JPS5840470B2 - Method for producing sarcosine oxidase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing sarcosine oxidase (hereinafter abbreviated as SO) by culturing bacteria belonging to the genus Arthrobacter in a nutrient medium.

SO is an enzyme that converts sarcosine glycine, formaldehyde, and hydrogen peroxide, and has long been known to exist in nature, especially in animal organs.

This enzyme is involved in the creatinine or choline metabolic system, and in particular undergoes a coupled reaction with other enzymes of the former system, namely creatinine amidohydrolase and creatine amidinohydrolase, to produce formaldehyde or hydrogen peroxide, which are known in the art. By quantifying with this method, it is possible to quantify freanine or creatine easily and specifically, so it has become extremely useful in the field of clinical diagnosis as an alternative to conventional non-specific chemical quantitative methods. .

Many microorganisms can metabolize sarcosine;
The SO enzyme system that converts sarcosine into glycine, formaldehyde, and hydrogen peroxide has only been reported for the genus Corynebacterium (Collection of Lecture Abstracts of the Japanese Society of Agricultural Chemistry, 1978). (page 86).

Therefore, the present inventors conducted a wide search for microorganisms capable of producing SO, and among the strains, they recognized that bacteria belonging to the genus Arthrobacter have strong SO activity. By extracting and purifying from
They established a method to efficiently produce highly pure and extremely stable SO.

That is, the present invention involves culturing a microorganism belonging to the genus Arthrobacter and having the ability to produce sarcosine oxidase in a nutrient medium,
This is a method for producing sarcosine oxidase, which is characterized by collecting sarcosine oxidase from a culture.

As the strain used in the present invention, a strain belonging to the genus Arthrobacter is used, and a representative strain is Arthrobacter ureafaciens FO12140.
is used.

The IFO number of the above strain is provided by the Fermentation Research Institute (In
5titude for Fermentation
.

0saka1Japan).

The vehicle is cultured in a nutrient medium, preferably a medium supplemented with choline to increase enzyme production ability, to increase SOx.
Since the enzyme is produced and accumulated in the bacterial cells, enzyme powder can be obtained by extraction, purification, and drying using known methods.

More specifically, the vehicle is cultured in an appropriate nutrient medium, such as a medium containing appropriate carbohydrates, nitrogen sources, inorganic salts, and choline and organic promoters to enhance enzyme production ability, and SO is removed from the cells. In this case, monosaccharides such as glucose and sucrose, and trisaccharides can be used as carbohydrates.

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

As the inorganic salts, ammonium phosphate, dipotassium phosphate, magnesium sulfate, etc. are used.

Good organic promoters include yeast extract, peptone, meat extract, and corn steep liquor.

The pH of the medium is set to around neutrality, and aerobic culture such as aeration and stirring is performed at around 30° C. for 1 to 2 days.

The SO-containing bacterial cells obtained by the above method are separated by filtration or centrifugation, suspended in an appropriate buffer, and then the enzyme is extracted by grinding or sonication.

After insoluble matter is separated from the extract by filtration or centrifugation, an enzyme preparation is obtained from the resulting filtrate or supernatant by a known method such as salting out with ammonium sulfate or solvent precipitation using acetone, alcohol, or the like.

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

As shown in the drawings, the enzyme obtained according to the present invention has an optimum pH value near neutrality (see Fig. 1), and an optimum temperature of about 35°C (see Fig. 2).

Regarding pH stability, it is stable in the range of pH 6.0 to 9.0 (see Figure 3), and regarding thermal stability, it is stable at 30°C.
It is almost stable up to this point, but beyond that it gradually loses its activity (see Figure 4).

The activity of this enzyme can be measured by allowing it to act on sarcosine and measuring the hydrogen peroxide produced.

That is, the generated hydrogen peroxide is decomposed by peroxidase (POD) in the presence of O-aminophenol, and at the same time O-
The enzymatic titer of SO is determined by quantitatively oxidizing aminophenol and colorimetrically quantifying the amount of pigment produced at 480 nm.

The composition and reaction conditions of the enzyme reaction solution are as follows.

(1) Composition of reaction solution React at 37°C for 15 minutes.

The reaction is stopped by adding 0.5rrlE of IN hydrochloric acid, and the produced dye is determined colorimetrically at 480 nm.

(3) Enzyme titer Enzyme titer is expressed as 1 unit, which is the amount of enzyme that decomposes 1 μmol of sarcosine per minute.

Next, the present invention will be explained using examples.

Example 1 Choline chloride 1%, peptone 0.2%, yeast extract 0.1
%, magnesium sulfate (heptahydrate) 0.1%, K2HPO
500ml of medium consisting of 40.1%, pH 7.2 per 50"
Dispense into 10 bottles of ml Yosaka Lokolben and heat at 120℃ for 15 minutes.
After heat sterilization for a minute, Arthrobacter ureafaciens IFO12140 was inoculated and cultured with shaking at 30°C for 40 hours.

After the cultivation was completed, the culture solution was collected from each strain, and the bacterial cells were separated by centrifugation to obtain 15P of wet bacterial cells.

Add this to 75ml of 0.05M phosphate buffer (pH 7.0).
), SO is extracted by ultrasonication, and insoluble matter is removed by centrifugation, and the resulting supernatant contains 70%
Saturated ammonium sulfate was added to precipitate SO.

The salt precipitate was collected by centrifugation, dissolved in 20 ml of 0.02M phosphate buffer (pH 7.0), and added to a column (diameter 4.5) packed with Sephadex G25 buffered with 0.002M phosphate buffer. (X, length 60 cIrL) was passed for desalting and the active fraction was collected.

As a result of freeze-drying the obtained desalted enzyme solution, 0.5 g of SO powder containing 0.13 units/■ was obtained.

Example 2 A desalted enzyme solution obtained in the same manner as in Example 1 was then added to a DEAE-cellulose column (diameter 4 (1; 772, length) equilibrated with 0.02M phosphate buffer (pH 7,0)). 30 cm) to adsorb SO, wash with the same buffer, create a concentration gradient with the same buffer and the same buffer with 0.5M salt dissolved, and gradually increase the salt concentration. S.O.
was eluted.

The eluted SO active fraction was collected, and after salting out and concentrating with 70% saturated ammonium sulfate, 0.02M! Molecular sieving was carried out through Sephadex G-200 buffered with J acid buffer, and the final Sephadex G200 molecular sieve solution was then freeze-dried to obtain a 15-inch SO sample.

Is the specific activity of this product 1.6 units/1rlI? The yield from the extract was 37%.

[Brief explanation of the drawing]

FIG. 1 shows the pH activity curve of the enzyme obtained according to the present invention. FIG. 2 shows the temperature activity curve of the enzyme obtained according to the present invention. FIG. 3 shows the pH stability curve of the enzyme obtained according to the present invention. FIG. 4 shows the thermostability curve of the enzyme obtained according to the present invention.

Claims (1)

[Claims] 1. A method for producing sarcosine oxidase, which comprises culturing a microorganism belonging to the genus Arthrobacter and having the ability to produce sarcosine oxidase in a nutrient medium, and collecting sarcosine oxidase from the culture.