JPS5889183A - Preparation of nad(p)-dependent cholesterol dehydrogenase - Google Patents

Abstract

PURPOSE:To prepare an NAD(P)-dependent cholesterol dehydrogenase, by culturing microorganisms belonging to Nocardia genus, etc. and capable of growing under aerobic conditions. CONSTITUTION:Microorganism belonging to Nocardia genus, Alcaligenes genus, or Proteus genus, and capable of growing under aerobic conditions, e.g. Nocardia sp. No.Ch2-1 (FERM-P No.6217), Alcaligenes sp. No.4 (FERM-P No.6216), Proteus vulgaris IAM 1025, etc. is cultured in a medium containing cholesterol under aerobic conditions, and the objective NAD(P)-dependent cholesterol dehydrogenase is separated from the cultured cells and the culture liquid.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to NAD(P)-dependent cholesterol dehydrogenase (Cbolesterol dehydrogenase).
se: Relating to [NAD■-CDHJ hereinafter].

Let me explain in more detail. Microorganisms are cultured under aerobic conditions, and NAD(P)-CDH is extracted from the microorganisms or culture fluid.
Concerning how to collect.

NAD(P)-CDH obtained by the method of the present invention is
.

The NAD(P)-CD referred to herein is useful as a method for quantifying cholesterol and as a reagent for quantifying cholesterol.
H refers to cotinamide adenine and NAD as coenzymes.
dinucleotide, ), cotinamide adenine, dinucleotide phosphate) from NADP, steals hydrogen from an electron donor (cholesterol), and transfers hydrogen from an electron acceptor (NAD).
or NADP).

It is already known that aerobic microorganisms produce cholesterol oxidase and cholesterol dehydratase. Furthermore, the fermentation in which Nocardia erythropolis catalyzes the oxidation of cholesterol is not dependent on NAD(P). The same can be said about Cterium-sterolicum (Japanese Patent Publication No. 48-11907).Furthermore, Oibacterium Sp, which is an obligate anaerobic microorganism.

There is a report that ATCC21408 produces NAD(P)-CDH (Japanese Unexamined Patent Publication No. 53-56090), but there is almost no description of the enzymatic properties, etc.
Despite the description of AD(P) dependence, NAD
Examples are described in which the reaction proceeds even in the absence of (P). Therefore, this enzyme is N as defined in the present invention.
It cannot be said to be an AD(P)-dependent dehydrogenase.

Conventionally, cholesterol oxidase is widely used to quantify cholesterol using an enzyme, but it requires peroxidase, etc. to produce a color system, and the operation is complicated. , ascorbic acid, etc., and this has the disadvantage that the error is variable.

In the determination of cholesterol, NAD(P)-CDH is used, in which the reaction does not proceed without the presence of NAD(P),
NA D(P) produced by the reaction shown in the reaction formula below
If H could be measured directly with a photometer, the operation would be simple and various problems of the above-mentioned method using cholesterol oxidase would be solved.

Cholesterol + NAD (P), :: Cholestenone + N
AD■H The present inventors searched widely in nature for an enzyme suitable for the above reaction, that is, a dehydrogenase that is highly specific for cholesterol and is dependent on NAD(P). Microorganisms growing below have a significant amount of NAD(P)
-CD H was found to be produced.

Among them, particularly excellent strains include Nocardia Sagi sp.
Nil Ch2-1 (Nocardia 5p-Nc
hCh2-1), Alcaligenes sp, Na4
(Alcaligenes sp, N[L4), and Proteus vulgaris IAM1025Prote
US vulgaris IAM 1025 is exemplified.

Next, the mycological properties of Nocardia sp, Nl1ch2-1 and Alcaligenes 5p-Na4 will be described below.

(1) Nocardia sp, NaCh2 1 (Nocar
dia sp.

Mycological properties of Arashi Ch2-1) ■ Morphological properties 1) Cell shape and size: At the initial stage of culture, it grows like a hyphae and branches. After that, irregular division occurs and the cells become rod-shaped.

The size is about 0.8-1.0μ×1.5-4.0μ. It does not form aerial hyphae or sporangium.

2) Durham staining: VvJ 3) Acid-fastness: Positive 4) Motility: None) Chemical composition analysis Cell wall contains meso-dianopimelic acid, arabic acid, -\
, Kara 2. -Contains hook 1-P.

Contains no aminopimelic acid or glycine.

Growth status in each medium 1) Broth agar plate medium: After 4 days of culture at 30°C, the diameter is 0.
．． 5-1. Form a circular colony of Q mm. The periphery is gilded or wavy. The surface is smooth and hemispherical, and the center may be convex. The color is pale cream and opaque.

No dye is released into the medium.

2) Sea-Close Nitrate Agar Medium The color of the colonies is white to light cream color, about the size of a raw back. Does not release water-soluble dyes.

3) Curcose, asparagine agar S The soil growth is medium, and the color of the colony is cream-colored. Does not release water-soluble dyes.

4) Glycerin and asparagine agar medium Growth is medium and the color of the colonies is white to cream colored. Does not release water-soluble dyes.

5) Starch inorganic salt agar medium Growth is medium and the color of the colonies is white to light cream color. Does not release water-soluble dyes.

6) Tyrosine agar medium Growth is medium and the colony color is white to light cream color. Does not release water-soluble dyes.

7) Nutrient agar medium It grows well and the color of the colony is cream-colored.

Does not release water-soluble dyes.

8) Yeast malt agar medium It grows well and the color of the colony is cream-colored.

Does not release water-soluble dyes.

9) Oatmeal agar medium Growth is medium and the colony color is white to light cream color. Does not release water-soluble dyes.

0 Physiological properties 1) Growth temperature: Grows at 15°C to 43°C.

It does not grow at 10°C or 145°C.

The optimum temperature is 30-35°C. Ru.

2) Nitrate reducing property: positive 3) Catalase di-positive 4) Oxidase: negative 5) Urease: positive 6) Starch hydrolysis: negative 7) Gelatin liquefaction: negative 8) Tyrosine hydrolysis: negative 9) Casein hydrolysis: negative 10 ) Xanthine hydrolysis: Negative 11) DNA degradation: Negative 12) Lidomus milk: No alkalinity, no peptonization, no coagulation.

13) Production of melanin-like pigment: none 14) Aesculin hydrolysis: positive 15) Tween 20.40.60.80 hydrolysis: all positive - 16) Penicillin resistance test: resistant 17) Attitude towards oxygen: aerobic 18) Inorganic Use of nitrogen source: Use both ammonium salt and nitrate.

19) Na&j growth range: Grows in 0-6%.

Will not grow at 7%.

20) Assimilation of various carbon sources (Pridham, Godelive agar medium) Assimilates D-glucose, D-fructose, mannose, glycerin, and trehalose. - Does not assimilate arabinose, D-xylose, saccharose, inosit, serumnose, raffinose, D-galactose, D-mannite, maltose, and sorbitol.

21) Production of acids from various bacteria - Glucose, Mannose, D-F Lactose, trehalose, glycerin Produces acid from water. wise arabino D-xylose, D-galactose su, maltose, saccharose, lac Toth, D-Sorvit, D-Manni acid from starch, inosit, and starch Not generated.

Hereinafter, the mycological properties of 1- are described in Bergey's Manua.
l of attack Determinative Bacteriolog
Based on the 8th edition, it contains citric acid, arabinose, and galactose, and does not contain diaminopimelic acid and glycine. It grows well in an aerobic mycelial form, and later divides into a closed form. This fungus is known as Nocardii because it is acidic and does not attach sporangia or aerial mycelia.
It is a bacterium belonging to A.

Therefore, this bacterium was identified by the present inventors as Nocardia sp-No.
.

Ch2 1 (Nocardia sp, No, Ch2
1) and submitted it to the Institute of Microbial Technology, Agency of Industrial Science and Technology (FERM-PP!lL6217).
It has been deposited.

■ Alcaligenes sp, N114 (Alcalig
enes sp.

N114) Mycological properties (c) Morphology 1) Cell shape and size: 0.4-0.6μ×對
It is a 0.8-1.2μ bacterium.

2) Presence or absence of cell pleomorphism: No pleomorphism was observed.

3) Presence or absence of interlocking: Has periflagella and moves.

4) Presence or absence of spores: Spores are not formed.

5) Durham staining: negative 6) Anti-acidity: negative 0 Growth status in each medium 1) Meat soup agar tenpei kanyo Round low knee with smooth surface and half lens ridges, entire margins, light cream color, semi- Transparent, glossy 2) Meat juice agar slant culture Medium growth, thread-like growth, pale cream color and translucent 3) Meat juice liquid culture Does not form a bacterial film, is slightly cloudy and has little sedimentation. There is.

4) Meat juice gelatin puncture culture: Gelatin does not liquefy.

5) Lidmus milk culture: Becomes alkaline but does not become peptonized or coagulate.

0 Physiological properties 1) Nitrate reduction: positive 2) Denitrification reaction: negative 3) MR test: negative 4) VR test: negative 5) Indole production: negative 6) Hydrogen sulfide production: patency 7) Starch Hydrolysis: negative 8) Citrate utilization: Koser's medium and Chri
It is used together with Stensen's medium.

9) Utilization of inorganic nitrogen sources: Utilize nitrates and ammonium salts.

10) Production of pigment: Does not produce water-soluble pigment.

11) Urease: positive 12) Oxidase: positive 13) Catalase: positive 14) Growth range PH: Grows at pH 5.0 to 10.0. Temperature: Grows between 5°C and 37°C.

Will not grow at 42°C. .

15) Attitude towards oxygen: aerobic 16) OF test (Hugh-Leifson method): Generate acid aerobically from fructose.

17) Presence or absence of acid and gas generation from sugars, Ayers;
Rupp and Johngon's medium produces acid or no gas from fructose and glycerin.

arabinose, xylose, glucose s, mannose, galactose, malt Sugar, sugar, lactose, trehalose, Rubit, Mannit, Inojit, Starch does not produce acids or gases.

18) Autotrophic growth: Does not grow in gases containing hydrogen gas, carbon dioxide gas, or oxygen gas.

19) Degradability of Tween 80: Positive 20)
Assimilation: D-fructose, k-phenylalanine,
Assimilates calcium levulinate and ζ-threonine. Does not assimilate mannose, maltose, mannitol, and betaine.

Bergey's manu based on the mycological properties of L.
al of Determinative Bacter
When compared with the description in ``Iology'' (8th edition), it was found that it is a short bacillus and moves by pericytial hairs, and Durham is negative.
Al
It is classified into the genus caligenes.

Iku, D-7 lactose assimilation, A,, agvama
r 1nus is mainly responsible for starch decomposition and maltose assimilation, A, pacif 1cus is mainly for threonine and betaine assimilation, and A, cupidus is mainly for oxidase and manganese assimilation. A, venustus is mainly responsible for the assimilation of levulinate, threonine, and betaine.
It differs from s mainly in its ability to assimilate mannitol, threonine, and phenylalanine. Therefore, this bacterium is called Alcaligenes sp.
.

% 4 (Alcal igenes sp, N[L
4) and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiology No. 6216 (FERM-PN [16216)].

Next, a method for producing NAD(P)-CDH using these bacteria will be described in detail. All of these bacteria constitutively have the ability to produce NADQ))-CDH, and contain common peptone, yeast extract, or nitrogen sources such as ammonium sulfate, carbon sources such as glucose and glycerol, and other inorganic salts. Although NAD(P)-CDH is produced even in a medium containing cholesterol, a larger amount of NAD(P)-CDH is produced by adding cholesterol to the medium. At this time, cholesterol may be added either at the start of the culture or during the culture. - In addition, other culture conditions can be carried out within the usual range. NAD(P)-cDm produced by these bacteria is accumulated not only in the bacterial cells but also in the culture solution, and the enzyme can be recovered from either of them. The crude enzyme obtained by salting out the culture filtrate or bacterial cell extract with ammonium sulfate or by precipitation with a solvent such as acetone or ethanol can be used as is or after further purification. It's also possible to do that. For example, purification can be carried out by known methods such as ion exchange crostography and molecular sieve crostography.

The NAD(P)-CDH obtained here can be advantageously used for the determination of cholesterol in cholesterol-containing substances.

The method for measuring the activity of NAD(P)-CDH used in the present invention is shown below.

The enzymatic activity of NAD(P)-CDH is calculated by measuring the amount of NADoH produced when urestyol and NAD(P) are reacted as substrates using a spectrophotometer as an increase in absorbance at 340 nm. i.e. 0.1M)! J
Susu-acid buffer (PH8,6) 2.65ml 1°28m
MNAD solution 0.1 mJ, (8% Triton X-100 solution 0.1511), 1% cholesterol solution 0.05 m
j and NAD(P)-CDH aqueous solution 0.05 m/m were mixed and reacted at 30°C.
Measure the increase in absorbance at m.

As a control, the same operation is performed using water instead of cholesterol with the above composition, and the increase in absorbance at 340 nm of the control solution is subtracted from that of the test solution. The amount of NAD(P)H produced is determined from the obtained value, and from this the amount of NA in the sample is determined.
Calculate D(P)-CDH activity.

Enzyme activity is displayed under conditions of PH8, 6, and 30°C.
One unit was an enzyme that produced 1 μmole of NAD(P)H per minute.

Next, the action of NAD[F]-CDH used in the present invention will be shown.

Cholesterol + NAD (P+, ==? Cholestenone +
NAD(PJH) All NAD(P)-CDHs in the present invention catalyze this reaction.

The general properties of NAD[F]\CDH used in the present invention are as follows:
rdiasp, N[lCh2-1)FERM-P621
7 and Alcaligenes sp, % 4 (Alcal
igenes STY, N [14) FERM-P6
The products produced by 216 are shown below.

0) Nocardia sp, l'IkLch2-1 (No
cardia sp.

Il! lCh2-1) Optimal PH for NAD (Pl-CDH 1) produced by FERM-P6217 Figure 1 shows the relationship between PH and activity at 30°C.

2) PH stability Figure 2 shows the relationship between pH at 37°C and stability.

3) Optimal temperature Figure 3 shows the relationship between temperature and activity at pH 8 and 6.

4) Thermal stability Figure 4 shows the relationship between temperature and stability at pH 7.0.

5) Substrate specificity The element reacts with steroids that have a hydroxyl group at the 3β position,
When cholesterol is 100, it has a slight effect on stigmastyrol 35, β-sitosterol 25, and others such as dehydroeviantrosterone and el-gostyol.

6) Complement Request NAD.

(2) Alcaligenes sp, N+14 (Alcal
igenes sp.

4) FERM-P6216 (7) Production-i NAD
(Pi-CDH 1) Optimal PH Figure 5 shows the relationship between PH and activity at 30°C.

2) PH stability Figure 6 shows the relationship between PH and stability at 37°C.

3) Optimal temperature Figure 7 shows the relationship between temperature and activity at pH 8.5.

4) Thermal stability Figure 8 shows the relationship between temperature and stability at PH7,Q.

5) Substrate specificity The element reacts with steroids that have a hydroxyl group at the 3β position, and when cholesterol is 100, β-sitosterol is 36
, stigmasterol 20, and others such as dehydroepiandrosterone and ergosterol.

6) Complement Request NAD.

Next, the determination of cholesterol using NAD■-CDI of the present invention will be described in detail.

When quantifying cholesterol, actually buffer solution, NA
D■, substrates (serum, cholesterol, etc.) and NAD (
Mix Pi-CDH and react for a certain period of time to produce NA
The increase in D(P)H is measured at a absorbance of 340 nm. Further, if necessary, hydrogen of the generated NAD[F]H may be guided to a coloring system such as a formazan dye using phenazine meso sulfate (PMS), diaphorase, or the like. Furthermore, cholesterol esterase,
It is also possible to add surfactants, stabilizers, etc. It can be carried out in the range of p4 to 10 during the reaction, but it is excellent P)
The f range is 7-9.

Next, to give an example of the quantitative composition of a reagent for cholesterol determination using NAD (Pi-CDH), the reaction system
/per NAD(P)-CDHo, 1 to 10 units, NA
D(P)-10-100mM1 Triton X-1001
, 0% or less, 01 to 10 units of cholesterol esterase (Boehringer) are advantageous. However, the present invention is not limited to these quantitative compositions. A particular advantage of the method of the invention is that the NAD(P)H formed can be measured directly and completely quantified.

Next, test examples and examples will be described.

Test Example: Cholesterol 5g/per strain 3 in the present invention
11 Meat extract 5g Otsu - Yeast extract 9.2 g/l.

Medium consisting of the composition of a junior antifoam agent (PH7T2) xo6
Inoculate a 500ml flask containing 30ml
The culture was incubated with shaking at °C for 40 hours. Collect bacteria by centrifugation (8000 rpm, 10 minutes) from 50 m of culture solution, and
Washed with M phosphate buffer PH7,Q, 5Qm/.

Bacterial cells were suspended in 59 mL of the same buffer solution and disrupted by ultrasonication. This crushed solution is centrifuged (110,000r
p, 10 minutes) to obtain a clear solution. N of the obtained clear liquid
AD(P)-CDH activity was measured and the following results were obtained.

Example I Nocardia sp, Ch2 1 FERM-P
Nn6217 with glucose 5g/7, meat extract 5g//
1. Inoculate the yeast extract into yeast extract and culture with shaking at 30°C for 24 hours.

This ceramic seed culture solution contains 5 g/l of cholesterol and Mg.
SO47H200, 2g/l, antifoaming agent 0-5 g/z
30 containing a medium (PH7,2) 201 having the composition of
/ Inoculate jarf 1-mentor, aerate at 30°C,
Stirring (0.5v/v/min, 20Orpm) L
The cells were cultured for 40 hours.

The culture solution was centrifuged, the resulting bacterial cells were suspended in 0.1 MIJ acid buffer PH79, and the bacterial cells were disrupted with glassase. This cell suspension was centrifuged (11,000 rpm,
10 minutes) to obtain a clear bacterial cell extract. Ammonium sulfate was added to the resulting clear solution to a saturation of 35% to precipitate the enzyme.

Collect the precipitate by centrifugation (8000 rpm, 10 minutes) 2
Dissolve in 0mM phosphate buffer PH7, 0, 100ml,
In a cellophane tube, 20mM phosphate solution P H7
, 9 for 24 hours.

Next, the obtained dialysate was mixed with 20mM J acid buffer PH7,
Equilibrated at 0. It was passed through a column packed with 20 Qml of DEAE/cellulose to adsorb the enzyme.

After washing the column with the same buffer, the buffer concentration was adjusted to 0.1M.
NAD(P)-CDH was eluted after a few seconds.

Both fractions containing NAD(P)-CDH were collected, concentrated, and then dialyzed against 20 mM IJ acid buffer P H7,Q.

This sample was passed through a column packed with 20 ml of hexylsepha 0-sulfur equilibrated with the same buffer solution for adsorption.

The column was washed with 20mM phosphate buffer PH7,9. Then NA was added in a similar buffer containing 9.5 M NaCl.
D■-CDI was eluted, active fractions were collected, concentrated, and
unit/ml of NAD (Pi-CDH solution 5.Qml
I got it. The overall activity yield was 30%.

Example 2 Alcaligenes sp, N114 FERM
-PN[16216 with cholesterol 10 g/l,
Glycerol 2 g/l 1 corn steep liquor - 5 g
/l 1KH2PO45g/l, Mton 507H20
It was cultured in a medium with a composition of 0-2g/11 defoaming agent and 05g/l, and the same operation as in Example 1 was performed to obtain a concentration of 40 units/m.
5.1 of NAD(P)-CDH solution. I got Qml. The overall activity yield was 40%.

Example 3 Using Proteus vulgaris IAM1025, an operation similar to Example 1 was performed, and 37 AAA vmt
of NAD[F]-CDI solution 4 times a day. The overall activity yield was 52%.

Example 4 Nocardia 111), N[l Ch21 F
ERM-PN [16217 with glucose 5 g/l,
500ml containing 200ml of a medium (PH7,2) consisting of meat extract, yeast extract Q, and a small amount of antifoaming agent.
Inoculate into mz'8 Sakaro flask and culture with shaking at 30°C for 24 hours. This seed culture solution contains 5 g of cholesterol/
l s glucose 2g/11 meat x'4 s 5 g/l
, lKH2PO4・5 g/l, MgSO4・7
A 301-volume jar fermentor containing 201 medium (PH 7, 2) consisting of 0.2 g/l of H2O and 0.5 g/l of antifoaming agent was inoculated, and the cells were incubated at 30°C with aeration and stirring (
The cells were cultured for 72 hours at 0.5v7v/min, 20Orpm) L/min. This culture solution was centrifuged (80
00 rpm, 10 minutes). Ammonium sulfate was added to the obtained culture filtrate to 40% saturation to precipitate the enzyme.

The precipitate was collected by centrifugation (8000 rpm, 10 minutes),
Dissolve in 100ml of 20mM phosphate buffer PH7.0, and in a cellophane tube, add 20mM phosphate buffer PH7.
, 0 for 24 hours.

Next, purification was carried out in a manner similar to Cloudy Examples 1 to 3,
30 units/ml NAD(P)-CDH solution: 32
I got m1. The overall activity yield was 32%.

Example 5 Alcaligenes sp, Na3 FERM
Using PN116216, perform the operation similar to Example 4, and add 25 units/ml NAD(P)-CDH solution to 4.2
ml was obtained. The overall activity yield was 38%.

Example 6 Using Proteus vulgaris IAM1025, an operation similar to Example 4 was performed to obtain 18 units/m
3.5 m4 of NAD(P)-CDH solution was obtained. The overall activity yield was 49%.

[Brief explanation of drawings]

Figure 1 shows Nocardia sp, N11Ch2-I FER
Figure 2 is a diagram showing the optimum pH at 30'C of NAD(P)-CDH produced by M-PNQ6217, and Figure 2 is a diagram showing the PH stability of this NAD(P)-CDH at 37°C. Figure 3 shows the optimum temperature of this NAD(F)-CDH, and Figure 4 shows the optimum temperature of this NAD[F]-CDH.
It is a figure showing the thermal stability of. Figure 5 shows Alcaligenes sp, Nch4 FERM-P
30°C of NAD[F]-CDH produced by Umbrella 6216
FIG. 6 is a diagram showing the optimum pH for this NAD[
Diagram showing the PH stability of CDH at 37°C
FIG. 7 is a diagram showing the optimum temperature of the present NAD(P)-CDH, and FIG. 8 is a diagram showing the thermal stability of the present NAD(P)-CDH. 1rj4 H Figure 2 5ti7 III υ 1υ 11H Figure 8 Temperature Figure 4 Temperature Figure 5 Figure 7 ° Temperature Figure 8 Temperature

Claims (1)

[Claims] 1. NAD[F]-dependent cholesterol dehydrogenase, which is characterized by culturing a microorganism that grows under aerobic conditions and collecting NAD (P)-dependent cholesterol dehydrogenase from the culture. Enzyme production method. 2. The NAD (F
Method for producing M-dependent cholesterol dehydrogenase. 3. The strains belonging to the genus Nocardia are Nocardia sp, N
Qch 2-1 (Nocardia sp, PJnC
h2-1) A method for producing NAD (FM-dependent cholesterol dehydrogenase) according to claim 2, in which FERM-Pt' is 6217. 4. The strain belonging to alkaline earth metals is Alcaligenes sp. Arashi 4 ( Alcaligenes sp, N
L14) A method for producing NAD(P)-dependent cholesterol dehydrogenase as described in Item 2 of the patent FERM-PNt1621'6. 5. The strain belonging to the genus Proteus is Proteus vulgaris IAMIQ25 (Proteus vulgaris IAMIQ25).
s IAM1025). The method for producing NAD(P)-dependent cholesterol dehydrogenase according to claim 2.