U.S. patent application number 09/871656 was filed with the patent office on 2001-10-25 for process for producing a cholesterol-reduced substance.
Invention is credited to Aisaka, Kazuo, Ando, Katsuhiko, Katsumata, Ryoichi, Kumazawa, Hideyo, Mizukami, Toru, Ochiai, Keiko, Ouchi, Kozo, Saitoh, Chiaki.
Application Number | 20010034041 09/871656 |
Document ID | / |
Family ID | 17814874 |
Filed Date | 2001-10-25 |
United States Patent
Application |
20010034041 |
Kind Code |
A1 |
Saitoh, Chiaki ; et
al. |
October 25, 2001 |
Process for producing a cholesterol-reduced substance
Abstract
According to the present invention, a process for producing a
practical cholesterol-reduced substance by converting cholesterol
contained in foods and feeds to coprostanol having very low
intestinal tract absorbability by utilizing enzymatic action is
provided. Cholesterol in a cholesterol-containing substance such as
meat, egg, milk, seafood and cooked processed foods containing the
same, or feeds for animals, poultry and pisciculture, and the like,
can be treated with three kinds of enzymes which are a cholesterol
dehydrogenase having an optimum pH in a neutral pH range and
4-cholesten-3-one dehydrogenase and coprostan-3-one dehydrogenase
each having an optimum pH in a weak acidic range, or microbial
cells containing the enzymes, for converting the cholesterol to
coprostanol to reduce the amount of the cholesterol.
Inventors: |
Saitoh, Chiaki;
(Ibaraki-ken, JP) ; Kumazawa, Hideyo; (Tokyo,
JP) ; Aisaka, Kazuo; (Tokyo, JP) ; Mizukami,
Toru; (Tokyo, JP) ; Ando, Katsuhiko; (Tokyo,
JP) ; Ochiai, Keiko; (Ebina-shi, JP) ;
Katsumata, Ryoichi; (Miyagi-ken, JP) ; Ouchi,
Kozo; (Hasuda-shi, JP) |
Correspondence
Address: |
ANTONELLI TERRY STOUT AND KRAUS
SUITE 1800
1300 NORTH SEVENTEENTH STREET
ARLINGTON
VA
22209
|
Family ID: |
17814874 |
Appl. No.: |
09/871656 |
Filed: |
June 4, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09871656 |
Jun 4, 2001 |
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09101327 |
Jul 7, 1998 |
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09101327 |
Jul 7, 1998 |
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PCT/JP97/04067 |
Nov 7, 1997 |
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Current U.S.
Class: |
435/52 ;
514/177 |
Current CPC
Class: |
A23L 13/48 20160801;
A23L 15/25 20160801; A23L 5/25 20160801; C12P 33/00 20130101; C12Y
101/99001 20130101; A23K 20/189 20160501; A23L 17/65 20160801; C12N
9/0006 20130101 |
Class at
Publication: |
435/52 ;
514/177 |
International
Class: |
C12P 033/00; A61K
031/575 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 7, 1996 |
JP |
8-294986 |
Claims
What is claimed is:
1. A process for producing a cholesterol-reduced substance,
comprising converting cholesterol in a cholesterol-containing
substance into coprostanol via cholesten-3-one and coprostan-3-one
by contacting said cholesterol-containing substance with
cholesterol dehydrogenase, 4-cholesten-3-one dehydrogenase and
coprostan-3-one dehydrogenase, wherein enzymatic action of said
cholesterol dehydrogenase, 4-cholesten-3-dehydrogenase and
coprostan-3-one dehydrogenase reduce amount of the cholesterol, and
wherein the cholesterol dehydrogenase is a cholesterol
dehydrogenase having a relative active value at pH=7.0 which is not
less than 80% of the maximum active value.
2. The process for producing a cholesterol-reduced substance
according to claim 1, wherein the 4-cholesten-3-one dehydrogenase
and the coprostan-3-one dehydrogenase are a 4-cholesten-3-one
dehydrogenase and a coprostan-3-one dehydrogenase each having an
optimum pH in a weak acidic pH range.
3. The process for producing a cholesterol-reduced substance
according to claim 1, wherein the cholesterol-containing substance
is selected from the group consisting of meat, egg, milk, seafood
and cooked processed foods containing the same, or feeds for
animals, poultry and pisciculture.
4. The process for producing a cholesterol-reduced substance
according to claim 1, wherein one or more compounds selected from
the group consisting of nicotinamide, phospholipase and phosphate
ion are added to the cholesterol-containing substance.
5. The process for producing a cholesterol-reduced substance
according to claim 1, wherein the enzymes have been isolated from
Eubacterium separated from lion feces.
6. The process for producing a cholesterol-reduced substance
according to claim 1, wherein the enzymes have been isolated from
Eubacterium separated from human feces.
7. The process for producing a cholesterol-reduced substance
according to claim 1, wherein the cholesterol dehydrogenase has an
optimum pH of 6.7 to 7.7.
8. The process for producing a cholesterol-reduced substance
according to claim 1, wherein the cholesterol dehydrogenase has an
optimum pH of 6.5 to 7.8.
9. The process for producing a cholesterol-reduced substance
according to claim 1, wherein the enzymes are provided
sequentially, in the order of cholesterol dehydrogenase,
4-cholesten-3-one dehydrogenase and coprostan-3-one
dehydrogenase.
10. A method for reducing the amount of cholesterol in a
cholesterol-containing substance, by enzymatic action of
cholesterol dehydrogenase, 4-cholesten-3-one dehydrogenase and
coprostan-3-one dehydrogenase on the cholesterol-containing
substance, comprising contacting the cholesterol-containing
substance with the cholesterol dehydrogenase, the 4-cholesten-3-one
dehydrogenase and the coprostan-3-one dehydrogenase to reduce the
amount of cholesterol in the cholesterol-containing substance by
converting the cholesterol to coprostanol via 4-cholesten-3-one and
coprostan-3-one, wherein the cholesterol dehydrogenase is a
cholesterol dehydrogenase having a relative active value at pH=7
which is not less than 80% of the maximum active value.
11. The method for reducing the amount of cholesterol according to
claim 10, wherein the 4-cholesten-3-one dehydrogenase and the
coprostan-3-one dehydrogenase are a 4-cholesten-3-one dehydrogenase
and a coprostan-3-one dehydrogenase each having an optimum pH in a
weak acidic pH range.
12. The method for reducing the amount of cholesterol according to
claim 10, wherein the cholesterol-containing substance is selected
from the group consisting of meat, egg, milk, seafood and cooked
processed foods containing the same, or feeds for animals, poultry
and pisciculture.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method for producing a
cholesterol-reduced substance, a cholesterol-reducing composition
and a novel cholesterol dehydrogenase, 4-cholesten-3-one
dehydrogenase and coprostane-3-one dehydrogenase for using the
above-mentioned purpose.
BACKGROUND ART
[0002] It is widely known that excess intake of food having high
cholesterol content increases the amount of cholesterol in serum
and that high cholesterol content in serum is a significant factor
in heart diseases. Therefore, processing techniques are required
for selectively reducing the amount of cholesterol in food without
deteriorating the quality of the food.
[0003] Among techniques for reducing the amount of cholesterol in
food, a method is known that decomposes cholesterol with
microorganisms (Japanese Laid-Open Patent Publication No.
267231/88) as a biochemical technique; however, this method
produces by-products, therefore, it is not a safe method. Further,
a method in which cholesterol is converted to epicholesterol by
using an enzyme is known (WO93/25702).
[0004] Beitz, et al., United States patent 4921710 describes a
method for converting cholesterol to coprostanol by using a
cholesterol reductase derived from plants, and suggests a method
for converting cholesterol to coprostanol by using a cholesterol
reductase derived from bacteria such as Eubacterium species ATCC
21408. Also, Beitz, et al., United States patent 5436004 describes
that the conversion ratio from cholesterol to coprostanol as 0.01%
when a cream is treated using the above-mentioned enzyme derived
from plants (see, column 5, table 1). However, such a low
conversion ratio to coprostanol can not be admitted as practical
level.
[0005] EYSSEN, British patent 1237483 describes that Eubacterium
species bacterium separated from feces of rats reduces cholesterol
to coprostanol, and also, in EYSSEN, Biochemica et Biophysica Acta,
348,279-284 (1974), it is estimated that the bacterium reduces
cholesterol to coprostanol via 4-cholesten-3-one.
[0006] Beitz, et al., Applied Microbiology and Biotechnology 43,
887 (1995) describes that Eubacterium species bacterium (ATCC
51222) converts cholesterol in micelle to coprostanol, that
4-cholesten-3-one and trace amounts of coprostane have been
detected in the conversion process, and that the reduction
mechanism of cholesterol using the above-mentioned bacterium might
be studied after pure preparations of the cholesterol reductase are
obtained.
[0007] However, up to now, it has not been confirmed that
cholesterol in a substance containing cholesterol is converted via
4-cholesten-3-one and coprostane-3-one to coprostanol by utilizing
enzymatic action of cholesterol dehydrogenase, 4-cholesten-3-one
dehydrogenase, coprostane-3-on dehydrogenase with using a coenzyme
NAD (P) and NAD (P) H, and enzymes which convert cholesterol in a
substance containing cholesterol to coprostanol via
4-cholesten-3-one and coprostane-3-one, respectively, have not been
isolated from bacterium which reduces cholesterol, and in addition,
no report have been proposed in which food is treated with these
converting enzymes and microorganism containing the same.
[0008] It is known that cholesterol dehydrogenase derived from
Nocardia, Alcaligenes, Proteus, which has optimum pH of around 9.0,
requires NAD (P) as a coenzyme, and is used for a quantitative
determination of cholesterol (Japanese Post-Examined Patent
Publication No.18064/90), however, this enzyme exhibits low
activity at neutral pH , so that it can not be admitted as
practical for food treatment.
[0009] Testing has been tried to obtain 4-cholesten-3-one
dehydrogenase from the feces of a rat, however, the dehydrogenase
is rapidly deactivated and can not be purified in this method,
therefore, the method can not be admitted as practical (European J.
of Biochemistry 37, 143 (1973)).
[0010] It is known that treatment of food with a phospholipase,
protease and lipase accelerates conversion by a cholesterol oxidase
(Japanese Laid-Open Patent publication No.76311/93), however,
effect in enzymatic conversion of cholesterol to coprostanol is not
known yet.
[0011] It is known that meat contains NAD (H) (Journal of Food
Science 37, 612 (1972)), and that nicotinamide inhibits
decomposition of NAD (Archives of Biochemistry and Biophisics 156,
143 (1973)). However, addition of nicotinamide in enzymatically
converting cholesterol to coprostanol is not known.
[0012] It is known that an introduction of the cholesterol oxidase
gene into lactic acid bacteria for decomposing food cholesterol
(Applied Microbiology and Biotechnology 37, 330 (1992)).
[0013] As an applied example of these enzymes, the above-mentioned
Beitz, et al., United States patent 5436004 suggests a treating
method for reducing the amount of cholesterol in serum in which a
cholesterol reductase derived from plants is orally administered.
Further, it is reported that if a bacterium (ATCC 51222) which
reduces cholesterol is orally administered to a rabbit suffering
from hypercholesterolemia, the cholesterol level in serum decreases
(Letters in Applied Microbiology 20, 137 (1995)).
[0014] Since absorbability of coprostanol through the intestinal
tract is very low (American J. of Physiology 251, G495 (1986)),
conversion of cholesterol to coprostanol is effective as a
cholesterol reducing method. A practical method is not known for
producing a cholesterol-reduced substance which reduces cholesterol
by enzymatically converting cholesterol in food via
4-cholesten-3-one and coprostane-3-one to coprostanol. Also, a
method for producing a practical enzyme which can be used in the
above-mentioned production method is not known.
DISCLOSURE OF THE INVENTION
[0015] The object of the present invention is to provide a novel
method for producing a practical cholesterol-reduced substance in
food and feed. Another object of the present invention is to
provide a novel cholesterol dehydrogenase having optimum pH in
neutral pH range which is suitable for the above-mentioned
practical method for producing a cholesterol-reduced substance, a
novel 4-cholesten-3-one dehydrogenase and coprostane-3-one
dehydrogenase having an optimum pH in a weak acidic pH range
possessed by meat, and microbial cells and a treated material
thereof containing these enzymes. A further object of the present
invention is to provide a cholesterol-reducing composition which
reduces cholesterol level in serum, comprising the above-mentioned
three kinds of novel enzymes or comprising microbial cells
containing these novel enzymes.
[0016] The present inventors have screened various microorganisms
such as stock microorganisms mainly including 300 kinds (species
categories) of actinomycetes, fungi and bacteria, aerobic bacteria
separated from 100 kinds of various soils, and anaerobic bacteria
from 7 feces samples of humans, 8 feces samples of mammals other
than human and 9 feces samples of birds, to find a cholesterol
reduction converting enzyme group having optimum pH in a neutral pH
range or weak acidic range for solving the above-mentioned
problems, and have intensively examined a culture medium for
enzymatic activity detection and a method for recovering microbial
cells, as a result, have accomplished the present invention.
[0017] Namely, the present invention relates to a process for
producing a cholesterol-reduced substance and a method for reducing
the amount of cholesterol, wherein cholesterol in a substance
containing cholesterol such as meat, egg, milk, seafood and cooked
processed food containing the same, or feed for animals, poultry
and pisciculture, and the like, is treated with three kinds of
enzymes consisting of cholesterol dehydrogenase having optimum pH
in neutral pH range and 4-cholesten-3-one dehydrogenase and
coprostan-3-one dehydrogenase having optimum pH in a weak acidic
range or microbial cells containing these enzymes, to convert
cholesterol to coprostanol for reducing the amount of
cholesterol.
[0018] Further, the present invention relates to the
above-mentioned three kinds of enzymes consisting of cholesterol
dehydrogenase having optimum pH in a neutral pH range and
4-cholesten-3-one dehydrogenase and coprostan-3-one dehydrogenase
having optimum pH in weak acidic range, produced by a strain
belonging to Eubacterium, a cholesterol-reducing composition
comprising these three kinds of enzymes, and a cholesterol-reducing
composition comprising a strain belonging to Eubacterium which
produce this enzyme group.
[0019] Further, the present invention relates to a process for
producing a cholesterol-reduced substance and a method for reducing
the amount of cholesterol wherein nicotinamide, phospholipase and
nicotinamide, or a phosphate ion are added when cholesterol in a
cholesterol-containing substance is converted to coprostanol by
utilizing the above-mentioned enzyme group.
[0020] The process for producing a cholesterol-reduced substance of
the present invention will be described in detail below.
[0021] In the present invention, examples of the
cholesterol-containing substance include meat, egg, milk, seafood
and cooked processed foods containing the same, or feeds for
animals, poultry and pisciculture, and the like, but are not
limited to them providing the substance containing cholesterol.
[0022] In the present invention, the cholesterol dehydrogenase is
an enzyme conducting the following reaction.
Cholesterol+NAD(P).fwdarw.4-cholesten-3-one+NAD(P)H
[0023] Further, in the present invention, the 4-cholesten-3-one
dehydrogenase is an enzyme conducting the following reaction.
4-Cholesten-3-one+NAD(P)H.fwdarw.coprostan-3-one+NAD(P)
[0024] Similarly, in the present invention, the coprostan-3-one
dehydrogenase is an enzyme conducting the following reaction.
Coprostan-3-one+NAD(P)H coprostanol+NAD(P)
[0025] In the present invention, the term "optimum pH" refers to a
pH range in which relative active value is not less than 80% of the
maximum active value, and the case in which pH 7.0 is included in
such a pH range is referred to as "having optimum pH in a neutral
pH range" in the present invention.
[0026] Similarly, the phrase "having optimum pH in a weak acidic
range" in the present invention refers to a case in which pH 5.5 is
included in the optimum pH range and the maximum active value
exists in the acidic side.
[0027] For converting cholesterol in a cholesterol-containing
substance to coprostanol via 4-cholesten-3-one and coprostan-3-one
by utilizing the enzymatic actions of cholesterol dehydrogenase,
4-cholesten-3-one dehydrogenase and coprostan-3-one dehydrogenase
sequentially in the present invention, any of an enzymatic
conversion method using these enzymes and a microbial conversion
method using microbial cells having these enzymatic activities can
be used.
[0028] As the enzymatic conversion method, there is listed, for
example, a method in which enzymes having activities of the
above-mentioned cholesterol dehydrogenase, 4-cholesten-3-one
dehydrogenase and coprostan-3-one dehydrogenase are added to a
cholesterol-containing substance for converting cholesterol to
coprostanol, and the enzyme which can be used in this enzymatic
conversion method is not limited to a purified enzyme and may also
be a crude enzyme which has not been purified but has enzymatic
activity.
[0029] It is particularly desirable in this enzymatic conversion
method to be sequentially treated with cholesterol dehydrogenase,
the treatment with 4-cholesten-3-one dehydrogenase and the
treatment with coprostan-3-one dehydrogenase continuously by
successively-adding the enzymes and the like, from practical points
of view such as simplification of the enzyme treatment process,
improvement of conversion rate, and the like.
[0030] As the above-mentioned microbial conversion method, there is
listed a method in which microbial cells having at least one
enzymatic activity of cholesterol dehydrogenase activity,
4-cholesten-3-one dehydrogenase activity and coprostan-3-one
dehydrogenase activity, or a treated material thereof are added to
a cholesterol-containing substance for converting cholesterol to
coprostanol, and it is desirable to use microbial cells having
these enzymatic activities simultaneously or a treated material
thereof. Further, a recombinant microbial cells having these
enzymatic activities or a treated material thereof can also be
used.
[0031] The material having the above-mentioned enzymatic activity
(hereinafter, referred to as "enzyme source") is usually added in
the form of a powder or aqueous solution to a
cholesterol-containing substance. Further, if necessary, coenzymes
such as NAD (P), NAD (P) H and the like, enzymes such as
phospholipase, lipase, protease and the like, or nicotinamide,
phosphate ion and the like can be used alone or in combination
together with the enzyme source.
[0032] In treatment for meat, for example, beef, pork, mutton or
chicken, the enzyme source is mixed with minced meat, dispersed in
sliced meat or injected in block meat. Further, the enzyme source
can also be injected in blood vessels before and after
slaughtering.
[0033] In treatment for milk, the enzyme source is added to milk,
or milk is passed through a carrier to which the enzyme source is
fixed. Further, in the case of fermented milk food, the enzyme
source can also be added at milk fermentation.
[0034] In the treatment for eggs, the enzyme source is injected in
the whole egg, or the enzyme source is mixed with yolk obtained by
cracking the egg.
[0035] Further, when meat, milk, egg or seafood is cooked, the
enzyme source may be added.
[0036] Regarding feeds for animals, poultry and pisciculture, it is
possible to use a raw material for feed which has been treated with
the enzyme source, or to mix the enzyme source in the process for
preparing a feed.
[0037] In the above-mentioned enzymatic treatment, treatment
conditions (temperature, time, pH) and the amount of enzyme to be
added under which enzymatic conversion to coprostanol is possible
are selected, and in general, the treatment is conducted at
reaction temperature of 2 to 70.degree. C. and pH of 4 to 9 for 0.5
to 1.times.10.sup.3 hours, and it is desirable to used a
cholesterol reductase having optimum pH in the pH range possessed
by the cholesterol-containing food itself to be treated in a
practical point of view.
[0038] The amount used of each enzyme which is cholesterol
dehydrogenase, 4-cholesten-3-one dehydrogenase or coprostan-3-one
dehydrogenase to be used is from 1 to 1.times.10.sup.5 units,
preferably from 1.times.10.sup.2 to 1.times.10.sup.4 units per gram
of cholesterol in food.
[0039] Optionally, NAD(P) or NAD(P)H which is a coenzyme of the
above-mentioned enzyme can be added in an amount of
1.times.10.sup.-4 to 2.times.10.sup.2 g per gram of
cholesterol.
[0040] Optionally, nicotinamide can also be added in an amount from
0.01 to 5% in terms of concentration in food. In meat which
exhibits strong activity of decomposing these coenzymes it is
preferable to added nicotinamide in an amount of 0.1% or more.
[0041] If required, a phosphate ion can also be added in an amount
from 5 to 25 mM.
[0042] If occasion requires, it is also possible to add a
phospholipase in an amount from 1 to 1.times.10.sup.5 units per
gram of a phospholipid and to add a lipase in an amount from 1 to
1.times.10.sup.5 units per gram of a lipid.
[0043] Specific examples of the novel enzymes of the present
invention include cholesterol dehydrogenase A, 4-cholesten-3-one
dehydrogenase A and coprostan-3-one dehydrogenase A, cholesterol
dehydrogenase B. 4-cholesten-3-one dehydrogenase B and
coprostan-3-one dehydrogenase B. The method for production and the
physical and chemical properties of these enzymes are described
below.
[0044] The microorganism used in producing the novel cholesterol
dehydrogenase A, 4-cholesten-3-one dehydrogenase A and
coprostan-3-one dehydrogenase A may be any microorganism having
ability to produce the above-mentioned cholesterol dehydrogenase A,
4-cholesten-3-one dehydrogenase A and coprostan-3-one dehydrogenase
A, and may also be a variant species or variant strain thereof. As
a specific example of the microorganism having ability to produce
the cholesterol dehydrogenase A, 4-cholesten-3-one dehydrogenase A
and coprostan-3-one dehydrogenase A, for example, Eubacterium sp.
CP 2 is listed.
[0045] The above-mentioned Eubacterium sp. CP 2 is a strain which
has been newly separated from the feces of lions by the present
inventors, and mycological properties thereof are as follows.
[0046] The present strain exhibits excellent growth by standing
culture in a solution under anaerobic conditions at 37.degree. C.
using the following basal medium. Tests regarding various
properties were investigated under this condition.
[0047] Basal medium; 1% Casitone, 1% yeast extract, 0.5% soluble
starch, 0.5% sodium pyruvate, 0.05% sodium thioglycolate, 0.05%
calcium chloride, 0. 0001% Resazurin, and 0.01% lecithin (pH
7.5)
[0048] (a) Morphological properties
[0049] {circle over (1)} Form of cell; short rod bacterium, and
sometimes shown as almond figure.
[0050] {circle over (2)} Size of cell; 0.5 to 0.7 .mu.m.times.0.7
to 1.0 .mu.m
[0051] {circle over (3)} Polymorphism; none
[0052] {circle over (4)} Mobility; none
[0053] {circle over (5)} Spore; none
[0054] (b) Cultural properties
[0055] No growth was observed in bouillon plate agar-media and
bouillon broth medium under aerobic or anaerobic conditions.
[0056] Cultural properties under a culturing condition and basal
medium in which the present strain can grow are shown below.
[0057] {circle over (1)} Basal medium agar plate culture (culturing
for 14 days)
[0058] i) Appearance of growth; forming weak small colony
[0059] ii) Color; transparent white
[0060] iii) Gloss; recognized
[0061] iv) Dispersible pigment; none
[0062] {circle over (2)} Basal medium solution culture (culturing
for 3 days)
[0063] i) Growth on surface; none
[0064] ii) Turbidity; grown in the form of white emulsion at the
bottom part
[0065] {circle over (3)} Gelatin stab culture in Basal medium
[0066] i) Condition of growth; excellent
[0067] ii) Liquefaction of gelatin; none
[0068] (c) Physiological properties in basal medium culture
[0069] {circle over (1)} Gram staining; positive
[0070] {circle over (2)} Reduction of nitrate; negative
[0071] {circle over (3)} Denitrification reaction; negative
[0072] {circle over (4)} MR test; negative
[0073] {circle over (5)} VP test; negative
[0074] {circle over (6)} Generation of indole; negative
[0075] {circle over (7)} Generation of hydrogen sulfide;
positive
[0076] {circle over (8)} Hydrolysis of starch; negative
[0077] {circle over (9)} Decomposition of esculin; positive
[0078] {circle over (10)} Utilization of an inorganic nitrogen
source in a medium which has been obtained by removing 1% casitone
and 1% yeast extract from the basal medium
[0079] i) Nitrate; negative
[0080] ii) Ammonium salt; negative
[0081] {circle over (11)} Generation of pigment; none
[0082] {circle over (12)} Urease; negative
[0083] {circle over (13)} Oxidase; negative
[0084] {circle over (14)} Catalase; negative
[0085] {circle over (15)} Growth range
[0086] i) Growth pH range; pH 6.0 to pH 7.7 (optimum growth; around
pH 7.3)
[0087] ii) Growth temperature range; 28 to 44.degree. C. (optimum
growth temperature; around 35.degree. C.)
[0088] {circle over (16)} Attitude against oxygen; strictly
anaerobic
[0089] {circle over (17)} O-F test (Hugh Leifson method);
negative
[0090] {circle over (18)} Generation of acid and gas
[0091] i) L-arabinose; acid (none), gas (none)
[0092] ii) D-xylose; acid (none), gas (none)
[0093] iii) D-glucose; acid (observed), gas (none)
[0094] iv) D-mannose; acid (observed), gas (none)
[0095] v) D-fructose; acid (none), gas (none)
[0096] vi) D-galactose; acid (observed), gas (none)
[0097] vii) Maltose; acid (none), gas (none)
[0098] viii)Sucrose; acid (none), gas (none)
[0099] ix) Lactose; acid (none), gas (none)
[0100] x) Trehalose; acid (none), gas (none)
[0101] xi) Dsorbitol; acid (none), gas (none)
[0102] xii) D-mannitol; acid (none), gas (none)
[0103] xiii) Inositol; acid (none), gas (none)
[0104] xiv) Glycerin; acid (none), gas (none)
[0105] xv) Starch; acid (none), gas (none)
[0106] xvi) Metabolite from saccharides; butyric acid or acetic
acid
[0107] (d) Other various properties; test according to An-IDENT
[0108] {circle over (1)} .alpha.-glucosidase; positive
[0109] {circle over (2)} .beta.-glucosidase; positive
[0110] {circle over (3)} Alkali phosphatase; positive
[0111] {circle over (4)} .alpha.-galactosidase; positive
[0112] {circle over (5)} Phenylalanineamino peptidase; negative
[0113] {circle over (6)} Decomposition of arginine; positive
[0114] {circle over (7)} Decomposition indoxyl acetate;
negative
[0115] The present strain was of a gram positive strictly anaerobic
short rod bacterium, did not form a spore, had no mobility, was
negative against all of a catalase, oxidase and urease and formed
acids from glucose and lactose, and main metabolite thereof was
butyric acid or acetic acid. The classificational position of the
strain having these mycological properties was compared with the
description of Bergey's Manual of Systematic Bacteriology vol. 2,
1986, as a result, the strain was identified as a bacterium
belonging to Eubacterium, and the CP 2 strain was named as
Eubacterium sp. CP 2 This strain was deposited in the name of FERM
BP-5501 to National Institute of Bioscience and Human-Technology
Agency of Industrial Science and Technology (Higashi 1-1-3, Tsukuba
City, Ibaraki Prefecture, Japan) on Apr. 12, 1996.
[0116] As the medium used for culturing a microorganism which
produces cholesterol dehydrogenase A, 4-cholesten-3-one
dehydrogenase A and coprostan-3-one dehydrogenase A of the present
invention, any of a synthetic medium or natural medium containing a
carbon source, nitrogen source, inorganic substance and the like
can be used.
[0117] As the carbon source, for example, various carbohydrates can
be used such as a soluble starch, lactose, pyruvic acid, glucose,
molasses and the like, and the amount used thereof is preferably
from 1 to 20 g/L.
[0118] As the nitrogen source, for example, ammonium sulfate,
ammonium phosphate, ammonium carbonate and ammonium acetate, or
nitrogen-containing organic compounds such as peptone, yeast
extract, corn steep liquor, casein decomposed material, meat
extract, and the like, can be used, and the amount used thereof is
preferably from 1 to 20 g/L.
[0119] As the inorganic substance, for example, sodium chloride,
calcium chloride, magnesium sulfate and the like are used, and the
amount used thereof is preferably from 0.1 to 2 g/L. The amount
used of a surfactant such as lecithin and the like is preferably
from 0.01 to 1 g/L. The amount used of sodium thioglycolate is
preferably from 0.1 to 1 g/L.
[0120] Culturing is conducted under an anaerobic condition by
standing culture or stirring culture. Culturing temperature may
advantageously be a temperature at which a microorganism grows and
produces cholesterol dehydrogenase A, 4-cholesten-3-one
dehydrogenase A and coprostan-3-one dehydrogenase A, and preferably
from 35 to 40.degree. C. Culturing period varies depending on
conditions, and culturing may advantageously be conducted until the
maximum amount of cholesterol dehydrogenase A, 4-cholesten-3-one
dehydrogenase A and coprostan-3-one dehydrogenase A are produced,
and usually from about 5 to 10 days.
[0121] Cholesterol dehydrogenase A that is produced by the CP 2
strain is a novel enzyme, and the physical and chemical properties
and method for purification thereof are as follows.
[0122] (a) Action: it catalyses the following reaction.
Cholesterol+NADP.fwdarw.4-cholesten-3-one+NADPH
[0123] (b) Substrate specificity
[0124] The present enzyme reacts with steroid having a hydroxyl
group at 3.beta. position, and has relative activity of 38, 74, and
30 regarding .beta. sitosterol, campesterol, stigmasterol when the
activity of cholesterol is 100.
[0125] (c) Optimum pH: 6.5 to 7.8
[0126] (d) Stable pH: 5.3 to 9.0
[0127] An enzyme solution was allowed to stand still at 37.degree.
C. for 15 minutes using various pH buffer solutions, and remaining
activity was measured, and pH range in which activity was not less
than 50% of the maximum active value was regarded as a stable pH
range. (The following is the same.)
[0128] (e) Measurement of titer:
[0129] With 0.2 ml of 3 mM cholesterol micelle solution containing
0.33% Triton X-100 is mixed 0.3 ml of a 20 mM
piperazine-N,N'-bis-(2-ethane sulfuric acid) (hereinafter, referred
to as "PIPES") buffer solution (pH 7.5) containing 0.5% Triton
X-100 and 1 mM dithiothreitol (hereinafter, referred to as "DTT"),
and 0.1 ml of a 10 mM NAD solution, the resulted mixture is added
0.05 ml of an enzyme solution, reacted for 30 minutes at 37.degree.
C., and then added 0.1 ml of chloroform to extract sterol and to
terminate the reaction.
[0130] Then, the quantity of 4-cholesten-3-one produced in the
reaction solution is determined using TLC/FID iatroscan. The
quantity of 4-cholesten-3-one produced in the reaction solution is
determined likewise using an inactivated enzyme which has been
previously heated as a control. The enzyme activity which produces
1 .mu.mol of 4-cholesten-3-one per one minute is regarded as 1
unit.
[0131] (f) Range of suitable reaction temperature:
[0132] In reaction at pH 7.5 for 30 minutes, increasing temperature
up to 40.degree. C. is attended by increasing activity.
[0133] (g) Range of temperature stability
[0134] After heating treatment at 40.degree. C. for 10 minutes, it
keeps activity of not less than 80% of that before the
treatment.
[0135] (h) Influence of inhibitor, metal ion:
[0136] When 1 mM p-chloro mercury phenylsulfonate (PCMB), iodine
acetamide and ethylenediamine tetraacetate (EDTA) are added and
reacted for 30 minutes at pH 7.5 and 37.degree. C., the relative
activities are 0, 82 and 105 respectively, if the enzyme activity
without adding inhibitor is defined as 100. Further, when the
present enzyme is reacted in the presence of 1 mM iron chloride and
copper chloride, the relative activities are 95 and 3.2,
respectively, in comparison with the case of no addition.
[0137] (i) Purification method:
[0138] Microbial cells are collected from the culture by
centrifugal separation, and the cells are suspended in a 20 mM
PIPES buffer solution (pH 7.5, containing 1 mM MDTT). The cells are
disrupted by ultrasonication, further, treated with ultrasonication
in the presence of 0.2% Triton X-100, solid components are removed
by centrifugal separation, to obtain a crude enzyme solution. The
crude enzyme solution is dialyzed against a 20 mM PIPES buffer
solution (pH 7.5, containing 1 mM DTT, 0.2% Triton X-100, 10%
glycerol), then, adsorbed on Blue Sepharose CL-6B (Pharmacia) which
has been equilibrated with the same buffer solution. Then, the
above-mentioned buffer solution flows with sodium chloride
concentration being increased continuously from 0 to 3.0 M, and
active fractions are collected. The resulted active fractions are
dialyzed against the same buffer solution, then, adsorbed on Red
Sepharose CL-6B (Pharmacia). Then, the above-mentioned buffer
solution flows with sodium chloride concentration being increased
continuously from 0 to 3.0 M, and active fractions are collected to
obtain a purified sample.
[0139] (j) Coenzyme:
[0140] .beta.-nicotinamide adenine dinucleotide phosphoric acid
(NADP) is used as a coenzyme.
[0141] (k) Molecular weight:
[0142] The molecular weight according to SDS polyacrylamide
electrophoresis method is about 57,500.
[0143] The 4-cholesten-3-one dehydrogenase A of the present
invention produced by the CP 2 strain is a novel enzyme, and the
physical and chemical properties and method for purification
thereof are as follows.
[0144] (a) Action: it catalyses the following reaction.
4-cholesten-3-one+NADH.fwdarw.coprostan-3-one+NAD
[0145] (b) Substrate specificity
[0146] The relative activities are 0, 41 and 0 for testosterone,
progesterone and prognerone when the activity of 4-cholesten-3-one
is defined as 100.
[0147] (c) Optimum pH: 5.4 to 6.5
[0148] (d) Stable pH: 5.5 to 7.2
[0149] (e) Measurement of titer:
[0150] With 0.2 ml of 3 mM 4-cholesten-3-one micelle solution
containing 0.33% Triton X-100 is mixed 0.3 ml of a 20 mM PIPES
buffer solution (pH 7.5) containing 0.5% Triton X-100 and 1 mM DTT,
and 0.1 ml of a 10 mM NADH solution, the resulted mixture is added
0.05 ml of an enzyme solution, and reacted for 30 minutes at
37.degree. C., and then added 0.1 ml of chloroform to extract
sterol and to terminate the reaction.
[0151] Then, the quantity of coprostan-3-one produced in the
reaction solution is determined using TLC/FID iatroscan. The
quantity of coprostan-3-one produced in the reaction solution is
determined likewise using an inactivated enzyme which has been
previously heated as a control. The enzyme activity which produces
1 .mu.mol of coprostan-3-one per one minute is regarded as 1
unit.
[0152] (f) Range of suitable reaction temperature:
[0153] In reaction at pH 6.0 for 30 minutes, increasing temperature
up to 40.degree. C. is attended by increasing the activity
[0154] (g) Range of temperature stability
[0155] After heating treatment at 40.degree. C. for 10 minutes, it
keeps activity of not less than 80% of that before the
treatment.
[0156] (h) Influence of inhibitor, metal ion:
[0157] When 1 mM PCMB, iodine acetamide and EDTA are added and
reacted for 30 minutes at pH 7.5 and 37.degree. C., the relative
activities are 47, 94 and 82 respectively, if the enzyme activity
without adding inhibitor is defined as 100. Further, when the
present enzyme is reacted in the presence of 1 mM iron chloride and
copper chloride, the relative activities are 110 and 0,
respectively, in comparison to the case of no addition.
[0158] (i) Purification method:
[0159] Microbial cells are collected from the culture by
centrifugal separation, and the cells are suspended in a 20 mM
PIPES buffer solution (pH 7.5, containing 1 mM MDTT). The cells are
disrupted by ultrasonication, further, treated with ultrasonication
in the presence of 0.2% Triton X-100, solid components are removed
by centrifugal separation, to obtain a crude enzyme solution. The
crude enzyme solution is dialyzed against a 20 mM PIPES buffer
solution (pH 7.5, containing 1 mM DTT, 0.2% Triton X-100, 10%
glycerol), then, adsorbed on Blue Sepharose CL-6B (Pharmacia) which
has been equilibrated with the same buffer solution. Then, the
above-mentioned buffer solution flows with sodium chloride
concentration being increased continuously from 0 to 3.0 M, and
active fractions are collected. The resulted active fractions are
dialyzed against the same buffer solution, then, adsorbed on
Resource Column (Pharmacia). Then, the above-mentioned buffer
solution flows with sodium chloride concentration being increased
continuously from 0 to 1.0 M, and active fractions are collected to
obtain a purified sample.
[0160] (j) Coenzyme:
[0161] Reduced type .beta.-nicotinamide adenine dinucleotide (NADH)
is used as a coenzyme.
[0162] (k) Molecular weight:
[0163] The molecular weight according to SDS polyacrylamide
electrophoresis method is about 37,500.
[0164] The coprostan-3-one dehydrogenase A of the present invention
produced by the CP 2 strain is a novel enzyme, and the physical and
chemical properties and method for purification thereof are as
follows.
[0165] (a) Action: it catalyses the following reaction.
coprostan-3-one+NADPH.fwdarw.coprostanol+NADP
[0166] (b) Substrate specificity
[0167] The relative activities are 13.2 for
5.alpha.-cholesten-3-one when the activity of coprostan-3-one is
defined as 100.
[0168] (c) Optimum pH: 5.2 to 5.7
[0169] (d) Stable pH: 4.0 to 7.5
[0170] (e) Measurement of titer:
[0171] With 0.2 ml of 3 mM coprostan-3-one micelle solution
containing 0.33% Triton X-100 is mixed 0.3 ml of a 40 mM
Britton-Robinson buffer solution (pH 6.0) containing 0.5% Triton
X-100 and 1 mM DTT, and 0.1 ml of a 10 mM NADPH solution, the
resulted mixture is added 0.05 ml of an enzyme solution, reacted
for 30 minutes at 37.degree. C., and then added 0.1 ml of
chloroform to extract sterol and to terminate the reaction. The
enzyme solution was mixed with equal amounts of activating
fractions and left for 15 minutes at 5.degree. C., then used.
[0172] Then, the quantity of coprostanol produced in the reaction
solution is determined using TLC/FID iatroscan. The quantity of
coprostanol in the reaction solution is determined likewise using
an inactivated enzyme which has been previously heated as a
control. The enzyme activity which produces 1 .mu.mol of
coprostanol per one minute is regarded as 1 unit.
[0173] (f) Range of suitable reaction temperature:
[0174] In reaction at pH 7.5 for 30 minutes, increasing temperature
up to 40.degree. C. is attended by increasing the activity.
[0175] (g) Range of temperature stability
[0176] After heating treatment at 40.degree. C. for 10 minutes in
the presence of 0.5 mM NADPH, it keeps activity of not less than
80% of that before the treatment.
[0177] (h) Influence of inhibitor, metal ion:
[0178] When 1 mM PCMB, iodine acetamide and EDTA are added and
reacted for 30 minutes at pH 7.5 and 37.degree. C., the relative
activities are 41, 82 and 101 respectively, if the enzyme activity
without adding inhibitor is defined as 100. Further, when the
present enzyme is reacted in the presence of 1 mM iron chloride and
copper chloride, the relative activities are 12 and 2.7,
respectively, in comparison with the case of no addition.
[0179] (i) Purification method:
[0180] Method 1;
[0181] Microbial cells are collected from the culture by
centrifugal separation, and the cells are suspended in a 20 mM
phosphoric acid-citric acid buffer solution (pH 6.0, containing 0.1
mM DTT). The cells are disrupted by ultrasonication, further,
treated with ultrasonication in the presence of 0.2% Triton X-100,
solid components are removed by centrifugal separation, to obtain a
crude enzyme solution. The crude enzyme solution is dialyzed
against a 20 mM PIPES buffer solution (pH7.5, containing 1 mM DTT,
0.2%Triton X-100, 10% glycerol), then, passed through Blue
Sepharose CL-6B (Pharmacia) which has been equilibrated with the
same buffer solution, and fractions which have not been adsorbed on
Blue Sepharose CL-6B are recovered, and dialyzed against a 20 mM
phosphoric acid-citric acid buffer solution (pH 6.0) (containing
0.5 mM NADPH, 1 mM DTT, 0.2% Triton X-100, 10% glycerol). The
solution is adsorbed on DEAE-Cellulofine which has been
equilibrated with the same buffer solution, then, the buffer
solution flows with sodium chloride concentration being increased
continuously from 0 to 1.0 M, and active fractions are collected
and dialyzed against the buffer solution. The active fractions are
adsorbed on Blue Sepharose CL-6B, and fractions which had not been
adsorbed were recovered. The fractions which had not been adsorbed
were heated at 95.degree. C. for 10 minutes to remove protein and
to obtain activating fractions. Then, the active fractions which
had been adsorbed on Blue Sepharose were eluted with the same
buffer solution containing 1 mM NADPH with sodium chloride
concentration being increased continuously from 0 to 1.0 M. To
these active fractions, the above-mentioned activating fractions
were added to prepare a purified sample.
[0182] Method 2;
[0183] Microbial cells are collected from the culture by
centrifugal separation, and the cells are suspended in a 20 mM
phosphoric acid-citric acid buffer solution (pH 6.0, containing 0.1
mM DTT). The cells are disrupted by ultrasonication, further,
treated with ultrasonication in the presence of 0.2% Triton X-100,
solid components are removed by centrifugal separation, to obtain a
crude enzyme solution. The crude enzyme solution is dialyzed
against a 20 mM phosphoric acid-citric acid buffer solution (pH
6.0, containing 1 mM DTT, 0.2% Triton X-100, 30% glycerol), then,
adsorbed on Blue Sepharose CL-6B (Pharmacia) which has been
equilibrated with the same buffer solution. Then, the
above-mentioned buffer solution flows with sodium chloride
concentration being increased continuously from 0 to 3.0 M, and
active fractions are collected. The resulted active fractions are
dialyzed against a 20 mM phosphoric acid-citric acid buffer
solution B (pH7.5, containing 1 mM DTT, 0.2% Triton X-100, 30%
glycerol), then adsorbed on DEAE-Cellulofine which has been
equilibrated with the same buffer solution B, then, the buffer
solution flows with sodium chloride concentration being increased
continuously from 0 to 1.0 M, and active fractions are collected
and dialyzed against the buffer solution. The active fractions are
dialyzed against the same buffer solution B, then adsorbed on
Resource Column (Pharmasia). Then, the above-mentioned buffer
solution B flows with sodium chloride concentration being increased
continuously from 0 to 1.0 M and active fractions are collected to
prepare a purified sample.
[0184] (j) Coenzyme:
[0185] Reduced type .beta.-nicotinamide adenine dinucleotide
phosphoric acid (NADPH) is used as a coenzyme.
[0186] (k) Molecular weight:
[0187] The molecular weight according to SDS polyacrylamide
electrophoresis method is about 29,000.
[0188] Then, the method for producing the novel cholesterol
dehydrogenase B, 4-cholesten-3-one dehydrogenase B and
coprostan-3-one dehydrogenase B is described below.
[0189] The microorganism used in producing the present enzymes may
be any microorganism having the ability to produce the
above-mentioned cholesterol dehydrogenase B, 4-cholesten-3-one
dehydrogenase B and coprostan-3-one dehydrogenase B, and may also
be a variant species or variant strain thereof. As a specific
example of the microorganism having the ability to produce the
cholesterol dehydrogenase B, 4-cholesten-3-one dehydrogenase B and
coprostan-3-one dehydrogenase B and belonging to Eubacterium, for
example, Eubacterium sp. CP 1 is listed.
[0190] The above-mentioned Eubacterium sp. CP 1 is a strain which
has been newly separated from the feces of humans by the present
inventors, and mycological properties thereof are as follows.
[0191] The present strain exhibits excellent growth by standing
culture in a solution under anaerobic conditions at 37.degree. C.
using the following basal medium. Tests regarding various
properties were investigated under this condition.
[0192] Basal medium; 1.2% a bovine brain extracted lipid, 1.8%
Trypticase, 0.05% yeast extract, 0.13% dipotassium phosphate, 0.22%
sodium chloride, 0.05% cholesterol, 0.04% L-cystine, 0.03% sodium
thioglycolate, 0.05% agar, and a small amount of 0.05% Methylene
Blue (pH 7.2).
[0193] (a) Morphological properties
[0194] {circle over (1)} Form of cell; long rod bacterium and grown
in the form of chain
[0195] {circle over (2)} Size of cell; 0.3 to 0.5 .mu.m.times.3.0
to 5.0 .mu.m
[0196] {circle over (3)} Polymorphism; none
[0197] {circle over (4)} Mobility; none
[0198] {circle over (5)} Spore; none
[0199] (b) Cultural properties
[0200] No growth was observed in the bouillon plate agar-media and
bouillon broth media under aerobic or anaerobic conditions.
[0201] Cultural properties under culturing condition and basal
medium in which the present strain can grow are shown below.
[0202] {circle over (1)} Basal medium agar plate culture (culturing
for 21 days)
[0203] i) Appearance of growth; forming powdery colony
[0204] ii) Color; white
[0205] iii) Gloss; none
[0206] iv) Dispersible pigment; none
[0207] {circle over (2)} Basal medium solution culture (culturing
for 5 days)
[0208] i) Growth on surface; none
[0209] ii) Turbidity; grows, but, discrimination from sediment in a
medium by turbidity is difficult
[0210] {circle over (3)} Gelatin stab culture in basal medium
[0211] i) Condition of growth; excellent
[0212] ii) Liquefaction of gelatin; none
[0213] (c) Physiological properties in basal medium culture
[0214] {circle over (1)} Gram staining; positive
[0215] {circle over (2)} Reduction of nitrate; negative
[0216] {circle over (3)} Litmus milk; coagulation negative
[0217] {circle over (4)} Generation of indole; negative
[0218] {circle over (5)} Generation of hydrogen sulfide;
negative
[0219] {circle over (6)} Hydrolysis of starch; positive
[0220] {circle over (7)} Decomposition of esculin; positive
[0221] {circle over (8)} Utilization of an inorganic nitrogen
source in a medium which has been obtained by removing trypticase
and yeast extract from a basal medium i) Nitrate; negative ii)
Ammonium salt; negative
[0222] {circle over (9)} Generation of pigment; none
[0223] {circle over (10)} Urease; negative
[0224] {circle over (11)} Oxidase; negative
[0225] {circle over (12)} Catalase; negative
[0226] {circle over (13)} Growth range
[0227] i) Growth pH range; pH 5 to pH 8 (optimum growth; around pH
7)
[0228] ii) Growth temperature range; 33 to 40.degree. C. (optimum
growth temperature; around 34.degree. C.)
[0229] {circle over (14)} Behavior against oxygen; strictly
anaerobic
[0230] {circle over (15)} O-F test (Hugh Leifson method);
negative
[0231] {circle over (16)} Generation of acid
[0232] i) L-arabinose; acid (small amount)
[0233] ii) D-xylose; acid (observed)
[0234] iii) D-glucose; acid (observed)
[0235] iv) D-mannose; acid (observed)
[0236] v) D-fructose; acid (small amount)
[0237] vi) D-galactose; acid (observed)
[0238] vii) maltose; acid (observed)
[0239] viii) Sucrose; acid (small amount)
[0240] ix) Lactose; acid (observed)
[0241] x) Trehalose; acid (none)
[0242] xi) D-sorbitol; acid (none)
[0243] xii) D-mannitol; acid (none)
[0244] xiii) Inositol; acid (none)
[0245] xiv) Glycerin; acid (none)
[0246] xv) Starch; acid (none)
[0247] xvi) Ribose; acid (observed)
[0248] xvii) Cellobiose; acid (observed)
[0249] xviii) Lactose; acid (observed)
[0250] xix) Melibiose; acid (observed)
[0251] xx) Raffinose; acid (observed)
[0252] xxi) Salicin; acid (observed)
[0253] xxii) Amygdalin; acid (observed)
[0254] xxiii) Melezitose; acid (none)
[0255] xxiv) Glycogen; acid (none)
[0256] xxv) Inulin; acid (none)
[0257] xxvi) Metabolite from saccharides; acetic acid
[0258] The present strain was gram positive strictly anaerobic long
rod bacterium, did not form spores, had no mobility, was negative
against all of a catalase, oxydase and urease and formed acids from
glucose, and main metabolite thereof was acetic acid. The
classificational position of the strain having these mycological
properties was compared with description of Bergey's Manual of
Systematic Bacteriology vol. 2, 1986, as a result, the strain was
identified as a bacterium belonging to Eubacterium, and the CP 1
strain was named as Eubacterium sp. CP 1. This strain was deposited
in the name of FERM BP-5500 to National Institute of Bioscience and
Human-Technology Agency of Industrial Science and Technology
(Higashi 1-1-3, Tsukuba City, Ibaraki Prefecture, Japan) on Apr.
12, 1996.
[0259] As the medium used for culturing a microorganism which
produces cholesterol dehydrogenase B, 4-cholesten-3-one
dehydrogenase B and coprostan-3-one dehydrogenase B of the present
invention, any of the synthetic mediums or natural mediums
containing a carbon source, nitrogen source, inorganic substance
and the like can be used.
[0260] As the carbon source, for example, various carbohydrates can
be used such as glucose, maltose, molasses and the like, and the
amount used thereof is preferably from 1 to 20 g/L.
[0261] As a nitrogen source, for example, ammonium sulfate,
ammonium phosphate, ammonium carbonate and ammonium acetate, or
nitrogen-containing organic compounds such as peptone, yeast
extract, corn steep liquor, casein decomposed material, meat
extract, and the like, can be used, and the amount used thereof is
preferably from 1 to 20 g/L. The amount of solvent-extract used
(chloroform: methanol=2:1) of a bovine brain lipid is preferably
from 1 to 20 g/L.
[0262] As an inorganic substance, for example, sodium chloride,
calcium chloride, magnesium sulfate, potassium monohydrogen
phosphate and the like are used, and the amount used thereof is
preferably from 0.1 to 2 g/L. The amount used of cystine and sodium
thioglycolate is preferably from 0.1 to 1 g/L.
[0263] Culturing is conducted under anaerobic conditions by
standing culture or stirring culture. Culturing temperature may
advantageously be a temperature at which a microorganism grows and
produces cholesterol dehydrogenase B, 4-cholesten-3-one
dehydrogenase B and coprostan-3-one dehydrogenase B, and preferably
from 35 to 40.degree. C. Culturing period varies depending on
conditions, and culturing may advantageously be conducted until the
maximum amount of these enzymes are produced, usually from about 3
to 10 days.
[0264] Cholesterol dehydrogenase B of the present invention that is
produced by the CP 1 strain is a novel enzyme, and the physical and
chemical properties and method for purification thereof are as
follows.
[0265] (a) Action: it catalyses the following reaction.
Cholesterol+NADP.fwdarw.4-cholesten-3-one+NADPH
[0266] (b) Substrate specificity
[0267] The present enzyme reacts with steroid having a hydroxyl
group at 3.beta. position, and has relative activity of 67, 50 and
39 regarding .beta. sitosterol, campesterol, stigmasterol when the
activity of cholesterol is defined as 100.
[0268] (c) Optimum pH: 6.7 to 7.7
[0269] (d) Stable pH: 5.0 to 10.5
[0270] (e) Measurement of titer:
[0271] With 0.2 ml of 3 mM cholesterol micelle solution containing
0.33% Triton X-100 is mixed 0.3 ml of a 50 mM phosphoric acid
buffer solution (pH7.5) containing 1.0% Triton X-100 and 1 mM DTT,
and 0.1 ml of a 10 mM NADP solution, to the resulted mixture is
added 0.05 ml of an enzyme solution, reacted for 30 minutes at
37.degree. C., and then added 0.1 ml of chloroform to extract
sterol and to terminate the reaction.
[0272] Then, the quantity of 4-cholesten-3-one produced in the
reaction solution is determined using TLC/FID iatroscan. The
quantity of 4-cholesten-3-one produced in the reaction solution is
determined likewise using an inactivated enzyme which has been
previously heated as a control. The enzyme activity which produces
1 .mu.mol of 4-cholesten-3-one per one minute is regarded as 1
unit.
[0273] (f) Range of suitable reaction temperatures:
[0274] In reaction at pH 7.5 for 30 minutes, increasing temperature
up to 40.degree. C. is attended by increasing activity.
[0275] (g) Range of temperature stability
[0276] After heating treatment at 40.degree. C. for 10 minutes, it
keeps activity of not less than 80% of that before the
treatment.
[0277] (h) Influence of inhibitor, metal ion:
[0278] When 1 mM PCMB, iodine acetic acid and EDTA are added and
reacted for 30 minutes at pH 7.5 and 37.degree. C., the relative
activities are 0, 98 and 102 respectively, if the enzyme activity
without adding inhibitor is 100. Further, when the present enzyme
is reacted in the presence of 1 mM iron chloride and copper
chloride, the relative activities are both 0 in comparison to the
case of no addition.
[0279] (i) Purification method:
[0280] Air is bubbled through the culture, and microbial cells
adsorbed on the bubble are recovered and concentrated. The
concentrated solution is subjected to centrifugal separation to
collect the cells, and the cells are suspended in a 20 mM
phosphoric acid buffer solution (pH 7.5, containing 1 mM DTT). The
cells are disrupted by ultrasonication, further, treated with
ultrasonication in the presence of 1.0% Triton X-100, solid
components are removed by centrifugal separation, to obtain a crude
enzyme solution. To the crude enzyme solution is added 1 mM EDTA, 1
mM iodine acetic acid and 0.5 mM phenylmethylsulfonyl fluoride
(PMSF), and the mixture is dialyzed against a 20 mM
Tris-hydrochloric acid buffer solution (pH 7.5, containing 1 mM
DTT, 10% glycerol, 1 mM EDTA, 1 mM iodine acetic acid, and 0.5 mM
PMSF). The crude enzyme solution is adsorbed on Blue Sepharose
CL-6B (Pharmacia) which has been equilibrated with the same buffer
solution containing 0.25% Triton X-100. Then, lipid components are
eluted with the same buffer solution containing 1.0% Triton X-100,
then, the above-mentioned buffer solution flows with sodium
chloride concentration being increased continuously from 0 to 3.0
M, and active fractions are collected. The resulted active
fractions are dialyzed against the above-mentioned buffer solution,
then, adsorbed on DEAE-Sepharose Fast Flow which has been
equilibrated with the same buffer solution containing 1.0% Triton
X-100. Then, the above-mentioned buffer solution flows with sodium
chloride concentration being increased continuously from 0 to 0.5
M, and active fractions are collected to obtain a purified
sample.
[0281] (j) Coenzyme:
[0282] .beta.-nicotinamide adenine dinucleotide phosphoric acid
(NADP) is used as a coenzyme.
[0283] The 4-cholesten-3-one dehydrogenase B of the present
invention produced by the CP 1 strain is a novel enzyme, and the
physical and chemical properties and method for purification
thereof are as follows.
[0284] (a) Action: it catalyses the following reaction.
4-cholesten-3-one+NADH.fwdarw.coprostan-3-one+NAD
[0285] (b) Substrate specificity
[0286] The relative activities are 0 for 5-cholestenon-3-one when
the activity of 4-cholesten-3-one is defined as 100.
[0287] (c) Optimum pH: 5.3 to 7.0
[0288] (d) Stable pH: 5.2 to 8.0
[0289] (e) Measurement of titer:
[0290] With 0.2 ml of 3 mM 4-cholesten-3-one micelle solution
containing 0.33% Triton X-100 is mixed 0.3 ml of a 20 mM
Tris-hydrochloric acid buffer solution (pH 7.5) containing 1.0%
Triton X-100 and 1 mM DTT, and 0.1 ml of a 10 mM NADH solution, the
resulted mixture is added 0.05 ml of an enzyme solution, reacted
for 30 minutes at 37.degree. C., and then added 0.1 ml of
chloroform to extract sterol and to terminate the reaction.
[0291] Then, the quantity of coprostan-3-one produced in the
reaction solution is determined using TLC/FID iatroscan. The
quantity of coprostan-3-one produced in the reaction solution is
determined likewise using an inactivated enzyme which has been
previously heated as a control. The enzyme activity which produces
1 .mu.mol of coprostan-3-one per one minute is regarded as 1
unit.
[0292] (f) Range of suitable reaction temperatures:
[0293] In reaction at pH 7.5 for 30 minutes, increasing temperature
up to 40.degree. C. is attended by increasing the activity.
[0294] (g) Range of temperature stability
[0295] After heating treatment at 37.degree. C. for 10 minutes, it
keeps activity of not less than 80% of that before the
treatment.
[0296] (h) Influence of inhibitor, metal ion:
[0297] When 1 mM PCMB, iodine acetamide and EDTA are added and
reacted for 30 minutes at pH 7.5 and 37.degree. C., the relative
activities are 0, 99 and 125, respectively, if the enzyme activity
without adding inhibitor is 100. Further, when the present enzyme
is reacted in the presence of 1 mM iron chloride and copper
chloride, the relative activities are 107 and 0, respectively, in
comparison with the case of no addition.
[0298] (i) Purification method:
[0299] Air is bubbled through the culture, and microbial cells
adsorbed on the bubble are recovered and concentrated. The
concentrated solution is subjected to centrifugal separation to
collect the cells, and the cells are suspended in a 20 mM
phosphoric acid buffer solution (pH 7.5, containing 1 mM DTT). The
cells are disrupted by ultrasonication, further, treated with
ultrasonication in the presence of 1.0% Triton X-100, solid
components are removed by centrifugal separation, to obtain a crude
enzyme solution. To the crude enzyme solution is added 1 mM EDTA, 1
mM iodine acetic acid and 0.5 mM PMSF, and the mixture was dialyzed
against a 20 mM Tris-hydrochloric acid buffer solution (pH 7.5,
containing 1mM DTT, 10% glycerol, 1 mM EDTA, 1 mM iodine acetic
acid, and 0.5 mM PMSF). The enzyme solution is adsorbed on Blue
Sepharose CL-6B (Pharmacia) which has been equilibrated with the
same buffer solution containing 0.25% Triton X-100. Then, lipid
components are eluted with the same buffer solution containing 1.0%
Triton X-100, then, the above-mentioned buffer solution flows with
sodium chloride concentration being increased continuously from 0
to 3.0 M, and active fractions are collected. The resulted active
fractions are dialyzed against the above-mentioned buffer solution,
then, adsorbed on DEAE-Sepharose Fast Flow which has been
equilibrated with the same buffer solution containing 1.0% Triton
X-100. Then, the above-mentioned buffer solution flows with sodium
chloride concentration being increased continuously from 0 to 0.5M,
and active fractions are collected to obtain a purified sample.
[0300] (j) Coenzyme:
[0301] Reduced type .beta.-nicotinamide adenine dinucleotide (NADH)
is used as a coenzyme.
[0302] The coprostan-3-one dehydrogenase B of the present invention
produced by the CP 1 strain is a novel enzyme, and the physical and
chemical properties and method for purification thereof are as
follows.
[0303] (a) Action: it catalyses the following reaction.
coprostan-3-one+NADPH.fwdarw.coprostanol+NADP
[0304] (b) Substrate specificity
[0305] The relative activities are 0 for 5a-cholesten-3-one when
the activity of coprostan-3-one is defined as 100.
[0306] (c) Optimum pH: 5.4 to 7.6
[0307] (d) Stable pH: 4.5 to 7.0
[0308] (e) Measurement of titer:
[0309] With 0.2 ml of 3 mM coprostan-3-one micelle solution
containing 0.33% Triton X-100 is mixed 0.3 ml of a 20 mM
Tris-hydrochloric acid buffer solution (pH 7.5) containing 1.0%
Triton X-100 and 1 mM DTT, and 0.1 ml of a 10 mM NADPH solution, to
the resulted mixture is added 0.05 ml of an enzyme solution,
reacted for 30 minutes at 37.degree. C., and then added 0.1 ml of
chloroform to extract sterol and to terminate the reaction.
[0310] Then, the quantity of coprostanol produced in the reaction
solution is determined using TLC/FID iatroscan. The quantity of
coprostanol in the reaction solution is determined likewise using
an inactivated enzyme which has been previously heated as a
control. The enzyme activity which produces 1 .mu.mol of
coprostanol per one minute is regarded as 1 unit.
[0311] (f) Range of suitable reaction temperature:
[0312] In reaction at pH 7.5 for 30 minutes, increasing temperature
up to 45.degree. C. is attended by increasing the activity.
[0313] (g) Range of temperature stability
[0314] After heating treatment at 45.degree. C. for 10 minutes, it
keeps activity of not less than 80% of that before the
treatment.
[0315] (h) Influence of inhibitor, metal ion:
[0316] When 1 mM PCMB, iodine acetamide and EDTA are added and
reacted for 30 minutes at pH 7.5 and 37.degree. C., the relative
activities are 0, 95 and 112, respectively, if the enzyme activity
without adding inhibitor is 100. Further, when the present enzyme
is reacted in the presence of 1 mM iron chloride and copper
chloride, the relative activities are 105 and 0, respectively, in
comparison to the case of no addition.
[0317] (i) Purification method:
[0318] Air is bubbled through the culture, and microbial cells
adsorbed on the bubbles are recovered and concentrated. The
concentrated solution is subjected to centrifugal separation to
collect the cells, and the cells are suspended in a 20 mM
phosphoric acid buffer solution (pH 7.5, containing 1 mM DTT). The
cells are disrupted by ultrasonication, further, treated with
ultrasonication in the presence of 1.0% Triton X-100, solid
components are removed by centrifugal separation, to obtain a crude
enzyme solution. To the crude enzyme solution is added 1 mM EDTA, 1
mM iodine acetic acid and 0.5 mM PMSF, and the mixture is dialyzed
against a 20 mM Tris-hydrochloric acid buffer solution (pH 7.5,
containing 1 mM DTT, 10% glycerol, 1 mM EDTA, 1 mM iodine acetic
acid, and 0.5 mM PMSF). The crude enzyme solution is adsorbed on
Blue Sepharose CL-6B (Pharmacia) which has been equilibrated with
the same buffer solution containing 0.25% Triton X-100. Then, lipid
components are eluted with the same buffer solution containing 1.0%
Triton X-100, then, the above-mentioned buffer solution flows with
sodium chloride concentration being increased continuously from 0
to 3.0 M, and active fractions are collected. The resulting active
fractions are dialyzed against the above-mentioned buffer solution,
then, adsorbed on DEAE-Sepharose Fast Flow which has been
equilibrated with the same buffer solution containing 1.0% Triton
X-100. Then, the above-mentioned buffer solution flows with sodium
chloride concentration being increased continuously from 0 to 0.5
M, and active fractions are collected to obtain a purified
sample.
[0319] (j) Coenzyme:
[0320] Reduced type .beta.-nicotinamide adenine dinucleotide
phosphoric acid (NADPH) is used as a coenzyme.
[0321] As the cholesterol-reduced composition of the present
invention, microbial cells or treated materials thereof, crude
purified enzymes, purified enzymes and the like containing these
three enzymes may be used without any treatment provided they have
activities of a cholesterol dehydrogenase, 4-cholesten-3-one
dehydrogenase and coprostan-3-one dehydrogenase, and further, those
in the form of tablets, powders, fine particles, granules,
capsules, syrups and the like molded with vehicles which are
acceptable for food or medicine may be used. The composition of the
present invention may be added as a composition to be added into
food or feed for reducing the amount of cholesterol in the food or
feed, or may be orally administered via oral route for reducing the
cholesterol level in serum. When a crude purified enzyme or
purified enzyme is used as the composition of the present
invention, the composition optionally may be advantageously
prepared so that nicotinamide, phosphate ion or phospholipase is
contained in the composition. Examples of the form of the oral
composition of the present invention include tablets, powders, fine
particles, granules, capsules, syrups, enteric agent, troches and
the like. In the case of addition or administration, as the
vehicle, any compound such as saccharides like sorbitol, lactose,
glucose, lactose, dextrin, starch, crystalline cellulose and the
like; inorganic compounds like calcium carbonate, calcium sulfate
and the like; distilled water, sesame oil, corn oil, olive oil,
cotton seed oil and the like, generally can be used. In preparing
the composition, additives such as binder, lubricant, disperser,
suspending agent, emulsifying agent, diluent, buffering agent,
antioxidant, bacterium inhibiting agent and the like may be
used.
[0322] The amount to be added may be advantageously controlled to
be the above-mentioned value necessary for the enzymatic conversion
or the microbial conversion in the method for producing a
cholesterol-reduced substance of the present invention.
[0323] The dosage varies depending on age, sex, administration
pattern, times of administration, form and the like, and regarding
dosage for oral administration for adults, it is suitable that the
amount of bacteria is 1.times.10to 1.times.10.sup.12 cell/day,
preferably 1.times.10.sup.8 to 1.times.10.sup.11 cell/day and the
amount of three enzymes is 10 to 1.times.10.sup.5 unit/day,
preferably 1.times.10.sup.2 to 1.times.10.sup.4 unit/day, and that
the composition is divided into 1 to 4 portions for one
day-administration. If necessary, dosage out of the above-mentioned
restriction can also be adopted.
BRIEF DESCRIPTION OF DRAWINGS
[0324] FIG. 1 is a graph showing results of measurement of
conversion enzyme activity at pH 6, 7 and 8 of various cholesterol
dehydrogenases derived from microorganisms.
[0325] FIG. 2 is a graph showing results of measurement of optimum
pH of cholesterol dehydrogenases derived from a CP 2 strain and
ATCC 51222 strain belonging to Eubacterium.
[0326] FIG. 3 is a graph showing results of measurement of optimum
pH of cholesterol dehydrogenase A, 4-cholesten-3-one dehydrogenase
A and coprostan-3-one dehydrogenase A.
[0327] FIG. 4 is a graph showing progress of concentration of a CP
1 strain belonging to Eubacterium and concentration of coprostanol
in a medium according to a bubbling method.
[0328] FIG. 5 is a graph showing results of measurement of optimum
pH of cholesterol dehydrogenase B, 4-cholesten-3-one dehydrogenase
B and coprostan-3-one dehydrogenase B.
[0329] FIG. 6 is a graph showing results of measurement of
enzymatic conversion activity of cholesterol dehydrogenase B when a
phosphate ion is added.
BEST MODE FOR CARRYING OUT THE INVENTION
[0330] The following embodiments and comparative embodiments
further illustrate the characteristics of the present invention,
but, do not limit the scope thereof. In all embodiments, all parts
are by weight unless otherwise stated. Embodiment 1 (screening of
cholesterol reducing conversion enzyme producing microorganism) The
present inventors have screened various microorganisms such as
stock microorganisms mainly including 300 kinds (species level) of
actinomycetes, fungi and bacteria, aerobic bacteria separated from
100 kinds of various soils, and anaerobic bacteria from 7 feces
samples of humans, 8 feces samples of mammals other than humans and
9 feces samples of birds, to find a cholesterol reducing conversion
enzyme group having optimum pH in neutral pH range.
[0331] As a result, cholesterol reducing conversion enzymatic
activity has been detected which converts cholesterol to
coprostanol in anaerobic microorganisms separated from feces, as
shown below.
[0332] Each feces was separately poured on to 6 kinds of media for
detection (see, Table 1) to which cholesterol had been added as a
substrate, and reducing conversion enzymatic activity of cultured
substance, namely, whether coprostanol had been generated from
cholesterol or not was investigated. The results are shown in
Tables 2 to 4. In Table 1, CHOL represents cholesterol and PL
represents phospholipid, respectively.
[0333] Formulations of nutrition media are as follows.
[0334] Nutrition medium 1:
[0335] 1.8% trypticase, 0.05% yeast extract, 0.13% dipotassium 50 ,
phosphate, 0.22% sodium chloride, 0.04% L-cystine, 0.03% sodium
thioglycolate, 0.05% agar (pH 7.2)
[0336] Nutrition medium 2:
[0337] 1.0% casitone, 1.0% yeast extract, 0.5% lactose, 0.5% sodium
pyruvate,-0.05% sodium thioglycolate (pH 7.5)
[0338] Nutrition medium 3:
[0339] 0.24% Lab-lemco powder (Oxoid), 1.0% proteose peptone, 0.5%
yeast extract, 0.4% disodium phosphate, 0.15% glucose, 0.05%
soluble starch, 0.02% cystine, 0.05% cysteine hydrochloride (pH 7.6
to 7.8).
1TABLE 1 No. Additive {circle over (1)} Nutrition +1.2 bovine
medium 1 brain lipid {circle over (2)} Nutrition 1.2% bovine +5%
soybean oil medium 1 brain lipid {circle over (3)} Nutrition +0.2%
CHOL medium 2 +0.1% PL {circle over (4)} Nutrition +0.2% CHOL +1.2%
bovine medium 2 +0.1% PL brain lipid {circle over (5)} Nutrition
+5% horse blood medium 3 {circle over (6)} Nutrition +1.2% bovine
+5% horse blood medium 3 brain lipid
[0340]
2 TABLE 2 Medium No. No. Mammal {circle over (1)} {circle over (2)}
{circle over (3)} {circle over (4)} {circle over (5)} {circle over
(6)} 1 Bear 2 Giraffe 3 Zebra 4 Elephant 5 Lion + 6 Cheetah 7 Koala
8 Tapir
[0341]
3 TABLE 3 No. Human {circle over (1)} {circle over (2)} {circle
over (3)} {circle over (4)} {circle over (5)} {circle over (6)} 1
Human 1 + 2 Human 2 3 Human 3 4 Human 4 5 Human 5 6 Human 6 7 Human
7
[0342]
4TABLE 4 No. Human {circle over (1)} {circle over (2)} {circle over
(3)} {circle over (4)} {circle over (5)} {circle over (6)} 1
Ostrich 2 Crane 3 Emu 4 Pheasant 5 Flamingo + 6 Japanese crested
ibis 7 Red-crested white crane 8 Japanese great tit + 9 Peacock
[0343] As can be seen from Tables 2 to 4, growth of microorganisms
having reducing conversion enzymatic activity was detected from
cultivated materials in 4 kinds of feces of human sample 1
(nutrition medium 1), lion (nutrition medium 4), red flamingo
(nutrition medium 3) and Japanese great tit (nutrition medium 2),
and the enzymatic activity could not be detected from feces samples
2 to 7 of humans, feces samples of mammals other than lion such as
bear, giraffe and the like, and feces samples of birds other than
flamingo and Japanese great tit such as ostrich, crane and the
like. As described above, reducing conversion enzymatic activity
could be detected from specific feces of specific kinds of animals,
and in addition, only in specific media.
[0344] Embodiments 2 (Selection of an Enzyme Having Optimum pH in
Neutral pH Range)
[0345] The culture in 4 kinds of feces of human sample 1, lion, red
flamingo and Japanese great tit in which conversion activity from
cholesterol to coprostanol was detected, and a culture of a
bacterium belonging to Eubacterium ATCC 51222 described in the
above-mentioned Beitz, et al., Applied Microbiology and
Biotechnology 43, 887 (1995), were, respectively, treated with
ultrasonication to disrupt the bacterial cells (Eubacterium species
ATCC 21408 described in Beitz, et al., United States patent No.
4921710 could not be obtained from ATCC). Conversion active
fractions in this disrupted cells were adsorbed on Blue Sepharose
and eluted, cholesterol dehydrogenase active fractions were
investigated in the cholesterol conversion system, and the activity
was measured at pH 6, pH 7 and pH 8, and the results are shown in
FIG. 1.
[0346] As is known also from FIG. 1, cholesterol dehydrogenase A
from the CP 2 strain derived from lion and cholesterol
dehydrogenase B from the CP 1 strain derived from human sample 1
exhibited maximum activity at near pH 7 which is in neutral pH
range and other samples exhibited maximum activity at near pH 8
which is in alkaline pH range. Thus, a cholesterol dehydrogenase
having an optimum pH in neutral pH range has not been known yet
until now, therefore, it is a novel enzyme.
[0347] Embodiment 3 (Production of Cells of Eubacterium sp. CP
2)
[0348] 20 g of casitone (Difco), 20 g of yeast extract (Difco), 10
g of soluble starch, 10 g of sodium pyruvate, 1 g of sodium
thioglycolate, 1 g of potassium chloride and 0.2 g of lecithin
(type 4S, Sigma) were dissolved in 2 L of de-ionized water, pH
thereof was controlled at 7.5, and the solution was poured into 3
liter Erlenmeyer flasks, separately. This medium was sterilized at
120.degree. C. for 15 minutes, then, the CP 2 strain was
inoculated, and standing culture in anaerobic condition was
conducted at a temperature of 37.degree. C. for 7 days. After
completion of the culture, 8.5 g of a precipitate was obtained from
2 L of the culture by centrifugal separation, and the precipitate
was used as cells-containing material.
[0349] Embodiment 4 (Production of Extracted Material from Cells of
Eubacterium sp. CP 2)
[0350] The cells-containing material obtained in Embodiment 3 was
suspended in 30 ml of 40 mM Britton-Robinson buffer solution (pH
6.5), and the cells suspension was treated with an ultrasonication
at 60 W for 10 minutes, to obtain extracted material from the
cells.
[0351] Embodiment 5 (Production of Cholesterol Dehydrogenase A)
[0352] The extracted material obtained in Embodiment 4 was treated
with ultrasonicaion for 30 seconds in the presence of 1 mM DTT and
0.2% Triton X-100, and then solid components were removed by
centrifugal separation to obtain a crude enzyme solution. The crude
enzyme solution was dialyzed against a 20 mM PIPES buffer solution
(pH 7.5, containing 1 mM DTT, 0.2% Triton X-100 and 10% glycerol),
then, adsorbed on Blue Sepharose CL-6B (Pharmacia) equilibrated
with the buffer solution. Then, the buffer solution was flown with
sodium chloride concentration being increased continuously from 0
to 3.0 M, and active fractions were collected. The resulted active
fractions were dialyzed against the afore-said buffer solution,
then, adsorbed on Red Sepharose CL-6B (Pharmacia). Then, the
above-mentioned buffer solution was flown with sodium chloride
concentration being increased continuously from 0 to 3.0 M, and
active fractions were collected to prepare a purified sample. The
specific activity of this purified sample was 4.49 unit/mg protein,
and the yield thereof was 37.4 %.
[0353] This purified sample was subjected to SDS polyacrylamide gel
electrophoresis, and dyed by using a silver staining kit (Daiichi
Kagaku K.K.), and a single band was recognized in the position of
molecular weight of about 58,800.
[0354] Comparative Embodiment 1 (Optimum pH of Cholesterol
Dehydrogenase)
[0355] ATCC 51222 strain and CP 2 strain was inoculated to the
medium described in Embodiment 3, respectively, and standing
culture in anaerobic condition was conducted at a temperature of
37.degree. C. for 7 days. After completion of the culture,
cells-containing material was obtained from the culture solution by
centrifugal separation, and this cells-containing material was
suspended in 50ml of 40mM Britton-Robinson buffer solution (pH
6.5), and the cell suspension was treated with an ultrasonicaion at
60 W for 10 minutes, to obtain extracted material from cells. A
purified sample of cholesterol dehydrogenase was obtained according
to the method described in Embodiment 3 from this extracted
material.
[0356] The relative activities of these enzymes at various pH
values are shown in FIG. 2. As is known from FIG. 2, the
cholesterol dehydrogenase of the CP 2 strain having an optimum pH
in a neutral side corresponding to pH of meat, egg and milk is more
excellent in food treatment than the cholesterol dehydrogenase of
ATCC 51222 strain having optimum pH in alkaline side.
[0357] Embodiment 6 (Production of 4-cholesten-3-one Dehydrogenase
A)
[0358] The extracted material obtained in Embodiment 4 was treated
with ultrasonication for 30 seconds in the presence of 1 mM DTT and
0.2% Triton X-100, and solid components were removed by centrifugal
separation to obtain a crude enzyme solution. The crude enzyme
solution was dialyzed against a 20 mM PIPES buffer solution (pH
7.5, containing 1 mM DTT, 0.2% Triton X-100 and 10% glycerol),
then, adsorbed on Blue Sepharose CL-6B (Pharmacia) equilibrated
with the buffer solution. Then, the buffer solution was flown with
sodium chloride concentration being increased continuously from 0
to 3.0 M, and active fractions were collected. The resulted active
fractions were dialyzed against the afore-said buffer solution,
then, adsorbed on Resource Column (Pharmacia). Then, the
above-mentioned buffer solution was flown with sodium chloride
concentration being increased continuously from 0 to 1.0 M, and
active fractions were collected to prepare a purified sample. The
specific activity of this purified sample was 3.31 unit/mg protein,
and the yield thereof was 40.1 %.
[0359] This purified sample was subjected to SDS polyacrylamide gel
electrophoresis, and dyed by using a silver staining kit (Daiichi
Kagaku K.K.), and a single band was recognized in the position of
molecular weight of about 37,500.
[0360] Embodiment 7 (Production of coprostan-3-one Dehydrogenase
A)
[0361] The extracted material obtained in Embodiment 4 was treated
with ultrasonication for 30 seconds in the presence of 1 mM DTT and
0.2% Triton X-100, and solid components were removed by centrifugal
separation to obtain a crude enzyme solution. The crude enzyme
solution was dialyzed against a 20 mM PIPES buffer solution (pH
7.5, containing 1 mM DTT, 0.2% Triton X-100 and 10% glycerol),
then, passed through Blue Sepharose CL-6B (Pharmacia) equilibrated
with the buffer solution, fractions which had not been adsorbed on
Blue Sepharose CL-6B were recovered, and dialyzed against 20 mM
phosphoric acid-citric acid buffer solution (pH 6.0) (containing
0.5 mM NADPH, 1 mM DTT, 0.2% Triton X-100 and 10% glycerol). The
enzyme solution was adsorbed on DEAE-Cellulofine equilibrated with
the buffer solution, then, the buffer solution was flown with
sodium chloride concentration being increased continuously from 0
to 1.0 M, and active fractions were collected, then, dialyzed
against the afore-said buffer solution, then, the active components
were adsorbed on Blue Sepharose CL-6B, and non-adsorbed fractions
were recovered. The non-adsorbed fractions were heated for 10
minutes at 95.degree. C. to remove protein to prepare activating
fractions. Then, elution was conducted using the above-mentioned
buffer solution containing 1 mM NADPH with sodium chloride
concentration being increased continuously from 0 to 3.0 M, and
active fractions were collected, and above-mentioned activating
fractions were added to prepare a purified sample. The specific
activity of this purified sample was 4.51 unit/mg protein, and the
yield thereof was 19.0 %.
[0362] Embodiment 8 (Production of coprostan-3-one Dehydrogenase
A)
[0363] The cells-containing material obtained in Embodiment 3 was
suspended in 30 ml of a 20 mM phosphoric acid-citric acid buffer
solution (pH 6.0, 1 mM DTT) and the cells suspension was treated by
an ultrasonicator (BRANSON SONIFIER Model. 250) for 5 minutes at
120 W, to obtain extracted material from the cells. This extracted
material was treated with ultrasonication for 30 seconds in the
presence of 1 mM DTT and 0.2% Triton X-100, and solid components
were removed by centrifugal separation to obtain a crude enzyme
solution. The crude enzyme solution was dialyzed against 20 mM
phosphoric acid-citric acid buffer solution A (pH 6.0, containing 1
mM DTT, 0.2% Triton X-100 and 30% glycerol), then, adsorbed on Blue
Sepharose CL-6B (Pharmacia) equilibrated with the buffer solution,
then, the above-mentioned buffer solution was flown with sodium
chloride concentration being increased continuously from 0 to 3.0
M, and active fractions were collected. The resulted active
fractions were dialyzed against 20 mM phosphoric acid-citric acid
buffer solution B (pH 7.5, containing 1 mM DTT, 0.2% Triton X-100
and 30% glycerol), then, adsorbed on DEAE-Cellulofine equilibrated
with the buffer solution B, then, the above-mentioned buffer
solution was flown with sodium chloride concentration being
increased continuously from 0 to 1.0 M, active fractions were
collected and dialyzed against the same buffer solution. The
resulting active fractions were dialyzed against the same buffer
solution B, then, adsorbed on Resource Column (Pharmacia). Then,
the buffer solution B was flown with sodium chloride concentration
being increased continuously from 0 to 1.0 M, and active fractions
were collected to prepare a purified sample. The specific activity
of this purified sample was 4.90 unit/mg protein, and the yield
thereof was 9.5 %.
[0364] This purified sample was subjected to SDS polyacrylamide gel
electrophoresis, and dyed by using a silver staining kit (Daiichi
Kagaku K.K.), and a single band was recognized in the position of
molecular weight of about 29,000.
[0365] Embodiment 9 (Optimum pH of Enzyme System from CP 2 Strain
Derived from Lion)
[0366] Now, it will be shown that an enzyme system from the CP 2
derived from lion is particularly excellent in meat treatment. This
enzyme system has optimum pH for reducing sequentially from
cholesterol dehydrogenase through 4-cholesten-3-one dehydrogenase
to coprostan-3-one dehydrogenase. And the optimum pH of cholesterol
dehydrogenase is in a neutral range, and the optimum pH values of
4-cholesten-3-one dehydrogenase and coprostan-3-one dehydrogenase
are within the pH range from 5.5 to 6.5 which is a weak acidic
range corresponding to the pH of meat. Therefore, when meat
treatment is conducted using this enzyme system, cholesterol in
meat can be continuously converted to coprostanol without stock of
intermediates such as 4-cholesten-3-one and coprostan-3-one. The
measurement results of the optimum pH values of these enzymes are
shown in FIG. 3.
[0367] Embodiment 10 (Enzymatic Conversion to Coprostanol in Aging
Period of Meat)
[0368] 10 g of beef 6 hours after slaughtering was made into minced
meat, and to this was added the cholesterol dehydrogenase obtained
in Embodiment 5 (0.2 unit/g meat), the 4-cholesten-3-one
dehydrogenase obtained in Embodiment 6 (0.2 unit/g meat) and the
coprostan-3-one dehydrogenase obtained in Embodiment 7 (0.2 unit/g
meat) and mixed, and the mixture was aged at 5.degree. C. for 7
days. Also, mixtures to which 0.5 ml of a 20% aqueous nicotinamide
solution had been further added, meat to which phospholipase (2
unit/g meat) had been further added and meat to which both of them
had been further added at the mixing were aged at 5.degree. C. for
7 days, respectively.
[0369] After aging, the meat was freeze-dried and lipid components
were extracted with a 25 ml extraction solvent
(chloroform:methanol=2:1). Regarding the resulting extracted
sample, the amount of converted sterol products was determined
using gas chromatography (manufactured by GL Science, TC-1701,
column .phi. 0.25 .mu.m.times.30 m). The conversion ratio of
cholesterol to coprostanol in the meat was measured, and the
results are shown in Table 5. In Table 5, PL-D represents
phospholipase D and COP represents coprostanol, respectively.
[0370] As can be seen from Table 5, conversion to coprostanol could
not be observed when only a conversion enzyme was added, and the
conversion increased synergistically when nicotinamide was added or
when both nicotinamide and phospholipase D were added.
5 TABLE 5 Teating type COP conversion (%) No addition 0 Enzyme 0
Enzyme + PL-D 0.84 Nicotinamide 0 Enzyme + nicotinamide 4.4 Enzyme
+ PL-D + nicotinamide 49.1
[0371] Embodiment 11 (Enzymatic Conversion to Coprostanol in
Processing Meat)
[0372] 10 g of beef 6 hours after slaughtering was made into minced
meat, and further homogenated. To this homogenated meat was added
and mixed the cells-extracted material described in Embodiment 4,
and the mixture was heated at 37.degree. C. for 2 hours. The
amounts to be added were 0.2 unit/g meat for cholesterol
dehydrogenase A, 0.4 unit/g meat for 4-cholesten-3-one
dehydrogenase A and 0.4 unit/g meat for coprostan-3-one
dehydrogenase A. Also, a mixture to which 0.5 ml of a 20% aqueous
nicotinamide solution had been further added at the mixing was
processed similarly.
[0373] After completion of the processing, the conversion ratio to
coprostanol in the resulted sample was measured according to the
method in Embodiment 10. The conversion ratio of cholesterol to
coprostanol in the resulting meat was measured, and the results are
shown in Table 6.
[0374] As is known also from Table 6, enzymatic conversion of
cholesterol to coprostanol in meat could be recognized only with
addition of an enzyme source. When nicotinamide was added, the
conversion ratio further increased.
6 TABLE 6 meat treating type COP conversion (%) No addition 0
Enzyme 12.1 Enzyme + nicotinamide 51.5
[0375] Embodiment 12 (Microbial Conversion of Cholesterol in
Processing Meet)
[0376] 10 g of commercially available beef was processed into meat
paste by a food cutter, then, to this was added 0.57 g of cells of
the CP 2 strain described in Embodiment 3. This mixture was
fermented at 37.degree. C. for 20 hours to obtain processed meat.
After completion of the processing, the conversion ratio to
coprostanol in the resulting sample was measured according to the
method in Embodiment 10. The conversion ratio of cholesterol to
coprostanol in the resulting meat was 82.3 %.
[0377] Embodiment 13 (Microbial Conversion of Cholesterol in
Milk)
[0378] To 10 ml of commercially available milk was added 0.42 g of
cells of the CP 2 strain described in Embodiment 3, and this
mixture was fermented at 37.degree. C. for 20 hours to obtain
processed milk. After completion of the processing, the conversion
ratio to coprostanol in the resulting sample was measured according
to the method in Embodiment 10. The conversion ratio to coprostanol
in the resulting meat was 64.4 %.
[0379] Embodiment 14 (Microbial Conversion of Cholesterol in
Egg)
[0380] With 10 ml of a 10% aqueous yolk solution was mixed 0.42 g
of cells of the CP 2 strain described in Embodiment 3, and this
mixture was fermented at 37-C for 20 hours to obtain a processed
egg. After completion of the processing, the conversion ratio to
coprostanol in the resulting sample was measured according to the
method in Embodiment 10. The conversion ratio of cholesterol to
coprostanol in the resulting egg was 27.8 %.
[0381] Embodiment 15 (Production of Cells of Eubacterium sp. CP
1)
[0382] Commercially available bovine brain Was homogenated and
freeze-dried, and extracted three times with a 3-fold amount of
extraction solvent (chloroform:methanol 2:1), then, the solvent was
removed to obtain a lipid extract. The yield was 51.9% in terms of
dry weight.
[0383] 48 g of this bovine brain lipid extract, 72 g of trypticase
(BBL), 2 g of yeast extract (Difco), 8.8 g of sodium chloride, 5.2
g of dipotassium hydrogen phosphate, 2 g of Bacto Agar (Difco), 1.6
g of L-cystine, 2 g of cholesterol, 1.2 g of sodium thioglycolate
were dissolved in 4 L of de-ionized water, and pH of the solution
was controlled to 7.2, and poured into two 3-L Erlenmeyer flasks
separately. This medium was sterilized at 120.degree. C. for 15
minutes, then, a CP 1 strain was inoculated, and stirring culture
in anaerobic condition was conducted at 37.degree. C. for 5
days.
[0384] The recovering of cells of the CP 1 strain from the culture
was conducted by centrifugal separation which is an ordinary method
of recovering microbial cells. However, the CP 1 strain was
adsorbed on an insoluble lipid and coagulated, the specific gravity
thereof decreased and the strain became inhomogeneous. Therefore,
the cells could not be recovered. Because it was suggested that the
surface of the cells was hydrophobic and since the cells were
adsorbed on an insoluble lipid, the cells were adsorbed on
hydrophobic air by a bubbling method and recovered. Namely, air was
bubbled in from the lower side of the culture filled into a column,
and a part of the bubble on which the cells concentrated was
recovered from the upper end of the column. This operation was
repeated several times for obtaining a cells-concentrate. Whether
the cells-concentrate was obtained or not was recognized by
measuring the activity of 4-cholesten-3-one dehydrogenase B in the
extracted material which was obtained by treating a sample solution
taken from the bubble part and-a remaining sample solution after
recovering the bubble part by an ultrasonicator at 60 W for 10
minutes. The results when the bubbling was conducted twice are
shown in FIG. 4. From FIG. 4, it is known that the bubbling method
is effective for recovering cells of the CP 1 strain.
[0385] Embodiment 16 (Production of Cholesterol Dehydrogenase B,
4-cholesten-3-one dehydrogenase B and coprostan-3-one Dehydrogenase
B by Eubacterium sp. CP 1 Strain)
[0386] The cells-concentrate obtained in Embodiment 15 is
centrifugally separated, the resulting cells are suspended in a 20
mM Tris-hydrochloric acid buffer solution (pH 7.5, containing 1 mM
dithiothreitol). The cells are disrupted by ultrasonication,
further, treated with ultrasonication in the presence of 1.0%
Triton X-100, then, solid components are removed by centrifugal
separation, to obtain a crude enzyme solution. To the crude enzyme
solution is added 1 mM EDTA, 1 mM iodine acetic acid and 0.5 mM
PMSF, and the mixture is dialyzed against a 20mM Tris-hycrochloric
acid buffer solution (pH 7.5, containing 1 mM dithiothreitol, 10%
glycerol, 1 mM EDTA, 1 mM iodine actic acid, 0.5 mM PMSF). The
crude enzyme solution was diluted to a 4-fold volume with the same
buffer solution, then, adsorbed on Blue Sepharose CL-6B (Pharmacia)
which has been equilibrated with the same buffer solution
containing 0.25% Triton X-100. Then, lipid components are eluted
with the same buffer solution containing 1.0% Triton X-100, then,
the above-mentioned buffer solution flows with sodium chloride
concentration being increased continuously from 0 to 3.0 M, and
active fractions are collected. The resulting active fractions are
dialyzed against the above-mentioned buffer solution, then,
adsorbed on Resouce (Pharmacia) which has been equilibrated with
the same buffer solution containing 1.0% Triton X-100. Then, the
above-mentioned buffer solution flows with sodium chloride
concentration increased continuously from 0 to 0.5 M, and active
fractions are collected to prepare a purified product.
[0387] In this purified product, the specific activity of
cholesterol dehydrogenase B was 0.116 unit/mg protein, and the
yield thereof was 32.1 %, the specific activity of
4-cholesten-3-one dehydrogenase B was 1.24 unit/mg protein, and the
yield thereof was 65.2 %, and the specific activity of
coprostan-3-one dehydrogenase B was 0.835 unit/mg protein, and the
yield thereof was 54.8 %. The results of measurement of optimum pH
values of these enzymes are shown in FIG. 5.
[0388] Embodiment 17 (Activation of Cholesterol Dehydrogenase B by
Phosphoric Acid)
[0389] Conversion reaction by cholesterol dehydrogenase B is
remarkably activated by a phosphate ion. As the phosphate ion
source, sodium polyphosphate, sodium metaphosphate, sodium
pyrophosphate, trisodium phosphate and the like can be used, and
the results when trisodium phosphate was used are shown in FIG. 6.
From this, it is known that addition of a phosphate ion is
desirable in conversion of cholesterol to coprostanol using
cholesterol dehydrogenase B.
[0390] Embodiment 18 (Enzymatic Conversion of Cholesterol in Meat
Using Enzymes of Eubacterium sp. CP 1)
[0391] 10 g of beef 6 hours after slaughtering was made into minced
meat, and to this was added each enzyme described in Embodiment 15
and 0.05 g of sodium phosphate, and the mixture was heated at
37.degree. C. for 2 hours. The amounts to be added were 0.2 unit/g
meat for cholesterol dehydrogenase B, 0.2 unit/g meat for
4-cholesten-3-one dehydrogenase B and 0.2 unit/g meat for
coprostan-3-one dehydrogenase B. Also, a mixture to which 0.5 ml of
a 20% aqueous nicotinamide solution had been further added at the
mixing was processed similarly.
[0392] After completion of the processing, the conversion ratio to
coprostanol in the resulting sample was measured according to the
method in Embodiment 10. The conversion ratio of cholesterol to
coprostanol in the resulted meat was measured, and the results are
shown in Table 7. As can be seen also from Table 7, enzymatic
conversion ratio of cholesterol to coprostanol in meat could be
recognized only with addition of enzymes. When nicotinamide was
added, the conversion ratio was further increased.
7 TABLE 7 Meat treating type COP conversion (%) No addition 0 Cell
extract 28.1 Cell extract + nicotinamide 68.1
[0393]
8 Embodiments 19 Tablets (300 mg per one tablet) are produced by
the usual method according to the following formulation. Dried
cells of the CP 1 strain (1 .times. 10.sup.10 cells) 10 mg Lactose
230 mg Corn Starch 30 mg Synthetic aluminum silicate 12 mg
Carboxymethylcellulose calcium 15 mg Magnesium stearate 3 mg
Embodiments 20 Tablets (300 mg per one tablet) are produced by the
usual method according to the following formulation. Dried cells of
the CP 2 strain (1 .times. 10.sup.10 cells) 10 mg Lactose 190 mg
Corn Starch 70 mg Synthetic aluminum silicate 12 mg
Carboxymethylcellulose calcium 15 mg Magnesium stearate 3 mg
Embodiments 21 Hard capsules (700 mg per one capsule) are produced
according to the following formulation. Cholesterol dehydrogenase A
100 mg (purified sample described in Embodiment 5)
4-Cholesten-3-one dehydrogenase A 150 mg (purified sample described
in Embodiment 6) Coprostan-3-one dehydrogenase A 100 mg (purified
sample described in Embodiment 8) Lactose 230 mg Corn Starch 100 mg
Hydroxypropylcellulose 20 mg
[0394] To 100 mg of cholesterol dehydrogenase A and 100 mg of
coprostan-3-one dehydrogenase A, respectively, and 150 mg of
4-cholesten-3-one dehydrogenase A are added 230 mg of lactose and
100 mg of corn starch and mixed, to this mixture is added an
aqueous solution of 20 mg of hydroxypropycellulose and kneaded.
Then, granules are produced by an ordinary method using an
extrusion granulator. These granules are filled in a gelatin hard
capsule to produce a hard capsule.
9 Embodiments 22 Powders (1000 mg per one piece) are produced by an
ordinary method according to the following formulation. Cholesterol
dehydrogenase B 100 mg (purified sample described in Embodiment 15)
4-Cholesten-3-one dehydrogenase B 150 mg (purified sample described
in Embodiment 15) Coprostan-3-one dehydrogenase B 100 mg (purified
sample described in Embodiment 15) Nicotinamide 50 mg Lactose 430
mg Corn Starch 170 mg
[0395] Industrial Applicability
[0396] According to the present invention, cholesterol
concentration can be reduced by converting cholesterol in a
substance selectively to coprostanol having low intestinal tract
absorbability without losing the property and condition of the
cholesterol-containing substance such as meat and the like.
[0397] Further, the cholesterol level in serum can be reduced by
orally administrating a composition of microbial cells-containing
the novel enzyme of the present invention and converting
cholesterol to coprostanol in the small intestine to inhibit
absorption of the cholesterol.
* * * * *