U.S. patent number 4,594,321 [Application Number 06/479,979] was granted by the patent office on 1986-06-10 for process for producing 3-deoxyguanosine.
This patent grant is currently assigned to Yamasa Shoyu Kabushiki Kaisha. Invention is credited to Tetsuro Fujishima, Shinji Sakata.
United States Patent |
4,594,321 |
Fujishima , et al. |
June 10, 1986 |
Process for producing 3-deoxyguanosine
Abstract
Glycosylation or transglycosylation of a specified guanine
derivative, namely 9-substituted or non-substituted guanine of
formula [I] with a 3-deoxyribose donor such as 3'-deoxyadenosine in
the presence of a nucleoside phosphorylase source such as of
microorganism origin is disclosed.
Inventors: |
Fujishima; Tetsuro (Choshi,
JP), Sakata; Shinji (Choshi, JP) |
Assignee: |
Yamasa Shoyu Kabushiki Kaisha
(Choshi, JP)
|
Family
ID: |
12988665 |
Appl.
No.: |
06/479,979 |
Filed: |
March 29, 1983 |
Foreign Application Priority Data
|
|
|
|
|
Apr 1, 1982 [JP] |
|
|
57-55078 |
|
Current U.S.
Class: |
435/89; 435/194;
435/824; 435/840; 435/87; 435/874; 435/88; 544/244; 544/276 |
Current CPC
Class: |
C12N
9/1077 (20130101); C12P 19/40 (20130101); Y10S
435/824 (20130101); Y10S 435/84 (20130101); Y10S
435/874 (20130101) |
Current International
Class: |
C12P
19/00 (20060101); C12P 19/40 (20060101); C12N
9/10 (20060101); C12P 019/30 (); C12P 019/38 ();
C12P 019/40 (); C12N 009/12 () |
Field of
Search: |
;435/87,88,89,193,194,824,840,874 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Tarr, H. L. A., Methods in Enzymology, vol. XII, Part A pp.
113-118, 1968. .
"The Condensed Chemical Dictionary, 5th Ed. Reinhold Publishing
Corp., 1956, pp. 348-349, 792-793..
|
Primary Examiner: Wiseman; Thomas G.
Assistant Examiner: Huleatt; Jayme A.
Attorney, Agent or Firm: Wenderoth, Lind & Ponack
Claims
What is claimed is:
1. A process for producing 3'-deoxyguanosine, which comprises
reacting a guanine derivative represented by the formula: ##STR2##
wherein R is selected from the group consisting of a hydrogen atom,
a ribose-1-ly group, a 2-deoxyribose-1-yl group and a
monophosphate, a diphosphate or a triphosphate thereof with a
3-deoxyribose donor at a reaction temperature of 40 to 75 C. and at
a pH of 5.0 to 9.0, in the presence of a nucleoside phosphorylase
source in the presence of a phosphoric acid ion donor, which source
is capable of providing 3'-deoxyguanosine by the reaction of the
guanine derivative with the 3-deoxyribose donor in the presence of
a phosphoric acid ion donor to obtain 3-deoxyguanosine, said
nucleoside phosphorylase source being derived from a microorganism
which belongs to the genus Pseudomonas, Brevibacterium, or
Achromobacter.
2. A process for producing 3'-deoxyguanosine according to claim 1,
wherein the 3-deoxyribose donor is selected from the group
consisting of 3'-deoxyadenosine, 3'-deoxyinosine, and mono-, di-
and triphosphates thereof and 3-deoxyribose-1-phosphate.
3. A process for producing 3'-deoxyguanosine according to claim 1,
wherein the nucleoside phosphorylase source is a culture, intact
cells or modified cells of a microorganism which belongs to a genus
Pseudomonas, Brevibacterium or Achromobacter and is capable of
producing 3'-deoxyguanosine from the guanine derivative of the
formula and the 3-deoxyribose donor, said modified cells being
dried cells, cells having denatured cell membranes, cells having
denatured cell walls, crushed cells, immobilized cells, or
enzymatically active cells.
4. A process for producing 3'-deoxyguanosine according to claim 1,
wherein the guanine derivative is guanine, guanosine,
2'-deoxyguanosine, guanosine-5'-monophosphate,
guanosine-5'-diphosphate, guanosine-5'-triphosphate or
2'-deoxyguanosine-5'-monophosphate.
5. A process for producing 3'-deoxyguanosine according to claim 1,
wherein the genus is Pseudomonas.
6. A process for producing 3'-deoxyguanosine according to claim 5,
wherein the microorganism belonging to the genus Pseudomonas is
Pseudomonas desmolytica J-4-2, ATCC 39310 or a microorganism
derived therefrom having an ability to produce the nucleoside
phosphorylase source.
7. A process for producing 3'-deoxyguanosine according to claim 5,
wherein the microorganism belonging to the genus Pseudomonas is
Pseudomonas desmolytica.
8. A process for producing 3'-deoxyguanosine according to claim 1,
wherein the genus is Brevibacterium.
9. A process for producing 3'-deoxyguanosine according to claim 8,
wherein the microorganism belong to the genus Brevibacterium is
Brevibacterium acetylicum AT-6-7, ATCC 39311 or a microorganism
derived therefrom having an ability to produce the nucleoside
phosphorylase source.
10. A process for producing 3'-deoxyguanosine according to claim 8,
wherein the microorganism belonging to the genus Brevibacterium is
Brevibacterium acetylicum.
11. A process for producing 3'-deoxyguanosine according to claim 1,
wherein the genus is Achromobacter.
12. A process for producing 3'-deoxyguanosine according to claim
11, wherein the microorganism belonging to Achromobacter is
Achromobacter eurydice BE-3-3, ATCC 39312 or a microorganism
derived therefrom having an ability to produce the nucleoside
phosphorylase source.
13. A process for producing 3'-deoxyguanosine according to claim
11, wherein the microorganism belonging to the genus Achromobacter
is Achromobacter eurydice.
Description
BACKGROUND OF THE INVENTION
This invention relates to an enzymatic process for producing
3'-deoxyguanosine.
3'-Deoxyguanosine is a compound presently attracting attention
which not only exhibits radiation sensitizing action in therapy of
cancers but also has an action to enhance the effect of various
anticancer agents when employed in combination therewith (see
Japanese Laid-open Publication No. 35516/1982).
In the prior art, as the method for preparation of
3'-deoxyguanosine, is known the method in which a chloromercuri of
2-acetamidohypoxanthine is condensed with
2,5-di-O-benzoyl-3-deoxy-D-ribofuranosyl bromide [The Journal of
Organic Chemistry, 30, 2851 (1965)]. However, this method is
believed to involve various drawbacks in commercial production such
as, for example, difficult availability of the starting material
3-deoxyribose, formation of isomers during the condensation
reaction, use of a harmful mercuric salt, and others.
SUMMARY OF THE INVENTION
The present inventors have made various investigations in order to
overcome these drawbacks of the prior art and consequently found
that 3-deoxyguanosine can be formed by causing a guanine derivative
to react with a 3'-deoxyribose donor in the presence of a
nucleoside phosphorylase source. The present invention has been
accomplished based on such a finding.
The present invention provides a process for producing
3'-deoxyguanosine, which comprises causing a guanine derivative
represented by the formula [I]: ##STR1## wherein R designates a
hydrogen atom or a ribose-1-yl group, a 2-deoxyribose-1-ly group or
a monophosphate, a diphosphate or a triphosphate thereof, to react
with a 3-deoxyribose donor in the presence of a nucleoside
phosphorylase source to obtain 3'-deoxyguanosine.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is to be described in detail below.
Guanine derivative
As the starting quanine derivatives, there may be employed one or
more of the compounds included within the aforementioned
definition. More specifically, illustrative of these compounds are
guanine (Gua), guanosine (Guo) or 2'-deoxyguanosine (2'-dGuo) or
monophosphates, diphosphates or triphosphates thereof. The above
nucleotide may have a phosphoryl group at any position of hydroxyl
groups in the sugar residue. Typical examples of these nucleotides
may include guanosine-5'-monophosphate (GMP),
guanosine-3'-monophosphate, guanosine-2'-monophosphate,
guanosine-5'-diphosphate (GDP), guanosine-5'-triphosphate (GTP),
2'-deoxyguanosine-5'-monophosphate (2'-dGMP),
2'-deoxyguanosine-3'-monophosphate,
2'-deoxyguanosine-5'-diphosphate (2'-dGDP),
2'-deoxyguanosine-5'-triphosphate (2'-dGTP) and the like, which may
be either in free acid form or in an appropriate salt form such as
sodium salt.
3-Deoxyribose donor
As the other starting material, a 3-deoxyribose donor, there may be
employed any compounds which are capable of enzymatically
introducing 3-deoxyribose moiety to the 9-position of the guanine
moiety of the compound of formula [I] through direct glycosylation
or transglycosylation. Typical examples include one or more of
3'-deoxyadenosine (3'-dAdo; cordycepin), 3'-deoxyinosine (3'-dIno),
or monophosphates, diphosphates or triphosphates thereof, or
3-deoxyribose-1-mmonophosphate. The above phosphates may have
phosphoryl group at any position of hydroxyl group in
3-deoxyribose, and may also be either in free acid form or in a
salt form. Illustrative of these nucleotides are
3'-deoxyadenosine-5'-monophosphate (3'-dAMP),
3'-deoxyinosine-5'-monophosphate (3'-dIMP) or
3-deoxyribose-1-phosphate.
Nucleoside phosphorylase source
The nucleoside phosphorylase which is in the nucleoside
phosphorylase source in the reaction of the present invention
refers comprehensively to a single enzyme or a plurality of enzymes
capable of providing 3'-deoxyguanosine by causing the guanine
derivative to react with the 3-deoxyribose donor in the presence of
a phosphoric acid ion donor. Accordingly, in the present invention,
the term "nucleoside phosphorylase source" includes the enzymes of
the phosphorylase type such as purine nucleoside phosphorylase,
pyrimidine nucleoside phosphorylase, etc., which can be used in
combination with enzymes such as nucleoside-N-glycosyl transferase,
nucleosidase, nucleotidase, phosphatase and others, which may
possibly participate in the reaction of the present invention. The
nucleoside phosphorylase source refers comprehensively to a
material containing such enzymes in any desired form, which is not
limited to its origin or source. That is, any enzyme material may
be applicable so long as it can accomplish the object of the
present invention, irrespective of whether it may be derived from
microorganisms or from animals, or whether it may be prepared in
any form. In particular, a nucleoside phosphorylase source of a
microorganism origin, namely a nucleoside phosphorylase source in
the form of a culture, a mass of intact cells or a modification of
cells of a microorganism, is preferred.
Preferred nucleoside phosphorylase sources of microorganism origin
are those based on microorganisms which belong to genera
Pseudomonas (hereinafter referred to as Ps.), Brevibacterium
(hereinafter referred to as Br.) and Achromobacter (hereinafter
referred to as Ach.).
Typical strains of such microorganisms are:
Ps. desmolytica J-4-2: FERM P-6307, ATCC 39310
Br. acetylicum AT-6-7: FERM P-6305, ATCC 39311
Ach. eurydice BE-3-3: FERM P-6304, ATCC 39312
Typical strains among these are the first three strains, namely
J-4-2 strain isolated from the soil in Nishiashikajima-Cho,
Choshi-Shi, Chiba-Ken, Japan; the AT-6-7 strain isolated from the
sand in the baseball ground of Koshion, Nishinomiya-Shi, Hyogo-Ken,
Japan; and the BE-3-3 strain isolated from the soil in Araoi-Cho,
Choshi-Shi, Chiba-Ken, Japan. The bacteriological properties of
these strains are set forth below.
1. J-4-2 strain
A. Morphology
(1) Form and size of cells: rod-shaped, 0.7-0.8.times.2.0-3.0
.mu.m;
(2) Motility, occurrence of flagella: motile with polar
flagella;
(3) Formation of spores: none
(4) Gram staining: negative
B. Growth on various culture media
(1) Boulillon-agar plate culture (28.degree. C.), 48 hours)
.circle.1 Form of colony: circular
.circle.2 Raising of colony surface: raised or umbonate
.circle.3 Size: 2-4 mm
.circle.4 Color tone: gray to buff when wet
(2) Bouillon-agar slant culture (28.degree. C., 48 hours)
.circle.1 Growth: good
.circle.2 Form of growth: filliform and slightly spreading
.circle.3 Formation of pigment: not clear
(3) Bouillon liquid culture (28.degree. C., 72 hours)
Growth: no membrane formation, turbid throughout the entire liquid,
sediment slightly formed.
(4) Bouillon-gelatin stab culture (20.degree. C., 6 days): not
liquefied
(5) Litmus-milk culture medium (28.degree. C., 4 days): not
changed.
C. Physiological properties:
(1) Reduction of a nitrate (28.degree. C., 5 days): reductive
(2) Formation of hydrogen sulfide (28.degree. C., 5 days):
formed
(3) Hydrolysis of starch: not hydrolyzed
(4) Catalase: positive
(5) Indole formation: negative
(6) Ammonia formation from peptone and arginine: negative
(7) Methyl red test: negative
(8) V-P test: positive
(9) Attitude to oxygen: aerobic
(10) O-F test (by the Hugh Leifson method): O type (Oxidation)
(11) Acid formation from sugars
positive: glucose, mannose, fructose, maltose, saccharose,
trehalose and mannitol;
negative: arabinose, xylose, galactose, lactose, sorbitol, inositol
and glycerine
(12) Growth pH range- 6.0-9.0
(13) Optimum growth temperature: 25.degree.-35.degree. C.
2. AT-6-7 strain
A. Morphology
(1) Form and size of cells: short rod-shaped, 0.8-1.0.times.1.0-1.2
.mu.m;
(2) Formation of spores: none
(3) Gram staining: positive
B. Growth on various culture media
(1) Bouillon-agar plate culture (28.degree. C., 48 hours)
.circle.1 Form of colony: circular
.circle.2 Raising of colony surface: flat, smooth
.circle.3 Size: 2-4 mm
.circle.4 Color tone: yellow to peach-yellow
(2) Bouillon-agar slant culture (28.degree. C., 48 hours)
.circle.1 Growth: good
.circle.2 Form of growth: echinulate
(3) Bouillon liquid culture (28.degree. C., 48 hours)
Growth: formation of ring on the surface, sediment slightly
formed.
(4) Bouillon-gelatin stab culture (20.degree. C., 6 days):
liquefied in stratiform
(5) Litmus-milk culture medium (28.degree. C., 4 days): slightly
coagulated, peptonization also observed
C. Physiological properties:
(1) Reduction of a nitrate (28.degree. C., 5 days): no
reductivity
(2) Formation of hydrogen sulfide (28.degree. C., 5 days): not
formed
(3) Hydrolysis of starch: hydrolyzed
(4) Catalase: positive
(5) Indole formation: not formed
(6) Ammonia formation from peptone and arginine: positive
(7) Methyl red test: negative
(8) V-P test: positive
(9) Attitude to oxygen: aerobic
(10) O-F test (by the Hugh Leifson method): F type
(Fermentation)
(11) Acid formation from sugars
positive: glucose, mannose, fructose, maltose, saccharose and
trehalose;
negative: arabinose, xylose, galactose, lactose, sorbitol, inositol
and glycerine
(12) Growth pH range: pH 6.0-9.0
(13) Optimum growth temperature: 25.degree.-37.degree. C.
BE-3-3 strain
A. Morphology
(1) Form and size of cells: rod-shaped, 0.8-0.9.times.1.4-1.8
.mu.m;
(2) Formation of spores: none
(3) Gram staining: negative
B. Growth on various culture media
(1) Bouillon-agar plate culture (28.degree. C., 48 hours)
.circle.1 Form of colony: rhizoid and lacerate
.circle.2 Raising of colony surface: flat and smooth
.circle.3 Size: 5-9 mm
.circle.4 Color tone: pale grayish brown to bluish gray
(2) Bouillon-agar slant culture (28.degree. C., 48 hours)
.circle.1 Growth: good
.circle.2 Form of growth: filiform
(3) Bouillon liquid culture (28.degree. C., 48 hours)
Growth: turbid throughout the entire liquid, sediment formed.
(4) Bouillon-gelatin stab culture (20.degree. C., 6 days): not
liquefied
(5) Litmus-milk culture medium (28.degree. C., 4 days):
substantially not changed
C. Physiological properties
(1) Reduction of nitrate (28.degree. C., 5 days): no
reductivity
(2) Formation of hydrogen sulfide (28.degree. C., 5 days):
formed
(3) Hydrolysis of starch: not hydrolyzed
(4) Catalase: positive
(5) Indole formation: not formed
(6) Ammonia formation from peptone and arginine: negative
(7) Methyl red test: negative
(8) V-P test: negative
(9) Attitude to oxygen: aerobic
(10) O-F test (by the Hugh Leifson method): O type (Oxidation)
(11) Acid formation from sugars
positive: glucose, mannose, fructose and trehalose;
negative: arabinose, xylose, galactose, maltose, saccharose,
sorbitol, lactose, inositol and glycerine
(12) Growth pH range: pH 6.0-9.0
(13) Optimum growth temperature: 25.degree.-37.degree. C.
The above bacteriological properties were examined with reference
to the taxonomical stnadards in Bergey's Manual of Determinative
Bacteriology, 7th edition (1957). As the result, J-4-2 strain was
identified to be a strain belonging to the genus Pseudomonas from
the various properties such as being a straight short-rod
bacterium, Gram-negative, having polar flagella, having no
spore-forming ability, being oxidative of glucose, etc. and was
designated as Pseudomonas desmolytica J-4-2. The AT-6-7 strain,
which is a short-rod bacterium almost approximate to a coccus,
Gram-positive, forms no filament and forms acids from
carbohydrates, was identified to be a strain belonging to the genus
Brevibacterium and designated as Brevibacterium acetylicum AT-6-7.
The BE-3-3 strain, which is Gram-negative, forms acids from hexose,
forms hydrogen sulfide with its cells being rod-shaped, was
identified to belong to the genus Achromobacter and designated as
Achromobacter eurydice BE-3-3.
The above three microorganism strains were identified according to
Bergey's Manual of Bacteriology, 7th edition, and it is possible
that they may belong to other species or genus when these strains
are to be identified to belong to other species or genus according
to different taxonomical standards due to some changes in
taxonomical standards in the future. However, the microorganisms as
designated above are inclusive of microorganisms which can at least
produce nucleoside phosphorylase source in conformity with the
object of the present invention and has the aforesaid
bacteriological properties or bacteriological properties equivalent
thereto, and can be unequivocally specified.
These three microorganism strains were deposited at the
Fermentation Research Institute, Agency of Industrial Science &
Technology on Jan. 13, 1982, under the following deposition numbers
(FERM P. No.). Further, these strains were sent directly from the
Fermentation Research Institute to the American Type Culture
Collection (ATCC), 12301 Parklawn Drive, Rockville, MD., U.S.A. for
deposition, and deposited on Mar. 2, 1983, under the following
deposition numbers (ATCC No.).
(1) Pseudomonas desmolytica J-4-2, FERM P-6307, ATCC 39311
(2) Brevibacterium acetylicum AT-6-7, FERM P-6305, ATCC 39311
(3) Achromobacter eurydice BE-3-3, FERM P-6304, ATCC 39312 The
mutant strains derived from the above microorganism strains through
induced mutation according to the mutagenic methods in general by a
physical treatment such as irradiation of UV-ray, X-ray or
.gamma.-ray or a chemical treatment with nitrosoguanidine or other
mutagens or natural mutation attributable to natural causes may be
also available in the present invention, so long as they do not
lose the ability to produce nucleoside phosphorylase source
suitable for the object of the present invention.
Further, when the gene for nucleoside phosphorylase source suitable
for the object of the present invention of the microorganism
strains preferably used in the present invention as described above
is integrated in a microorganism other than the genera Pseudomonas,
Brevibacterium and Achromobacter if the characteristic of such a
gene is phenotypically expressed, the method of employing the
culture, the intact cells of such a microorganism or the
modification thereof for the object of the present invention may
also be included within the present invention.
In cultivation of these microorganisms to produce a nucleoside
phosphorylase source, the culture medium and the method of culture
employed are not particularly limited, so far as growth of these
microorganisms is concerned.
As a culture medium, there may be employed one containing
appropriate amounts of a carbon source and a nitrogen source
assimilable by these microorganisms, optionally added with an
inorganic salt, minute amounts of a growth promoter, defoaming
agents, etc. More specifically, as carbon sources, there may be
employed one or more of those selected suitably in view of
assimilability by the microorganism employed from carbon sources in
general, including sugars such as glucose, fructose, maltose,
galactose, ribose, saccharose, starch, starch hydrolysate,
molasses, waste molasses, etc. or derivatives thereof such as fatty
acid esters thereof; natural carbohydrates such as wheat, wheat
bran, rice, etc.; alcohols such as glycerol, mannitol, methanol,
ethanol, etc.; fatty acids such as gluconic acid, pyruvic acid,
acetic acid, citric acid, etc.; hydrocarbons such as normal
paraffins, kerosene, etc.; amino acids such as glycine, glutamic
acid, glutamine, alanine, asparagine, etc.; and so on. As nitrogen
sources, there may be employed one or more of those selected
suitably in view of assimilability by the microorganism employed
from nitrogen sources in general, including organic nitrogenous
materials such as meat extract (bouillon), peptone, yeast extract,
dry yeast, soybean hydrolysate, soybean powder, milk casein,
casamino acid, various amino acids, corn steep liquor, cotton seed
meal or its hydrolysate, fish meal or its hydrolysate, hydrolysates
of other animals, vegetables, microoganisms, etc.; inorganic
nitrogen compounds such as ammonia, ammonium salts such as ammonium
nitrate, ammonium sulfate, ammonium chloride, ammonium phosphate,
ammonium carbonate, ammonium acetate and the like, nitric acid
salts such as sodium nitrate, urea, and so on. Further, as
inorganic salts, there may suitably be added one or more, in minute
amounts, or phosphates, hydrochlorides, sulfates, carbonates,
nitrates, acetates and others of magnesium, manganese, iron, zinc,
copper, sodium, calcium, potassium, etc. If necessary, there may
also be added a defoaming agent such as a vegetable oil or a
surfactant, a minute amount of a growth promoter such as vitamins
B.sub.1, B.sub.2, nicotinic acid, pantothenic acid, biotin,
P-aminobenzoic acid, etc. When employing a microorganism exhibiting
nutrient requirements, substances satisfying its growth must be
added into the culture medium as a matter of course.
Cultivation may be performed in a liquid medium containing the
above culture medium components by selecting a culture method
suitable for the microorganism employed from conventional culture
methods such as shaking culture, aerating stirring culture,
stationary culture, continuous culture and others.
The cultural conditions may be suitably chosen depending on the
microorganism and the culture medium employed, but generally by
adjusting before start-up of cultivation at pH of about 6 to 8 and
carrying out cultivation under the temperature condition of about
25.degree. to 35.degree. C. The culture duration may be a period
sufficient for growth of the microorganism employed, being
generally 1 to 3 days.
After culturing the microorganism as described above, the culture,
the intact microbial cells collected from the culture according to
a conventional method such as centrifugation, sedimentation
separation, agglomeration separation, or a modification of
microbial cells obtained by applying a suitable treatment on the
living or intact cells may be used as the nucleoside phosphorylase
source of the present invention. The "culture" herein refers to a
product under the state where the culture medium and the cultured
microbial cells after cultivation are still unseparated from each
other. The "modification of cells" refers to dried microbial cells,
microbial cells whose cell wall membrane having been modified,
crushed microbial cells, immobilized microbial cells, extracts of
microbial cells, protein fractions having nucleoside phosphorylase
activity of extract of microbial cells or purified product thereof,
immobilized product of the protein fractions or purified product
thereof, and the like. Methods for obtaining the modification of
microbial cells are to be illustrated below. Modifications of
microbial cells can be obtained by applying on intact microbial
cells singly or in combination physical treatment means such as
freezing-thawing, lyophilization, air drying, acetone drying,
heating under acidic or alkaline conditions, grinding, ultrasonic
treatment, osmotic treatment, etc. or chemical or biochemical
treatments such as enzymatic treatments with lysozyme, cell wall
lysing enzymes. etc., contact treatments with solvents such as
toluene, xylene, butyl alcohol or surfactants, or by applying on
the extract of microbial cells singly or in combination enzyme
separation and purification means such as salting-out, isoelectric
precipitation, precipitation with organic solvents, various
chromatographies, dialysis and others, or further by applying on
intact microbial cells, extracts of microbial cells or purified
products thereof an enzyme or cell immobilization means such as
inclusion method, crosslinking method, adsorption method onto a
carrier, etc.
Glycosylation or transglycosylation
The reaction in accordance with the present invention, namely
enzymatic reaction of a guanine derivative of formula [I] with a
3-deoxyribose donor, which is glycosylation when the substituent R
is hydrogen and is otherwise transglycosylation, is carried out by
bringing a guanine derivative and a 3-deoxyribose donor into
contact with the nucleoside phosphorylase source as described above
in an aqueous medium. The kinds of the enzyme substrates are
selected according to the kind of the enzyme source employed.
In a preferred embodiment of the present invention, there are two
methods available to effect the contact.
The first method is one in which a guanine derivative and a
3-deoxyribose donor are caused to be present in the culture medium
during cultivation of the aforesaid microorganism, thereby
accumulating 3'-deoxyguanosine in the culture medium.
The above method may be carried out by adding necessary amounts of
a guanine derivative and a 3-deoxyribese donor in the culture
medium prior to cultivation and cultivating the microorganism
therein, or by adding these substances at once at an appropriate
period of time during cultivation, or by carrying out cultivation
while adding intermittently or continuously these substances.
The second method may be carried out by bringing a culture, intact
microbial cells or a modification of cells into contact with an
aqueous medium containing a guanine derivative and a 3-deoxyribose
donor under the conditions capable of forming
3'-deoxyguanosine.
An aqueous medium in which the contact between the reactants
concerned is to take place may be water or various buffers
preferred for enzymatic reactions (e.g. phosphate buffers,
imidazole-hydrochloric acid buffer, veronal-hydrochloric acid
buffer, Tris-hydrochloric acid buffer), which contains a phosphate
ion generating source and may also contain various substances, if
desired.
The enzymatic reaction of the present invention is mainly based on
the action of phosphorylase, and therefore a phosphate ion must
exist in the reaction system. In the case where a phosphate ion
does not exist in the reaction system, an addition of a phosphate
ion generating substance is necessary. As the phosphate ion
generating substance, there may be employed any compound
dissociable into phosphate ion in an aqueous medium, such as
phosphoric acid itself, inorganic phosphoric acid salts such as
salts of alkali metals, for example, sodium, potassium and the
like, alkaline earth metals, for example, calcium, magnesium and
the like or ammonium. These phosphate generating sources may be
employed in amounts of about 1.0 to 2.5-fold moles per mole as
phosphate ions per mole of the guanine derivative. As substances
other than the phosphate ion generating source which may be
contained in the aqueous medium, there may be employed sugars such
as glucose, sacchrose and the like, organic solvents such as
methanol, ethanol, propanol, butanol, pentanol, toluene, xylene,
ethyl acetate and the like, various surfactants, metal salts and so
on.
As the method to bring a nucleoside phosphorylase source into
contact with a guanine derivative and a 3-deoxyribose donor in an
aqueous medium, there may be employed the method in which the
nucleoside phosphorylase source is suspended or dissolved in an
aqueous medium containing these reaction substrates, optionally
with stirring or shaking, or the method in which these reaction
substrates are added at once, intermittently or continuously into a
suspension or a solution of the nucleoside phosphorylase source in
a reaction medium, or the method in which the nucleoside
phosphorylase source is packed in a column optionally admixed with
a suitable diluent or carrier or immobilized onto a membrane and an
aqueous medium containing the reaction substrates is passed
therethrough.
During the reaction, the substrate concentration is not
particularly limited, and the reaction may be carried out under a
suspended state of the substrates. But each reaction substrate is
used usually at a concentration within the range from 5 to 50 mM,
preferably about 15 to 35 mM for a guanine derivative and about 15
to 30 mM for 3-deoxyribose donor. The nucleoside phosphorylase
source may be employed in an amount, which can easily be determined
by those skilled in the art by considering the particular source
material employed, the concentrations of the reaction substrates,
the reaction efficiency and economy.
The reaction conditions, which are not particularly limited and may
be determined while considering the optimum temperature and the
optimum pH for the enzymatic action of the nucleoside phosphorylase
source, stability of the substrates and reaction efficiency, may
generally comprise a temperature of 40.degree. to 75.degree. C.,
preferably 50.degree. to 70.degree. C. and a pH 5.0 to 9.0,
preferably 6.0 to 8.0. When pH is changed during the reaction, an
acid or an alkali can be used to correct the pH to a preferred
level. When a nucleoside phosphorylase source derived from the
aforesaid three microorganism strains of genera Pseudomonas,
Brevibacterium and Achromobacter, the optimum temperature is around
50.degree. to 70.degree. C. and the reaction can be carried out at
a relatively higher temperature, whereby there is the advantage
that no countermeasure against microorganism contamination is
required to be considered.
The reaction time, which may be determined while confirming the
conversion of the reaction substrates to the desired product, may
be generally about 15 to 45 hours, preferably 24 to 36 hours, in a
batch system. In a columnar system, the reaction may be carried out
under appropriate conditions set analogously as in the batch
system.
After the enzymatic reaction, the nucleoside phosphorylase source
may be removed by separation in a conventional manner, and the
residual product is subjected to the step for isolation and
purification of 3'-deoxyguanosine.
Isolation and purification of 3'-deoxyguanosine may be performed
according to any of the methods known in the art by using
separation/purification methods singly or in combination such as
various chromatographies, for example, ion-exchange chromatography,
adsorption chromatography, partition chromatography, gel
filtration, etc., the counter-current partition method, the
recrystallization method and others.
Examples of the preferred embodiments
The present invention is to be described in further detail below by
referring to Examples, each of which is illustrative of an
embodiment of the present invention and not limitative of the scope
of the present invention. In Examples, analysis of
3'-deoxyguanosine was conducted by high performance liquid
chromatography. When analyzed by means of the device and under the
conditions shown below, 3'-deoxyguanosine is eluted at a retention
time around 12.90 minutes and its quantity can be calculated from
the calibration curve.
Device: Shimadzu High Performance Liquid Chromatograph LC-3A model
(produced by Shimadzu Corporation)
Column: Sorbax ODS, 4.6 mm.times.250 mm (Shimadzu Du Pont Co.)
Eluant: 20 mM Tris-hydrochloric acid buffer containing 5%
acetonitrile (pH 7.5)
Flow rate: 1 ml/minute
Column operation temperature: room temperature
EXAMPLE 1
Two liters of a 2% bouillon culture medium were sterilized at
120.degree. C. for 15 minutes and cooled. Then, 100 ml of a
previously precultured culture broth of Brevibacterium acetylicum
AT-6-7 (FERM P-6305) was added to the culture broth and cultivation
was carried out at 28.degree. C. for 22 hours.
After completion of the cultivation, the cells were collected by
centrifugation and added into 200 ml of a sterilized water to be
suspended therein. Into 200 ml of a substrate solution (pH 7.0)
containing 25 mM GMP (disodium salt), 25 mM 3'-dAdo and 30 mM
monosodium dihydrogen phosphate was added the above cell suspension
and the reaction was carried out at 60.degree. C. for 36 hours.
The reaction mixture after removal of the cells by centrifugation
was analyzed by high performance liquid chromatography to show that
the yield of 3'-deoxyguanosine was 36.58%. The yield of
3'-deoxyguanosine is defined as the molar ratio (%) of
3'-deoxyguanosine formed to 3'-deoxyadenosine added.
EXAMPLE 2
Cultivation was carried out in the same manner as in Example 1
except that Pseudomonas desmolytica J-4-2 (FERM P-6307) was used,
and cells were collected and suspended in a sterilized water to
obtain 200 ml of a cell suspension.
Into 200 ml of a substrate solution (pH 7.0) containing 25 mM Guo,
25 mM 3'-dAdo and 35 mM monopotassium dihydrogen phosphate was
added the above cell suspension and the reaction was carried out at
60.degree. C. for 36 hours. The cells were removed by
centrifugation and the reaction mixture was analyzed to show that
the yield of 3'-deoxyguanosine was 42.09%. The reaction mixture was
diluted to one liter (pH 9.0), treated with an anion exchange resin
"Diaion SA-12A" (trade name; produced by Mitsubishi Kasei Kogyo
Co., Ltd.) (borate form) and the solution which had passed through
the column and the water washings were combined and adsorbed on a
cation exchange resin "Diaion PK-216" (trade name; produced by
Mitsubishi Kasei Kogyo Co., Ltd.) (free acid form), followed by
elution. The fractions of 3'-deoxyguanosine were neutralized,
concentrated and cooled. The crude crystals precipitated were
recrystallized from hot water to obtain 449 mg of 3'-deoxyguanosine
crystals.
EXAMPLE 3
After cultivation of the same microorganism as in Example 2
conducted in the same manner except for using each 100 ml of a
bouillon medium, cells were collected from each culture broth and
10 ml of a sterilized water was added to respective cells to
prepare each cell suspension. To each of the suspensions was added
each 10 ml of the solutions containing 20 mM 3'-dAdo, 25 mM
monopotassium dihydrogen phosphate and 20 mM of the guanine
derivative (Table 1), and the reaction was carried out at
60.degree. C. for 24 hours. After the reaction, the supernatant
obtained by centrifugation was analyzed to give the
3'-deoxyguanosine yield as shown in Table 1.
TABLE 1 ______________________________________ 3'-Deoxyguanosine
yield Guanine derivative (%) ______________________________________
Gua 2.14 Guo 26.36 GMP 21.72
______________________________________
When the same experiments were performed for Brevibacterium
acetylicum AT-6-7 (FERM P-6305) and Achromobacter euridice BE-3-3
(FERM P-6304), the similar results as shown above were obtained
with respect to the reactivities for guanine derivatives.
EXAMPLE 4
Example 3 was repeated by use of Brevibacterium acetylicum AT-6-7
(FERM P-6305) as the nucleoside phosphorylase source, GMP as the
guanine derivative and the respective enzymatic reaction
temperature of 40.degree. to 80.degree. C. (Table 2), under
otherwise the same conditions as in Example 3, and
3'-deoxyguanosines formed were analyzed to give the results as
shown in Table 2.
TABLE 2 ______________________________________ 3'-Deoxyguanosine
yield Reaction temperature (%)
______________________________________ 40.degree. C. 5.81
50.degree. C. 12.32 60.degree. C. 20.86 70.degree. C. 4.32
80.degree. C. 0 ______________________________________
When the same experiments were performed for the BE-3-3 strain
(FERM P-6304) and the J-4-2 strain (FERM P-6307), substantially the
similar results were obtained with respect to the effect of the
reaction temperature.
EXAMPLE 5
Example 3 was repeated by use of Achromobacter euridice BE-3-3
(FERM P-6304) as the nucleoside phosphorylase source, GMP as the
guanine derivative and the respective enzymatic reaction pH's of
6.0 to 9.0 (Table 2), under otherwise the same conditions as in
Example 3, and 3'-deoxyguanosines formed were analyzed to give the
results as shown in Table 3.
TABLE 3 ______________________________________ 3'-Deoxyguanosine
yield Reaction pH (%) ______________________________________ 6.0
19.70 7.0 21.26 8.0 19.46 9.0 11.28
______________________________________
When the same experiments were performed for the AT-6-7 strain
(FERM P-6305) and the J-4-2 strain (FERM P-6307), substantially the
similar results were obtained with respect to the effect of pH.
EXAMPLE 6
After cultivation was carried out in the same manner as in Example
1 by use of the same nucleoside phosphorylase source as in Example
5, the cells were collected and suspended in a sterilized water to
obtain each 1 ml of cell suspensions. As the substrate solutions,
there were prepared various combinations of aqueous solutions (pH
7.0) containing as a 3-deoxyribose donor 15 mM of 3'-dAdo, 3'-dAMP
or 3'-dIno, as a guanine derivative 15 mM of GMP, GDP, GTP, 2'-dGuo
or 2'-dGMP and 20 mM of monopotassium phosphate. Such cell
suspensions and substrate solutions were mixed, respectively, and
the reactions conducted at 60.degree. C. for 24 hours. As the
result, the yields of 3'-deoxyguanosine obtained were as shown in
Table 4.
TABLE 4 ______________________________________ 3'-deoxy- Substrate
solution guanosine 3'-Deoxyribose Guanine yield donor derivative
(%) ______________________________________ 3'-dAdo GMP 62.70
3'-dAdo GDP 56.02 3'-dAdo GTP 14.87 3'-dAdo 2'-dGuo 43.82 3'-dAdo
2'-dGMP 34.50 3'-dAMP GMP 63.48 3'-dAMP GDP 44.74 3'-dAMP GTP 7.66
3'-dAMP 2'-dGuo 27.30 3'-dAMP 2'-dGMP 24.64 3'-dIno GMP 31.04
______________________________________
* * * * *