JPH0218064B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to NAD(P)-dependent cholesterol dehydrogenase (hereinafter abbreviated as "NAD(P)-CDH"). More specifically, the present invention relates to a method of culturing microorganisms under aerobic conditions and collecting NAD(P)-CDH from the microbial cells or culture solution. NAD(P)-CDH obtained by the method of the present invention
is useful as a method for quantifying cholesterol and a reagent for quantifying cholesterol. Here
NAD(P)-CDH consists of NAD (nicotinamide adenine, dinucleotide) and NADP as coenzymes.
(nicotinamide adenine, dinucleotide phosphate) and requires an electron donor (cholesterol)
It absorbs hydrogen from the electron acceptor (NAD or
An enzyme that catalyzes the addition reaction to NADP).
It is already known that aerobic microorganisms produce cholesterol oxidase and cholesterol dehydratase. It has also been reported that Nocardia erythropolis produces an enzyme that catalyzes the oxidation of cholesterol (Ann.Clin.Biochem., 10
Vol. 79, 1973). For this enzyme,
No dependence on NAD(P) was observed. Also, Mycobacterium cholestericum (J.Biol.
Chem., vol. 206, p. 511, 1953) and Brevibacterium sterolicum (Special Publication No. 1983-1190). Furthermore, Oibacterium sp. ATCC21408, an obligate anaerobic microorganism,
reported that it produces NAD(P)-CDH (JP-A-Sho
53-56090), but there are almost no descriptions of enzymatic properties, etc., and even though there is a description of NAD(P) dependence, the reaction proceeds even in the absence of NAD(P). An example is given. Therefore, this enzyme cannot be said to be an NAD(P)-dependent dehydrogenase as defined in the present invention. Conventionally, a method using cholesterol oxidase has been widely used for quantifying cholesterol using enzymes, but it requires peroxidase or the like to introduce it into a coloring system, and the operation is complicated. Moreover, it has the disadvantage that it is influenced by bilirubin, ascorbic acid, etc. in the blood, which tends to cause errors. In the determination of cholesterol, the reaction does not proceed without the presence of NAD(P) - NAD(P)-CDH
is produced by the reaction shown in the reaction formula below.
If NAD(P)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 +
NAD(P)H The present inventors have discovered that an enzyme suitable for the above reaction, that is, NAD(P)
When we searched for dehydrogenases that depend on the enzymes in nature, we unexpectedly discovered that microorganisms that grow under aerobic conditions produce significant amounts of NAD(P)-CDH. Among them, Nocardia sp.No.Ch2-1 (Nocardia sp.No.Ch2-1) is a particularly excellent strain.
1), Alcaligenes sp.No.4 (Alcaligenes sp.No.
4), and Proteus vulgaris
IAM1025Proteus vulgaris IAM1025 is exemplified. Next, the mycological properties of Nocardia sp. No. Ch2-1 and Alcaligenes sp. No. 4 will be described below. (1) Nocardia sp.No.Ch2-1 (Nocardia sp.No.
Ch2-1) Mycological properties (A) 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 0.8 ~
It is about 1.0μ x 1.5 to 4.0μ. It does not form aerial hyphae or sporangium. 2 Gram staining: Positive 3 Acid-fastness: Positive 4 Motility: None (B) Chemical composition analysis The cell wall contains meso-dianopimelic acid, arabinose, and galactose, but does not contain L,L-diaminopimelic acid and glycine. (C) Growth status in each medium 1 Broth agar plate medium: After culturing at 30°C for 4 days, form circular colonies with a diameter of 0.5 to 1.0 mm. The periphery is complete or wavy. The surface is smooth and hemispherical, and the center may be convex. The color is light cream and opaque. No dye is released into the medium. 2. Seuclose oxalate agar medium Growth is medium and the color of the colonies is white to light cream color. Does not release water-soluble dyes. 3 Glucose, asparagine agar medium Growth is medium and the color of the colonies is cream-colored. Does not release water-soluble dyes. 4 Glycerin, asparagine agar medium Growth is medium and the color of the colonies is white to light cream. Does not release water-soluble dyes. 5 Starch inorganic 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 color of the colonies is white to light cream. Does not release water-soluble dyes. 7 Nutrient agar medium Growth is good and the colony is cream colored.
Does not release water-soluble dyes. 8 Yeast malt agar medium Growth is good and the color of the colonies is cream-colored.
Does not release water-soluble dyes. 9 Oatmeal agar medium Growth is medium and the color of the colonies is white to light cream. Does not release water-soluble dyes. (D) Physiological properties 1 Growth temperature: Grows at 15°C to 43°C. 10℃,
Does not grow at 45℃. The optimal temperature is 30-35°C. 2 Nitrate reducing ability: Positive 3 Catalase: 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 Decomposition: Negative 12 Litmus milk: No alkalinity, no peptonization, no coagulation. 13 Formation of melanin-like pigments: none 14 Aesculin hydrolysis: positive 15 Tween20, 40, 60, 80 hydrolysis: all positive 16 Penicillin resistance test: resistant 17 Attitude towards oxygen: aerobic 18 Utilization of inorganic nitrogen sources: ammonium salts, Use with nitrate. 19 NaCl growth range: Grows in 0-6%. 7
% does not grow. 20 Assimilation method of various carbon sources (Pridham, Godelive agar medium) Assimilates D-glucose, D-fructose, mannose, glycerin, and trehalose. L-arabinose, D-xylose, sutucarose, inosito, L-rhamnose,
Does not assimilate raffinose, D-galactose, D-mannite, maltose, and sorbitol. 21 Production of acids from various sugars Acid is produced from D-glucose, mannose, D-fructose, trehalose, and glycerin. L-arabinose, D-xylose, D-galactose, maltose, sutucarose, lactose, D-sorbit, D-
Does not produce acid from mannitrate, inosyte, or starch. The above mycological properties are summarized in Bergey's Manual of
As a result of the study with reference to Determinative Bacteriology 8th edition, it was found that the cell wall contains meso-diaminopimelic acid, arabinose, galactose, L,
Does not contain L-diaminopimelic acid or glycine, grows well in aerobic mycelial form and later divides into fungi, is acid-resistant, does not adhere to sporangia or aerial mycelium, etc. This fungus is from
It is a bacterium belonging to Nocardia. Therefore, the present inventors identified this bacterium as Nocardia sp.No.
Named Ch2-1 (Nocardia sp.No.Ch2-1),
It has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERM-P No. 6217. (2) Alcaligenes sp.No.4 (Alcaligenes sp.No.4)
4) Mycological properties (A) Morphology 1 Cell shape and size: 0.4-0.6μ x 0.8-
It is a 1.2μ bacterium. 2 Presence or absence of cell pleomorphism: No pleomorphism was observed. 3 Motility: Has periflagella and is motile. 4 Presence or absence of spores: Spores are not formed. 5 Gram staining: Negative 6 Acid-fast: Negative (B) Growth status in each medium 1 Broth agar plate culture Circular rowny with smooth surface, semi-lenticular ridges, entire rim, light cream color, translucent, glossy 2 Meat juice agar slant culture Medium growth, filamentous growth, pale cream color and translucent 3 Meat juice liquid culture No bacterial film, slightly cloudy with some sediment. 4 Meat juice gelatin puncture culture: Gelatin does not liquefy. 5 Litmus milk culture: Becomes alkaline but does not peptonize or coagulate. (C) Physiological properties 1 Nitrate reduction: Positive 2 Denitrification reaction: Negative 3 MR test: Negative 4 VP test: Negative 5 Indole production: Negative 6 Hydrogen sulfide production: Weak positive 7 Starch hydrolysis: Negative 8 Citrus Utilization of acid salts: Koser's medium and
Used together with Christensen's medium. 9 Use of inorganic nitrogen sources: Use nitrates and ammonium salts. 10 Pigment production: Does not produce water-soluble pigments. 11 Urease: Positive 12 Oxidase: Positive 13 Catalase: Positive 14 Growth range PH: Grows at PH5.0 to 10.0. Temperature: Grows between 5°C and 37°C. Does not grow at 42℃. 15 Attitude towards oxygen: aerobic 16 OF test (Hugh-Leifson method): produces acid aerobically from fructose. 17 Whether acid and gas are produced from sugars Ayers, Rupp and Johnson medium produces acid or no gas from fructose and glycerin. arabinose, xylose, glucose,
Mannose, galactose, maltose, sucrose, lactose, trehalose, sorbitol, mannite, inosyte, and starch do 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 Tween80: Positive 20 Assimilation: Assimilates D-fructose, L-phenylalanine, calcium levulinate, and L-threonine. Does not assimilate mannose, maltose, mannite, and betaine. From the above mycological properties, Bergey's manual of
According to the description in Determinative Bacteriology (8th edition), it is a short bacillus, moves by peribacterial setae, is gram negative, is aerobic, has no auxotrophy, and uses ammonium salts and nitrates. Because it does not degrade casein and gelatin and is oxidase positive, etc.
It is classified in the genus Alcaligenes. For species, Alcaligenes eutrophus, A.
paradoxus, A. ruhlandii and A. latus mainly in autotrophic growth. In addition, A.faecalis mainly has the ability to grow at 42℃ and assimilate D-fructose, A.aquamarinus mainly has the ability to decompose starch and assimilate maltose, and A.pacificus mainly has the ability to decompose starch and assimilate maltose. Assimilation of threonine and betaine, A.
A. cupidus is mainly responsible for oxidase and assimilation of mannite and mannose, and A. venustus is
It differs from A. aestus mainly in its ability to assimilate levulinate, threonine, and betaine, and mainly in its ability to assimilate mannite, threonine, and phenylalanine. Therefore, this bacterium was transformed into Alcaligenes sp.No.4 (Alcaligenes sp.No.4).
4) and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERM-P No. 6216. Next, a method for producing NAD(P)-CDH using these bacteria will be described in detail. All of these bacteria have the ability to constitutively produce NAD(P)-CDH, and use common sources such as peptone, yeast extract, or ammonium sulfate, carbon sources such as glucose and glycerol, and other inorganic salts. Although NAD(P)-CDH is produced in a medium containing NAD(P)-CDH, adding cholesterol to the medium produces even more NAD(P)-CDH.
Produces NAD(P)-CDH. 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)-CDH 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 it is for the determination of cholesterol, or can be used after further purification. can. For example, purification can be achieved by known methods such as ion exchange chromatography and molecular sieve chromatography. 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 determined when cholesterol and NAD(P) are used as substrates.
The amount of NAD(P)H produced is measured and calculated using a spectrophotometer as an increase in absorbance at 340 nm. i.e. 0.1M Tris-HCl buffer (PH8.6) 2.65
ml, 0.1 ml of 28mMNAD solution, (8% Triton
100 solution 0.15ml), 1% cholesterol solution 0.05ml
and 0.05 ml of NAD(P)-CDH aqueous solution, 30
The reaction is carried out at °C, and the increase in absorbance at 340 nm is measured for 1 minute after the start of the reaction. As a control, perform the same operation using water instead of cholesterol with the above composition, and subtract the increase in absorbance at 340 nm of the control solution from that of the test solution. The amount of NAD(P)H produced is determined from the obtained value, and the NAD(P)-CDH activity in the sample is calculated from this. Enzyme activity is displayed under the conditions of PH8.6 and 30℃.
One unit was an enzyme that produced 1 μmole of NAD(P)H per minute. Next, the action of NAD(P)-CDH used in the present invention will be shown. Cholesterol + NAD (P) cholestenone +
NAD(P)H All NAD(P)-CDHs in the present invention catalyze this reaction. The general properties of NAD(P)CDH used in the present invention are described below.
sp.No.Ch2-1) FERM-P6217 and Alcaligenes sp.No.4 (Alcaligenes sp.No.4) FERM-P6216
Indicates what the company produces. (1) Nocardia sp.No.Ch2-1 (Nocardia sp.No.
Ch2-1) NAD (P) produced by FERM-P6217
-CDH 1 Optimal PH: around 9.0 (shown in Figure 1) 2 PH stability: PH6 at 37℃ for 15 minutes
It is stable at ~7. (As shown in Figure 2.) 3. Optimum temperature: 25-35℃ (As shown in Figure 3.) 4. Thermal stability: 35% at PH7.0 for 15 minutes.
Stable below ℃. (It is shown in Figure 4.) 5 Substrate specificity This substance reacts with steroids having a hydroxyl group at the 3β position, and when cholesterol is 100, stigmasterol 35, β-sitosterol
25, other dehydroepiandrosterone,
It has a slight effect on ergosterol, etc. 6 Complement Request NAD. (2) Alcaligenes sp.No.4 (Alcaligenes sp.No.
4) NAD(P)-CDH produced by FER-P6216 1 Optimal PH: Around 8.5 (shown in Figure 5) 2 PH stability: Stable at PH 67 at 37°C and 15%. (See Figure 6.) 3. Optimum temperature: 25-35°C (See Figure 7.) 4. Thermal stability: Stable up to 30°C under conditions of PH7.0 and 15 minutes. (It is shown in Figure 8.) 5 Substrate specificity This element reacts with steroids having a hydroxyl group at the 3β position, and when cholesterol is 100, β-
sitosterol 36, stigmasterol 20,
It also has a slight effect on dehydroepiandrosterone, ergosterol, etc. 6 Complement Request NAD. Next, the determination of cholesterol using NAD(P)-CDH of the present invention will be described in detail. When quantifying cholesterol, you actually need a buffer, NAD (P), and a substrate (serum, cholesterol, etc.)
Then, NAD(P)-CDH is mixed and reacted for a certain period of time, and the increase in the generated NAD(P)H is measured by absorbance at 340 nm.
Measure with. Also, if necessary, the generated NAD
It is also possible to introduce the hydrogen of (P)H into a coloring system such as a formazan dye using phenazine mesosulfate (PMS), diafluorase, or the like. Furthermore, it is also possible to add cholesterol esterase, surfactant, stabilizer, etc. to the reaction system. The pH during the reaction can be carried out in the range of 6 to 10, but an excellent PH range is 7 to 9. Next, an example of the quantitative composition of a cholesterol quantification reagent using NAD(P)-CDH is as follows.
0.1 to 10 units of NAD(P)-CDH per 3 ml of reaction system,
NAD(P) 10-100mM, Triton X-100 1.0%
Hereinafter, 0.1 to 10 units of cholesterol esterase (manufactured by 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 For three strains of the present invention, cholesterol 5g/, meat extract 5g/, yeast extract 0.2
g/, a medium consisting of a small amount of antifoaming agent (PH
7.2) The cells were inoculated into a 500ml Sakaro flask containing 100ml and cultured with shaking at 30°C for 40 hours. Collect 50ml of the culture solution by centrifugation (8000rpm for 10 minutes) and dilute to 0.1M.
Washed with 50 ml of phosphate buffer pH 7.0. Next, the bacterial cells were suspended in 50 ml of the same buffer solution and disrupted using ultrasound. This crushed solution was subjected to salt center separation (10,000 rpm, 10 minutes) to obtain a clear solution. The NAD(P)-CDH activity of the resulting clear liquid was measured and the following results were obtained.

[Table] Example 1 Nocardia sp.Ch2-1FERM-PNo.6217 was added to glucose 5g/, meat extract 5g/, yeast extract
The cells were inoculated into a 500ml Sakaro flask containing 200ml of a medium (PH7.2) consisting of 0.2g/a small amount of antifoaming agent, and cultured with shaking at 30°C for 24 hours. This seed culture solution was mixed with 5g of cholesterol/MgSO ₄ 7H ₂ O0.2
The bacteria were inoculated into a 30-volume jar fermenter containing 20 medium (PH7.2) with a composition of 0.5 g/g/defoamer and 0.5 g/v/defoamer, and aerated and stirred at 30°C (0.5 v/v/
200 rpm) for 40 hours. The culture solution was centrifuged, the resulting bacterial cells were suspended in 0.1M phosphate buffer PH70, and the bacterial cells were disrupted with glass beads. This cell suspension was centrifuged (1000 rpm, 10 minutes) to obtain a clear 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)
It was dissolved in 100 ml of 20mM phosphate buffer PH7.0 and dialyzed against 20mM phosphate buffer PH7.0 for 24 hours using cellophane tubes. Next, the obtained dialysate was added to 20mM phosphate buffer PH.
Equilibrated at 7.0. It was passed through a column packed with 200 ml of DEAE/cellulose to adsorb the enzyme. After washing the column with the same buffer, adjust the buffer concentration to 0.1M.
NAD(P)-CDH was eluted. N.A.D.
After collecting the fractions containing (P)-CDH and concentrating them,
Dialyzed against 20mM phosphate buffer PH7.0. This was passed through a column packed with 20 ml of hexyl-Sepharose equilibrated with the same buffer solution and adsorbed. The column was washed with 20mM phosphate buffer pH7.0. then in a similar buffer containing 0.5M NaCl.
Elute NAD(P)-CDH, collect active fractions, concentrate, and 5.0 ml of 50 units/ml NAD(P)-CDH solution.
I got it. The overall activity yield was 30%. Example 2 Alcaligenes sp.No.4FERM-PNo.6216 with cholesterol 10g/, glycerol 2g/, corn steep liquor 5g/, KH ₂ PO ₄ 5g/,
The cells were cultured in a medium containing 0.2 g of MgSO ₄ 7H ₂ O and 0.5 g of antifoaming agent.
Obtained 5.0ml. The overall activity yield was 40%. Example 3 Using Proteus vulgaris IAM1025, Example 1
37 units/ml of NAD(P)
-4 ml of CDH solution was obtained. Overall activity yield is 52%
It was hot. Example 4 Nocardia sp.No.Ch2-1FERM-PNo.6217 with glucose 5g/, meat extract, yeast extract 0.2ml,
200ml medium (PH7.2) consisting of a small amount of antifoaming agent
Inoculate the bacteria into a 500ml Yosaka flask containing
Incubate with shaking for 24 hours. Add this seed culture solution to cholesterol 5g/, glucose 2g/, meat extract 5g/
g/, KH ₂ PO ₄ 5g/, MgSO ₄・7H ₂ O0.2
The bacteria were inoculated into a 30-volume jar fermenter containing 20 medium (PH7.2) with a composition of 0.5 g/g/defoamer and 0.5 g/v/defoamer, and aerated and stirred at 30°C (0.5 v/v/
200 rpm) for 72 hours. This culture solution was sterilized by centrifugation (8000 rpm, 10 minutes). Add 40% ammonium sulfate to the resulting culture solution.
% saturation to precipitate the enzyme. Collect the precipitate by centrifugation (8000 rpm, 10 minutes),
Dissolve in 100ml of 20mM phosphate buffer (PH7.0) and add 20mM phosphate buffer (PH7.0) with cellophane tube.
Dialysis was performed for 24 hours. Next, purification was performed in the same manner as in Examples 1 to 3, and a 30 unit/ml NAD(P)-CDH solution was purified at 32
I got ml. The overall activity yield was 32%. Example 5 Using Alcaligenes sp.No.4FERM-PNo.6216,
Perform the operation similar to Example 4 to obtain a concentration of 25 units/ml.
4.2 ml of NAD(P)-CDH solution was obtained. The overall activity yield was 38%. Example 6 Using Proteus vulgaris IAM1025, Example 4
18 units/ml of NAD(P)
-3.5 ml of CDH solution was obtained. Overall activity yield is 49%
It was hot.

[Brief explanation of drawings]

Figure 1 shows Nocardia sp.No.Ch2-1FERM-PNo.
This is a diagram showing the optimum pH at 30°C of NAD(P)-CDH produced by 6217, and Figure 2 shows the optimum pH of NAD(P)-CDH produced by 6217.
It is a diagram showing the PH stability of CDH at 37°C,
FIG. 3 is a diagram showing the optimum temperature of the present NAD(P)-CDH, and FIG. 4 is a diagram showing the thermal stability of the present NAD(P)-CDH. Figure 5 shows Alcaligenes sp.No.4FERM-PNo.
This is a diagram showing the optimum pH at 30°C of NAD(P)-CDH produced by 6216.
It is a 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.

Claims (1)

[Scope of Claims] 1. NAD(P)-dependent cholesterol dehydrogenase-producing bacteria belonging to the genus Nocardia, Alcaligenes, and Proteus are cultured, and NAD(P) is obtained from the culture.
1. A method for producing NAD(P)-dependent cholesterol dehydrogenase, which comprises collecting NAD(P)-dependent cholesterol dehydrogenase. 2 A strain belonging to the genus Nocardia is Nocardia sp.
No.Ch2−1 (Nocardia sp.No.Ch2−1) FERM−
P No. 6217 as stated in claim 1
A method for producing NAD(P)-dependent cholesterol dehydrogenase. 3 The strain belonging to the genus Alcaligenes is Alcaligenes sp. No. 4 (Alcaligenes sp. No. 4) FERM-P
NAD as claimed in claim 1 which is No.6216
(P) Method for producing dependent cholesterol dehydrogenase. 4 The bacterial strain belonging to the genus Proteus is Proteus vulgaris IAM1025 (Proteus vulgaris).
IAM1025) according to claim 1
A method for producing NAD(P)-dependent cholesterol dehydrogenase.