JPH0249720B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to NAD-dependent cholesterol dehydrogenase (hereinafter abbreviated as "NAD-CDH"). To explain in more detail, NAD-CDH produced from microbial cells or culture fluid by culturing microorganisms under aerobic conditions.
Cholesterol quantification method and NAD using
-Regarding a reagent for quantifying cholesterol containing CDH. NAD-CDH here refers to an enzyme that requires NAD (nicotinamide adenine, dinucleotide) as a coenzyme, steals hydrogen from an electron donor (cholesterol), and catalyzes a reaction that adds it to an electron acceptor (NAD). means. 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 was observed. Also, Mycobacterium cholestericum (J.Biol.Chem.,
206, p. 511, 1953), Brevibacterium
The same can be said about Sterolicum (Special Publication 1190-1190). Furthermore, Oibacterium sp. ATCC21408, an obligate anaerobic microorganism, has NAD.
-Report that CDH will be produced (Japanese Patent Application Laid-Open No. 53-56090)
However, not only are there almost no descriptions of enzymatic properties, etc., and even though there is a description of NAD dependence, there are cases where the reaction proceeds even in the absence of NAD. Therefore, this enzyme cannot be said to be an NAD-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.
-If NADH produced by the reaction shown in the reaction formula below can be directly measured with a photometer using CDH,
It is simple to operate and also solves various problems of the above-mentioned method using cholesterol oxidase. Cholesterol + NAD cholestenone +
NADH 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 NAD-dependent. but,
It was found that it produced a significant amount of NAD-CDH. Among them, particularly excellent strains include Nocardia sp.No. Ch2-1 (Nocardia sp.No. Ch2-1),
Alcaligenes sp. No. 4 and Proteus vulgaris IAM1025
vulgarig IAM1025) is exemplified. Next, the mycological properties of Nocardia sp. No. Ch2-1 and Alcaligenes sp. No. 4 will be described below. (1) Mycological properties of Nocardia sp. No. Ch2-1 (A) Morphological properties 1) Cell shape and size: At the initial stage of culture, it grows in a hyphal form and branches. Thereafter, irregular division occurs and the cells become rod-shaped. The size is about 0.8-1.0μ x 1.5-4.0μ. It does not form aerial hyphae and does not form spores. 2) Gram staining: Positive 3) Acid-fastness: Positive 4) Motility: None (B) Chemical composition analysis Meso-diaminopimelic acid, meso-diaminopimelic acid, in the cell wall
Contains arabinose and galactose LL-
Contains no diaminopimelic acid or glycine. (C) Growth status in each medium 1) Broth agar plate medium: After culturing at 30°C for 4 days, circular colonies with a diameter of 0.5 to 1.0 mm are formed. The periphery is either complete 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) Seuucrose nitrate agar medium Growth is medium and the color of the colonies is white to pale cream. 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 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 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 colony color is white to light cream color. Does not release water-soluble dyes. (D) Physiological properties 1) Growth temperature: Grows at 15°C to 43°C.
Does not grow at 10℃ or 45℃. Optimum temperature is 30~
It is 35℃. 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 degradation: Negative 12) Litmus milk: No alkalinity, no peptonization, no coagulation. 13) Melanin-like pigment formation: none 14) Aesculin hydrolysis: positive 15) Tween20.40.60.80 hydrolysis: all positive 16) Penicillin resistance test: resistant 17) Attitude towards oxygen: aerobic 18) Inorganic nitrogen source Use: ammonium salt,
Use with nitrate. 19) NaCl growth range: Grows in 0-6%.
Will not grow at 7%. 20) Assimilation of various carbon sources (Pridham, Godelive agar) Assimilates D-glucose, D-fructose, mannose, glycerin, and trehalose. Does not assimilate L-arabinose, D-xylose, sutucarose, inosycetate, L-rhamnose, 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. Does not produce acid from L-arabinose, D-xylose, D-galactose, maltose, sutucarose, lactose, D-sorbitol, D-mannite, inosyte, or starch. The above mycological properties are summarized in Bergey's Manual of
As a result of examining Determinative Bacteriology 8th edition as a reference, it was found that the cell wall contains meso-diaminopimelic acid, arabinose, and galactose, but does not contain L,L-diaminopimelic acid and glycine, and that it grows well in an aerobic mycelium-like manner. This bacterium belongs to Nocardia because it later divides into rod-like forms, is acid-resistant, and does not attach sporangia or aerial hyphae. Therefore, the present inventors discovered that Nocardia sp.
(Nocardia sp.) No. Ch2-1 and 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 Mycological properties of Alcaligenes sp. No.4 (A) Morphology 1) Cell shape and size: 0.4-0.6μ×
It is a bacillus with a size of 0.8 to 1.2μ. 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-fastness: Negative (B) Growth status in each medium 1) Broth agar plate culture Round colonies with smooth surface, semi-lens-shaped ridges, entire rim, light cream color, and 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 become peptonized 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: Weakly positive 7) Starch hydrolysis: Negative 8) Utilization of citrate: Koser's medium and
Use together with Christensen's medium. 9) Use of inorganic nitrogen sources: Use nitrates and ammonium salts. 10) Pigment generation: Does not produce water-soluble pigments. 11) Urease: Negative, weakly 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): Generates acid aerobically from fructose. 17) Presence or absence of acid and gas generation from sugars
Ayers, Rupp and Johnson's medium produces acid from fructose and glycerin, but no gas. arabinose, xylose, glucose, mannose, galactose, maltose,
Sucrose, lactose, trehalose, sorbitol, mannitrate, 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 ability: D-fructose, L-phenylalan, calcium levulinate, L-
Capitalize on Threon. Does not assimilate mannose, maltose, mannite, and betaine. Based on 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. Regarding the species, I consulted the literature and found Alcaligenes eutrophus.A.paradoxus, A.
ruhlandii and A.latus mainly in autotrophic growth. In addition, A.faecalis is different from A.faecalis mainly in terms of growth at 42℃ and ability to assimilate D-fructose.
A. aquamarinus differs from A. pacifrius mainly in its ability to decompose starch and assimilate maltose, and A. pacifrius mainly in terms of its ability to assimilate threonine and betaine.
cupidus mainly contains oxidase and mannitase,
In terms of assimilation of mannose, A. venustus differs mainly in terms of assimilation of levulinate, threonine, and betaine, and A. aestus differs mainly in assimilation of mannose, threonine, and feralanine. Each one is different. Therefore, the present inventor identified this bacterium as Alcaligenes sp.
(Alcaligenes sp.) No. 4, and was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology as Bacteria Collection No. 6216 (FERM-).
P No. 6216). Bergey's manual of Determinative
Bacteriology (8th edition) J.Bact.110(1)402-429 (1972) Translated by Kazuichi Sakazaki: Handbook of medical bacterial identification (2nd edition)
Kindai Publishing, Tokyo, 1974 Next, the method for producing NAD-CDH using these bacteria will be described in detail. All of these bacteria have the ability to constitutively produce NAD-CDH, and can be used in a medium containing ordinary sources such as peptone, yeast extract, or ammonium sulfate, carbon sources such as glucose and glycerol, and other inorganic salts. But N.A.D.
-It produces CDH, but by adding cholesterol to the medium it produces even more NAD-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. produced by these bacteria
NAD-CDH is accumulated not only in bacterial cells but also in the culture solution, and the enzyme can be recovered from either of them. The crude enzyme obtained by salting 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 for the determination of cholesterol, or it can be further purified before use. You can also do it. For example, purification can be carried out by known methods such as ion exchange chromatography and molecular node chromatography. The NAD-CDH obtained here can be used to quantify cholesterol in cholesterol-containing substances, and can be advantageously used, for example, to quantify cholesterol in blood and blood. The method for measuring the activity of NAD-CDH used in the present invention is shown below. The enzymatic activity of NAD-CDH is related to cholesterol and
The amount of NADH produced when reacting with NAD as a substrate is measured and calculated as an increase in absorbance at 340 nm using a spectrophotometer. That is, 2.65 ml of 0.1M Tris-HCl buffer (PH8.6), 0.1 ml of 28mMNAD solution, (0.15 ml of 8% Triton X-100 solution), 1
% cholesterol solution and 0.05 ml of NAD-CDH aqueous solution are mixed and reacted at 30°C, and the increase in absorbance at 340 mm for 1 minute after the start of the reaction is measured. As a control, perform the same operation with the above composition but using water instead of cholesterol, and subtract the increase in absorbance of the control solution at 340 mm from that of the test solution. The amount of NADH produced is determined from the obtained value, and the NAD-CDH activity in the sample is calculated from this. Enzyme activity is displayed under the conditions of PH8.6 and 30℃.
An enzyme that generates 1 μmole of NADH per minute was defined as one unit. Next, the action of NAD-CDH used in the present invention will be shown. Cholesterol + NAD cholestenone +
NADH All NAD-CDHs in the present invention catalyze this reaction. The general properties of NAD-CDH used in the present invention are as follows: Nocardia sp. No. Ch2-1 (Nocardia sp.
No.Ch2-1) and Alcaligenes sp.No.4
(Alcaligenes sp. No. 4) is shown below. (1) NAD produced by Nocardia sp.No. Ch2-1.Nocardia sp.No. Ch2-1
-CDH 1) Optimum PH: around 9.0 (shown in Figure 1) 2) PH stability: PH6 at 37℃ for 15 minutes
It is stable at ~7. (See Figure 2.) 3) Optimum temperature: 25-35℃ (See Figure 3.) 4) Thermal stability: PH7.0, 15 minutes.
Stable below 35℃. (It is shown in Figure 4.) 5) Substrate specificity This enzyme reacts with steroids that have a hydroxyl group at the 3β position, and when cholesterol is 100, it reacts with stetugumasterol 35, β-sitosterol 25, other dehydroepianes, and drosterone. , has a slight effect on ergosterol, etc. 6) Requires coenzyme NAD. (2) Alcaligenes sp.No.4 (Alcaligenes sp.No.
4) NAD-CDH produced by
6-7 is stable. (As shown in Figure 6.) 3) Optimum temperature: 25-35°C (As shown in Figure 7.) 4) Thermal stability: Stable down to 0°C under conditions of PH7.0 and 15 minutes. (This is shown in Figure 8.) 5 Substrate specificity Hydroenzyme reacts with steroids that have hydroxyl properties at the 3β position, and when cholesterol is 100, β
-Sitosterol 36, Stegmasterol
20, other dehydroepiandrosterone,
It has a slight effect on ergosterol, etc. 6 Requires coenzyme NAD. Next, the determination of cholesterol using NAD-CDH of the present invention will be described in detail. When quantifying cholesterol, a buffer, NAD, substrate (serum, cholesterol, etc.) and NAD-CDH are mixed, reacted for a certain period of time, and the increase in NADH produced is measured at absorbance at 340 nm.
Further, if necessary, the hydrogen of the generated NADH can be guided to a coloring system such as a formazan dye using phenazine meso sulfate (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, to give an example of the quantitative composition of a reagent for cholesterol determination using NAD-CDH, per 3 ml of reaction system, 0.1 to 10 units of NAD-CDH, 10 units of NAD-CDH,
~100mM (Triton X-100 1.0% or less), cholesterol esterase (Boehringer) 0.1
~10 units 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 NADH produced can be measured directly and can be completely quantified. Next, test examples and examples will be described. Test Example 1 For the three strains of the present invention, cholesterol 5g/, 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 500 ml Sakaro flask containing 100 ml, and cultured with shaking at 30°C for 40 hours. Collect 5 ml of the culture solution by centrifugation (8000 rpm for 10 minutes) and dilute to 0.1 M
Washed with 50 ml of phosphate buffer pH7.0. Next, the bacterial strain was suspended in 50 ml of the same buffer solution, and the bacterial cells were disrupted using ultrasound. This crushed solution was centrifuged (10,000 rpm for 10 minutes) to obtain a clear solution. The NAD-CDH activity of the resulting clear liquid was measured and the following results were obtained.

[Table] Test example 2 Cholesterol (manufactured by Katayama Chemical Co., Ltd.) 0.67 mg/ml,
NAD or NADP (manufactured by Oriental Yeast Co., Ltd.) 1.0
mg/ml, Triton X-100 (manufactured by Katayama Chemical Co., Ltd.) 4.0
25 ml of 0.1 M Tris-HCl buffer containing mg/ml
After preheating at ℃ for 5 minutes, add 0.1 ml of diluted various cholesterol dehydrogenase solutions and react at 25℃.
The reactivity of various cholesterol dehydrogenases to each coenzyme was investigated by measuring the increase in absorbance at 340 nm.

[Table] As a result, all of the cholesterol dehydrogenases of the present invention had a higher reactivity to NAD.
It can be seen that the reactivity to NADP is extremely low. Example 1 Nocardia sp.Ch2-1 (FERM-P No.6217)
was inoculated into a 500 ml Sakaro flask containing 200 ml of a medium (PH 7.2) consisting of 5 g of glucose, 5 g of extract, 0.2 g of yeast extract, and a small amount of antifoaming agent, and shaken at 30°C for 24 hours. Cultivate. This seed culture solution was added to 5g/g of cholesterol and 2g/g of glucose.
, meat extract 5g/, KH ₂ PO ₄ 5g/,
The cells were inoculated into a 30-volume jar fermenter containing 20 medium (PH7.2) consisting of 0.2 g of MgSO ₄ .7H ₂ O/0.5 g of antifoaming agent, and aerated and stirred at 30°C (0.5 V /V/min, 200 rpm) for 40 hours. The culture solution was centrifuged, and the resulting bacterial cells were suspended in 0.1 M phosphate buffer pH 7.0, and the bacterial cells were disrupted using 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 for 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.
The enzyme was adsorbed through a column packed with 200 ml of DEAE/cellulose equilibrated at 7.0. After washing the column with the same buffer, the buffer concentration was increased to 0.1M to elute NAD-CDH. Fractions containing NAD-CDH were collected, concentrated, and then dialyzed against 20 mM phosphate buffer pH 7.0. This was passed through a column packed with 20 ml of hexyl-Sepharose equilibrated with the same buffer solution and adsorbed. this column
Washed with 20mM phosphate buffer PH7.0. next
NAD-CDH was eluted with a similar buffer containing 0.5M NaCl, and the active fractions were collected, concentrated, and concentrated to 50 units/ml.
5.0 ml of NAD-CDH solution was obtained. The overall activity yield was 30%. Example 2 Alcaligenes sp. No. 4 (FERM-P No. 6216) was added to cholesterol 10g/glycerol 2g/
, corn steep liquor 5g/, KH ₂ PO ₄ 5
g/, MgSO ₄ 7H ₂ O0.2g/, antifoaming agent 0.5g/
The cells were cultured in a medium having the following composition, and the same operations as in Example 1 were performed to obtain 5.0 ml of a 40 units/ml NAD-CDH solution. The overall activity yield was 40%. Example 3 Using Proteus vulgaris IAM1025, Example 1
37 units/ml of NAD-
4 ml of CDH solution was obtained. The overall activity yield was 52%. Example 4 Using NAD-CDH obtained in Example 1, cholesterol in standard serum was quantified. 0.1M Tris-HCl buffer PH8.6, 2.71ml, 8% Triton X-100 0.1ml, NAD 200mg/ml solution 0.1ml,
0.05 ml of cholesterol esterase (manufactured by Boehringer) 100 units/ml solution and 0.02 ml of standard serum were mixed and reacted at 30°C, and the increase in absorbance at 340 nm was measured. The reaction reached its end point within 3 minutes. As a control, the same operation was performed using water instead of NAD-CDH in the above reaction composition.
The increase in absorbance at was subtracted from that of the test solution. The total cholesterol amount in this standard serum is
From the calibration curve prepared in advance, cholesterol
A value of 289 mg/dl was obtained. Comparative measurements performed using a commercially available assay reagent containing cholesterol oxidase yielded a value of 290 mg/dl of cholesterol. Example 5 Using the NAD-CDH obtained in Examples 2 to 3, the same procedure as in Example 4 was carried out, and substantially the same results were obtained. Example 6 Using the NAD-CDH obtained in Example 1, cholesterol in various serum samples was quantified in the same manner as in Example 4, and the following results were obtained. Commercially available cholesterol Serum NAD-CDH Ruoxidase (Amano Pharmaceutical) 1 289mg/dl 290mg/dl 2 150 146 3 219 221 4 143 143 5 173 170

[Brief explanation of drawings]

Figure 1 shows Nocardia sp. No. Ch2-1FERM-P
This is a diagram showing the optimal pH at 30℃ of NAD-CDH produced by No. 6217, and Figure 2 shows the optimum pH of NAD-CDH produced by No. 6217.
FIG. 3 is a diagram showing the optimum temperature of the present NAD-CDH, and FIG. 4 is a diagram showing the thermal stability of the present NAD-CDH. Figure 5 shows Alcaligenes sp.No.4FERM-P
This is a diagram showing the optimum pH at 30°C of NAD-CDH produced by No. 6216, and Fig. 6 is a diagram showing the optimum pH of NAD-CDH produced by No. 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-CDH, and FIG. 8 is a diagram showing the thermal stability of the present NAD-CDH.

Claims (1)

[Claims] 1. A method for quantifying cholesterol, characterized by using NAD-dependent cholesterol dehydrogenase. 2. The method for quantifying cholesterol according to claim 1, which uses NAD-dependent cholesterol dehydrogenase obtained from a culture of microorganisms grown under aerobic conditions. 3. The method for quantifying cholesterol according to claim 2, wherein the microorganism that grows under aerobic conditions is at least one species of the genus Nocardia, the genus Alcaligenes, and the genus Proteus. 4 A strain belonging to the genus Nocardia is Nocardia sp.
No.Ch2-1 (Nocardia sp.No.Ch2-1) FERM-
The method for quantifying cholesterol according to claim 3, which is P No. 6217. 5 The strain belonging to the genus Alcaligenes is Alcaligenes sp. No. 4 (Alcaligenes sp. No. 4) FERM-P
The method for quantifying cholesterol according to claim 3, which is No. 6216. 6 The bacterial strain belonging to the genus Proteus is Proteus vulgaris IAM1025 (Proteus vulgaris).
IAM1025) The method for quantifying cholesterol according to claim 3. 7 NAD-dependent cholesterol dehydrogenase,
A reagent for quantifying cholesterol, characterized in that it contains or consists of NAD. 8. The reagent for quantifying cholesterol according to claim 7, which contains NAD-dependent cholesterol dehydrogenase obtained from a culture of a microorganism grown under aerobic conditions. 9. The reagent for cholesterol determination according to claim 8, wherein the microorganism that grows under aerobic conditions is at least one species of the genus Nocardia, the genus Alcaligenes, and the genus Proteus. 10 Nocardia is a strain belonging to the genus Nocardia.
sp.No.Ch2-1 (Nocardia sp.No.Ch2-1) FERM
-P No. 6217, the reagent for quantifying cholesterol according to claim 9. 11 The strain belonging to the genus Alcaligenes is Alcaligenes sp. No. 4 (Alcaligenes sp. No. 4) FERM-
The reagent for quantifying cholesterol according to claim 9, which is P No. 6216. 12 The bacterial strain belonging to the genus Proteus is Proteus.
vulgaris IAM1025 (Proteus vulugaris
10. The reagent for quantifying cholesterol according to claim 9, which is IAM1025).