JPS5830033B2 - Cholesterol Estella Zeno Seizouhou - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a nutrient medium containing Pseudomonas knob sp.
The present invention relates to a method for producing cholesterol esterase by culturing l6109.

Cholesterol exists in body fluids, especially in serum, in the form of free or fatty acid esters, and the determination of total cholesterol and ester cholesterol in serum has important clinical diagnostic implications, such as hypertension,
The frequency of measurement has increased in recent years for the purpose of diagnosing conditions such as arteriosclerosis.

Recently, a specific direct enzyme method using cholesterol oxidase has been developed to quantify free cholesterol.
Oxidase cannot act on ester-type cholesterol, so when measuring ester-type cholesterol, it is first necessary to break down the ester bond to make it a free form on which cholesterol oxidase can act.

For this purpose, saponification, for example by reaction with alcoholic potassium hydroxide, or cholesterol
The ester must be hydrolyzed by an esterase.

Providing a method for directly quantifying cholesterol using an enzymatic method, especially a method for quantifying total cholesterol (free cholesterol + ester cholesterol) in serum, replaces conventional quantitative methods that were complex and difficult, such as applying the Lieberman-Purchart reaction. In turn, it benefits specific and easy quantification, which also makes it readily applicable to automated analysis.

Among them, the provision of cholesterol esterase can simplify the chemical saponification procedure in the determination of total cholesterol in serum, and can provide the greatest benefit for direct and rapid determination in automated analysis.

Cholesterol esterase is an enzyme that hydrolyzes fatty acid toester-bonded cholesterol to produce fatty acids and free cholesterol, and is said to be widely present in nature, especially in the organs of higher animals.

Currently, this enzyme is prepared primarily from animal liver or pancreas.

However, when using animal organs as a source, there are difficulties in securing the source and refining the source, so if you aim to constantly mass produce at low cost, you will have to actively seek other sources.

With the aim of industrial production of cholesterol esterase, which is used for the determination of cholesterol by an enzymatic method, the present inventors have sought microorganisms as a source of cholesterol esterase, and are currently searching for cholesterol esterase producing bacteria in the natural world. A strain of bacteria belonging to the genus Pseudomonas was isolated from wastewater in a ditch and was identified as having a strong ability to produce cholesterol esterase.The bacteria was cultured in liquid and extracted and purified from the culture. By doing so, we established a method for efficiently producing highly pure and extremely stable cholesterol esterase.

The method of decomposing ester-type cholesterol using this enzyme and measuring the liberated cholesterol is described in detail in Japanese Patent Application No. 50-82420 (% 1982-6593).

Next, the mycological properties of the bacterial strain used in the present invention will be described.

(a) Morphology (1) Bacillus (logarithmic growth phase is 0.7-0.8X2.0
~2.8 microns, gradually becoming smaller as the culture progresses).

Single or double chain.

It is motile and has polar flagella.

No spores.

Durham stain double negative.

Acid-fast: negative.

(b) Growth status (1) Meat juice agar slant culture bicircular.

Gold rim. Convex circular shape. The surface is smooth and yellowish brown.

(2) Meat juice agar slant culture: filamentous.

The periphery is smooth and slightly shiny.

Growth is normal. (3) Meat juice liquid culture: Growth is normal.

Forms a film at the interface.

Sediment forms at the bottom. (4) Growth in meat juice gelatin puncture culture is normal.

Liquefaction is similar.

(5) l mass milk: slightly alkaline.

Liquefies, but forms a slight precipitate.

(c) Physiological properties Nitrate reduction: positive.

Denitrification reaction: positive.

MR test: positive.

VP test: negative.

Indole production binegative.

Hydrogen sulfide formation: Positive.

(7) Starch hydrolysis: Negative.

(8) Utilization of citric acid: Positive.

(9) Utilization of inorganic nitrogen sources: Both nitrate and ammonium salt were positive.

α0) Production of pigment (Kriegler's medium): Production of green-yellow fluorescent pigment.

aυ Urease: Positive.

α2 oxidase: positive.

α3) Catalase: Positive.

(14) Growth pH: 5~9°a! 19 Growth temperature: 10°G 20°C 30°C 37°G 42°C (
-1-) (+-+) (-F-1-)-F-)
(+kanto) (-1-+7+-)47℃ (c) (16) Attitude toward oxygen: aerobic.

α7) 0-F test dioxide.

α ladder Gas production from sugars: negative.

agl Acid production from sugars: (-1-) is positive, (
→ is negative.

L-arabinose (→ D-xylose (1) D-glucose (t) D-mannose (t) D-fructose (middle) Maltose (+) Sucrose (
g) Lactose (g) Trehalose (-1-
) to D-sorbitol) D-mannide-small → inositol (→glycerin (to starch)) D-galactose ), Pseudomonas 1 aeruginosa (P
seudnmonas aeru-ginosa)
or Pseudomonas fluorescens (Pseud
However, when comparing growth temperature, denitrification reaction, carbon source availability, etc., it does not match any of the bacteria, so here we introduce a new species, Pseudomonas knob sp./i6109 (
Pseudomonasnov, sp, A10
9).

Next, the comparison is shown as a list.

Ae J Shi Fu Kanashi 4109 Ginosa sensense flagella Hair >11>1 Growth part (41℃) ++ Gelatin liquefaction + + +A109 Anil Fu is Reginosa sensu starch liquefaction denitrification reaction 1000 ± Carbon source utilization method Glucose + + + Meso-inositol − + trehalose
＋ ＋Geraniol
+ Lecithinase activity + ±
This strain has been assigned microbial accession number 3025 (FERM-PA3) to the Institute of Microbial Technology, Agency of Industrial Science and Technology.
025).

The above strain Pseudomonas knob sp. 109 (P
To explain the method for producing cholesterol esterase using S. pseudomonas nov, 5p-A109), this bacterium accumulates cholesterol esterase inside and outside the bacterium by culturing it in a nutrient medium, so known methods can be used. Enzyme powder can be obtained by purification and drying.

More specifically, this bacterium is cultured in a suitable medium, such as a medium containing suitable carbohydrates, nitrogen sources, inorganic salts, and natural oils and fats and organic enhancers to enhance enzyme production.
It causes cholesterol esterase to accumulate, and the carbohydrates used here include mono- and trisaccharides such as gelcole and sucrose, starch, and starch hydrolysates.

Organic nitrogen sources such as yeast extract, peftone, meat extract, and corn steep liquor are effective as nitrogen sources.

As the inorganic salts, ammonium chloride, dipotassium phosphate, magnesium sulfate, ferrous sulfate, common salt, etc. are used.

As the natural oil or fat, any of animal oil, vegetable oil, and industrially produced fatty acid ester can be used.

Suitable organic substances include yeast extract, pefton, meat extract, and corn steep liquor.

The pFI of the medium is set to around neutrality, and aerobic culture such as aeration and stirring is performed at around 28° C. for 1 to 4 days.

The bacterial cells are separated by filtration or centrifugation of the culture solution containing cholesterol esterase obtained by the above method, and ammonium sulfate salting out is performed from the F solution or supernatant.
Alternatively, an enzyme preparation can be obtained by a known method such as solvent precipitation using acetone, alcohol, or the like.

In addition, to extract the cholesterol esterase accumulated inside the bacterial cells, a cell-free enzyme solution is prepared by known methods such as bacterial cell grinding, autolysis, and ultrasonication, and then purified in the same way as enzymes outside the bacterial cells. It can be provided to

In order to obtain a more highly purified enzyme preparation, an adsorption/elution method using ion exchange, an electrophoresis method, or the like may be used.

As shown in the figure, the enzyme obtained by the present invention has an optimal pH value near neutrality (see Figure 1), and an optimal temperature of around 45% (see Figure 2). .

Regarding thermal stability, it is stable up to 55°C, but it gradually loses its activity at higher temperatures (see Figure 3).

The enzymatic activity of cholesterol esterase can be measured, for example, by quantifying the amount of free cholesterol produced by reaction with serum.

Free cholesterol is oxidized and decomposed into △4-cholestenone and hydrogen peroxide by the action of cholesterol oxidase, so the final △4-cholestenone or hydrogen peroxide is It can be measured by quantitative determination.

Δ4-Cholestenone can be determined by measuring its specific absorption in the ultraviolet region at 245 mμ.

The amount of hydrogen peroxide can be determined by reacting with a reagent that combines peroxidase and a chromogen, such as 4-aminophenazine and phenol, to increase the degree of color development to 505 mμ.
, D, can be obtained by measuring.

However, in this case, the net enzyme activity of cholesterol esterase must be determined by subtracting the amount of cholesterol already present in the free form in the serum.

To explain the display of the activity of cholesterol esterase in the present invention, one unit of cholesterol esterase uses serum as a substrate at pH 7.0 and 37°C.
The amount of enzyme required to generate 1 micromole of free cholesterol per minute was defined as the amount of enzyme required to generate 1 micromole of free cholesterol per minute.

Examples of the present invention are shown below.

Example 1 Medium consisting of 1.0% polypeptone, 0.5% yeast extract, 0.1% meat extract, 0.1% dipotassium phosphate, 0.05% magnesium sulfate, 1.0% beef tallow, pH 7.0 1
3M jar fermenter containing 51 (300r-p
Pseudomonas knob sp. 4109 according to the present invention was inoculated into Pseudomonas knob sp. v) Ammonium sulfate salting out was carried out, the salted out product was separated by F, desalted by the ultraviolet filtration method, and then lyophilized. As a result, 2
40 units/9 cholesterol esterase powder 1
4g was obtained.

,ゝExample 2 600 g of bacterial cell wet material separated after culturing in Example 1
Cholesterol esterase was extracted by ultrasonication, insoluble matter was removed by centrifugation, and the supernatant was
% (w/v) of ammonium sulfate, and the resulting salted-out product was separated from P and dried under vacuum to obtain cholesterol esterase powder 51 containing 124 units/g.

[Brief explanation of drawings]

Figure 1 shows the activity of cholesterol esterase obtained by the method of the present invention at each pH, Figure 2 shows the activity at each temperature, and Figure 3 shows the relative activity of the cholesterol esterase obtained by treatment at each temperature for 10 minutes. This is what is shown.

Claims (1)

[Claims] 1. A method for producing cholesterol esterase, which comprises culturing Pseudomonas knob sp. 4109 in a nutrient medium, and assisting cholesterol esterase from the culture.