione synthetase, 10
mM glycine and 1 mM .gamma.-glutamylcysteine were added to the
reaction buffer. The reaction temperature was 30.degree. C. When
.gamma.-glutamylcysteine synthetase and glutathione synthetase
perform enzymatic reactions, adenosine diphosphate is produced, and
at the same time, an amount of reduced nicotinamide adenine
dinucleotide (NADH) equivalent to that of the adenosine diphosphate
is converted into the oxidized form (NAD.sup.+). Since NADH absorbs
light at 340 nm and NAD.sup.+ does not absorb light at 340 nm, the
activity of the two enzymes can be determined by measuring the
decrease in absorbance at 340 nm over time. FIG. 3 is a graph
showing the result of measurement of .gamma.-glutamylcysteine
synthetase activity, and FIG. 4 is a graph showing the result of
measurement of glutathione synthetase activity. When the enzyme
activity was calculated, the change in the concentration of NADH
was determined using a molar extinction coefficient (6,220
M.sup.-1cm.sup.-1) at 340 nm, and it was assumed that one
equivalent of the product of each enzyme was produced when one
equivalent of NADH was consumed. The calculated amount of product
per unit time (nmole product/min) is referred to as enzyme
activity.
Example 6: Confirmation of Glutathione Production by
.gamma.-Glutamylcysteine Synthetase and Glutathione Synthetase
[0059] In this example, it was confirmed that glutathione was
produced from L-glutamate, L-cysteine, glycine and adenosine
triphosphate by .gamma.-glutamylcysteine synthetase and glutathione
synthetase. A reaction buffer containing 10 mM sodium phosphate
(Na.sub.2HPO.sub.4), 2 mM potassium phosphate (KH.sub.2PO.sub.4),
10 mM magnesium chloride (MgCl.sub.2), 0.8 mM adenosine
triphosphate (ATP), 10 mM L-glutamate, 10 mM L-cysteine, and 10 mM
glycine was used. To prevent oxidation of the resulting
glutathione, the reaction was performed at 30.degree. C. under
anaerobic conditions. A glutathione detection kit (GSH-Glo.TM.
Glutathione Assay, Promega) was used to detect glutathione. The
activity of each enzyme was measured using the method described in
Example 5, and relative activity of each enzyme was adjusted based
on the measured activity. FIG. 5 is a graph showing the amount of
glutathione produced depending on the relative activity of
.gamma.-glutamylcysteine synthetase. At this time, the relative
activity of glutathione synthetase was fixed at 1, and the relative
activity of .gamma.-glutamylcysteine synthetase was varied based on
the relative activity of glutathione synthetase. As a result, it
was confirmed that glutathione was produced by the two enzymes, and
the minimum activity ratio (i.e., GSH-I:GSH-II of FIG. 5) between
the two enzymes with the highest glutathione production was about
12:1. Thus, in the following example, the ratio of
.gamma.-glutamylcysteine synthetase to glutathione synthetase was
used at 12:1.
Example 7: Confirmation of Glutathione Production by Chromatophore
Membrane Vesicles, .gamma.-Glutamylcysteine Synthetase and
Glutathione Synthetase Under Light Conditions
[0060] In this example, it was confirmed that glutathione was
produced by chromatophore membrane vesicles,
.gamma.-glutamylcysteine synthetase, and glutathione synthetase in
the conditions in which adenosine triphosphate (ATP) was not
supplied but light was irradiated. A reaction buffer containing 10
mM sodium phosphate (Na.sub.2HPO.sub.4), 2 mM potassium phosphate
(KH.sub.2PO.sub.4), 10 mM magnesium chloride (MgCl.sub.2), 0.4 mM
adenosine diphosphate (ADP), 10 mM L-glutamate, 10 mM L-cysteine,
and 10 mM glycine was used, and the reaction was performed at
30.degree. C. under anaerobic conditions. An incandescent lamp with
a luminous intensity of 15 Watts/m.sup.2 was used as a light
source. FIG. 6 is a graph showing the amount of glutathione
produced depending on the relative activity of chromatophore
membrane vesicles. At this time, the relative activity of
.gamma.-glutamylcysteine synthetase to the relative activity of
glutathione synthetase was fixed at 12:1. As a result, it was
confirmed that the amount of glutathione produced (or the rate of
glutathione produced) was proportional to the amount of
chromatophore membrane vesicles. In addition, when the relative
activity of chromatophore membrane vesicles was 25 or more, the
amount of glutathione produced was close to maximum.
Example 8: Measurement of Activity of Bifunctional Glutathione
Synthetase on Glutathione Production
[0061] In this example, it was confirmed that glutathione was
produced by bifunctional glutathione synthetase in a reaction
solution containing L-glutamate, L-cysteine, glycine, and adenosine
triphosphate. The composition of the reaction solution was 10 mM
sodium phosphate (Na.sub.2HPO.sub.4), 2 mM potassium phosphate
(KH.sub.2PO.sub.4), 10 mM magnesium chloride (MgCl.sub.2), 100 mM
L-glutamate, 10 mM L-cysteine, 25 mM glycine, and 0.8 mM adenosine
triphosphate (ATP). The reaction was performed at 30.degree. C.
FIG. 7 is a graph showing the activity (nmole glutathione/min) of
bifunctional glutathione synthetase for producing glutathione,
wherein the activity was determined by measuring the amount of
glutathione produced over time in each reaction solution containing
a different amount of bifunctional glutathione synthetase. Since a
lag phase was present at an early stage of the reaction catalyzed
by bifunctional glutathione synthetase, the activity was calculated
based on the reaction rate of the linear region.
Example 9: Confirmation of Glutathione Production by Chromatophore
Membrane Vesicles and Bifunctional Glutathione Synthetase Under
Light Conditions
[0062] In this example, it was confirmed that glutathione was
produced from L-glutamate, L-cysteine, and glycine when
chromatophore membrane vesicles and bifunctional glutathione
synthetase were used together under conditions in which adenosine
diphosphate was added instead of adenosine triphosphate and light
was irradiated. 0.4 mM adenosine diphosphate was added in place of
0.8 mM adenosine triphosphate in the reaction solution of Example
8. The reaction was performed under anaerobic conditions in which
temperature is maintained at 30.degree. C. and light was irradiated
by an incandescent lamp with a luminous intensity of 15
Watts/m.sup.2. FIG. 8 is a graph showing the amount of glutathione
produced depending on the relative activity of chromatophore
membrane vesicles while the relative activity of bifunctional
glutathione synthetase was fixed at 1. It was confirmed that the
amount of glutathione produced was proportional to the amount of
chromatophore membrane vesicles.
[0063] The present invention has been described in detail with
reference to preferred embodiments. It will be apparent to those
skilled in the art that the preferred embodiments are only
illustrative and that the scope of the present invention is not
limited thereto. Accordingly, the actual scope of the present
invention will be defined by the appended claims and equivalents
thereof.
Sequence CWU 1
1
6137DNAArtificial Sequencegamma-glutamylcysteine synthetase forward
primer (gshA-F) 1aaaaaaggtc tctgcgcttg atcccggacg tatcaca
37238DNAArtificial Sequencegamma-glutamylcysteine synthetase
reverse primer (gshA-R) 2aaaaaaggtc tcttatcatc aggcgtgttt ttccagcc
38334DNAArtificial Sequenceglutathione synthetase forward primer
(gshB-F) 3aaaaaaggtc tctaatgatc aagctcggca tcgt 34438DNAArtificial
Sequenceglutathione synthetase reverse primer (gshB-R) 4aaaaaaggtc
tctgcgctct gctgctgtaa acgtgctt 38539DNAArtificial
Sequencebifunctional gamma-glutamylcysteine synthetase/ glutathione
synthetase forward primer (gshF-F) 5aaaaaaggtc tcagcgcatg
attatcgatc gactgttac 39641DNAArtificial Sequencebifunctional
gamma-glutamylcysteine synthetase/ glutathione synthetase reverse
primer (gshF-R) 6aaaaaaggtc tcgtatcatt ataattctgg gaacagttta g
41
* * * * *