JP2749577B2 - Dye-modified lysozyme-sensitive bacteria and lysozyme activity assay using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a lysozyme-sensitive bacterium modified with a dye. Lysozyme is widely distributed in the animal and plant kingdoms, and particularly in humans, is abundantly contained in tears, saliva, serum, leukocytes, and the like. Lysozyme is known as an enzyme that hydrolyzes a copolymer of N-acetylmuramic acid and N-acetylglucosamine present in a bacterial cell wall to cause lysis. BACKGROUND ART Human lysozyme is widely distributed in organs and body fluids, and recently, attention has been paid to the relationship between its fluctuation and pathological conditions. Lysozyme concentration in human serum is
It is maintained at 7 to 20 mg / ml, but its concentration is significantly increased in acute leukemia, myeloid leukemia and the like. In addition, it has been reported that a large amount of lysozyme is secreted into pancreatic juice with canceration of pancreatic cells. Furthermore, it has been reported that urinary excretion of lysozyme increases due to decreased renal function. For these reasons, measuring blood levels of lysozyme will aid in the diagnosis of the disease. Various methods for measuring the activity of lysozyme have been developed so far, but the method that has sufficient sensitivity and is actually used in clinical practice is the lysis method. However, this method is extremely difficult to operate, and unless a professional biochemist
There is a major drawback that high reproducible results cannot be obtained. In addition, due to the nature of the measurement method, a large amount of sample cannot be measured at a time, which is extremely inefficient in practical use. Therefore, development of a simple and sensitive method for measuring the activity of lysozyme is strongly desired. Thus, the present inventors have conducted intensive studies to develop a simple and highly sensitive method for measuring the activity of lysozyme, and as a result, the use of a lysozyme-sensitive bacterium modified with a dye makes it extremely simple, reproducible, and sensitive. As a result, it has been found that the lysozyme activity can be measured as high as possible. That is, an object of the present invention is to provide a dye-modified lysozyme-sensitive bacterium obtained by modifying a lysozyme-sensitive bacterium with a dye. The dye-modified lysozyme-sensitive bacterium according to the present invention can be obtained by modifying a lysozyme-sensitive bacterium with a dye. Lysozyme-sensitive bacteria that can be used in the present invention include:
It is not limited to any particular substance, and any substance having sensitivity to lysozyme can be used. Such lysozyme-sensitive bacteria include, for example, genus Planococcus, Staphylococcus, Enterococcus, Streptococcus, Gram-positive cocci belonging to Micrococcus, etc., Bacillus, Clostridium, Corynebacterium, Lactobacillus, Listeria. Gram-positive bacteria such as Gram-positive bacilli belonging to the genus, Nocardia, Actinomyces and the like are included. Among such lysozyme-sensitive bacteria, particularly preferred are:
It is a bacterium belonging to the genus Micrococcus, and among them, in particular, Micrococcus lysodicus
sodeikticus) and Micrococcus luteus (Micrococc
us luteus) and the like, but it should be emphasized again that the present invention is not limited to these. The dye used in the present invention is brilliant blue, and rimazole brilliant blue is preferably used. Furthermore, the method of modifying lysozyme-sensitive bacteria with a dye can be achieved only by mixing according to a conventional method, and the production method is extremely simple. If the cells thus obtained are used as a substrate for lysozyme quantification, the modified dye is easily released in, for example, an acidic, neutral or basic region,
As a result, lysozyme can be easily measured qualitatively and quantitatively, so that a very practical method for measuring lysozyme activity can be obtained. Hereinafter, the present invention will be described in more detail with reference to examples. Example 1 Micrococcus lysodetics (Micrococcus ly
300 mg of freeze-dried cells of sodeikticus) are suspended in 20 ml of distilled water, and the suspension is added with Limazole Brilliant Blue R200.
mg was dissolved in 20 ml of distilled water and added. Then, 4 g of sodium sulfate was added little by little to the mixture over 30 minutes.
Thereafter, a solution obtained by dissolving 200 mg of trisodium phosphate in 2 ml of distilled water was added, and the resulting reaction solution was further stirred for 30 minutes. After completion of the reaction, the reaction solution was centrifuged at 2600 rpm for 10 minutes to remove the supernatant. After adding 20 ml of distilled water to the obtained precipitate and dispersing it with a mixer, it was further centrifuged to remove the supernatant. After repeating this operation several times and washing, the obtained precipitate was dispersed in 5 ml of distilled water and freeze-dried. 32 mg of the lyophilized Micrococcus lysoditicus cells modified with rimazole brilliant blue were dispersed in 20 ml of a predetermined buffer, and centrifuged at 2600 rpm for 10 minutes to remove the supernatant. 20 ml of the buffer solution was added again to the obtained precipitate, and the mixture was dispersed with a mixer. 1 ml of this solution and 200 ml of bovine serum albumin (5 mg / ml) were placed in a test tube and pre-incubated for 30 minutes. Next, 1 ml of a lysozyme solution at 01.2 μg / ml was added and incubated for 40 or 90 minutes, and then 100 ml of 6N hydrochloric acid was added to stop the reaction. After centrifuging the obtained reaction solution at 15000 rpm for 2 minutes, the supernatant was measured for absorbance at 600 nm with a spectrophotometer. Therefore, after incubating chicken lysozyme with bovine serum albumin (BSA), the effect of bovine serum albumin was examined at pH 7 and 37 ° C.
Even at 083 mg / ml or more, it was found that the absorbance at 600 nm of the supernatant was constant without being affected by the BSA concentration in the reaction solution. The conditions for using the cells obtained as described above for quantifying lysozyme in serum were set as follows. 1.
1 ml of a 6 mg / ml bacterial cell solution (0.05 M phosphate buffer containing 0.2 M salt, pH 7, ionic strength 0.3) and a 5 mg / ml BSA solution (0.
0.05 M phosphate buffer containing 2 M salt, pH 7, ionic strength 0.
3) 200 ml was taken into a test tube and pre-incubated at 48 ° C for 30 minutes. Next, 1 ml of human serum diluted twice with 0.6 M saline was added, and incubation was performed for 50 minutes or 90 minutes. After stopping the reaction by adding 100 ml of 6N hydrochloric acid, the mixture was centrifuged at 15,000 rpm for 2 minutes, and the resulting supernatant was measured for absorbance at 600 nm. When the amount of lysozyme in the serum of 5 adult males was measured by such a method, the lysozyme concentration in the serum was 0.9%.
It was measured in the range of 1.31.3 μg / ml. In addition, it turned out that this result was equivalent to the result measured by the lysis method (pH7, 30 degreeC) conventionally used for comparison. Example 2 Micrococcus luteus modified by Limazol brilliant blue lyophilized in the same manner as in Example 1 was obtained using Micrococcus luteus instead of Micrococcus lysodetics. The dried cells also showed the same sensitivity to lysozyme in serum as that obtained in Example 1.

Claims (1)

(57) [Claims] A dye-modified lysozyme-sensitive bacterium, wherein the lysozyme-sensitive bacterium is modified with brilliant blue. 2. The dye-modified lysozyme-sensitive bacterium according to claim 1, wherein the brilliant blue is rimazole brilliant blue. 3. A method for measuring lysozyme activity, which comprises using a dye-modified lysozyme-sensitive bacterium obtained by modifying a lysozyme-sensitive bacterium with brilliant blue, and measuring the activity of lysozyme in the absence of a surfactant and an emulsifier. 4. 4. The method for measuring lysozyme activity according to claim 3, wherein the dye is rimazole brilliant blue.