JPH0762027B2 - Substrate for measuring new lysozyme activity - Google Patents

Description

The present invention relates to a novel lysozyme activity measuring substrate, a method for producing the same, and a lysozyme activity measuring reagent.

The measurement of lysozyme activity in serum and urine has important significance in clinical diagnosis, and is used for diagnosis of ulcerative colitis, liver disease, monocytic leukemia and the like.

Currently, the activity of lysozyme is measured by dry cells of Micrococcus lysode-ikticus as a substrate, and is carried out by a turbidimetric method or a lyso-plate method utilizing its lytic action.

However, these methods are not always consistent in the quality of the dry cells of Micrococcus lysodicticus, which is the substrate, and are greatly affected by the ionic strength and pH of the reaction solution when measuring lysozyme activity. There is a drawback that large variations occur.

From these things, JP-A-56-135494 and JP-A-58-15
According to Japanese Patent No. 996, p-nitrophenol-bound N-
Acetylchitotetraose (p-nitrophenyl-tetra-N-acetyl-β-chitotetraoside) and N-acetylchitotetraose (4-methylumbelliferyl-tetra-N-acetyl) bound with 4-methylumbelliferone -Β
-Chitotetraoside) is used as a substrate for lysozyme, and a method for measuring p-nitrophenol and 4-methylumbelliferone produced by the action of lysozyme has been published. However, when p-nitrophenyl-tetra-N-acetyl-β-chitotetraoside is used as a substrate, the reaction rate with lysozyme is slow and the sensitivity is low, which requires a long time for measurement. In the case of 4-methylumbelliferyl-tetra-N-acetyl-β-chitotetraoside,
Since it uses a fluorometer for activity measurement, it has the drawback of lacking versatility.

As a result of various studies to overcome these drawbacks of the conventional method, the present inventors have found that p-nitrophenyl-penta-N-acetyl-β-chitopentaoside exhibits a high reaction rate in the reaction with lysozyme. The present invention has been completed.

That is, the present invention is a novel p-nitrophenyl-penta-N-
The present invention relates to acetyl-β-chitopentaoside, a method for producing the same and a reagent for measuring lysozyme activity.

The present invention is structural formula Represented by p-nitrophenyl-penta-N-acetyl-
β-chitopentaoside is a novel compound having the following physicochemical properties.

Molecular _{formula: C 46 H 70 O 28 N} 6 · 2H 2 O Molecular weight: 1191.14 (theoretical) decomposition point: 204-210 ° C. Solubility: Water soluble, methanol, organic solvent-insoluble crystalline forms such as chloroform: tasteless, Odorless, yellow-white powdery crystals p-nitrophenyl-penta-N-acetyl-β-chitopentaoside can be produced by hydrolyzing chitin or chitosan with N-acetylchitopentaose or chitopentaose. It can be produced by using hydrochloride as a starting material, reacting this with acetic anhydride to give a peracetyl derivative, reacting with p-nitrophenol, and de-O-acetylating the reaction product.

For example, according to the method of JP-A 61-21102-3, chitosan prepared from crab shell is partially hydrolyzed with concentrated hydrochloric acid, and the decomposed product is adsorbed on a cation exchange resin column.
Chromatography is performed by a concentration gradient elution method with hydrochloric acid to obtain chitopentaose hydrochloride. Next, chitopentaose hydrochloride is gradually added to an acetic anhydride solution containing anhydrous zinc chloride and stirred at a reaction temperature of 50 to 60 ° C. for 1 to 2 hours to carry out a peracetylation reaction. The reaction mixture is neutralized with sodium hydrogen carbonate, extracted with a chloroform-methanol solution, and the solution is removed to obtain chitopentaose heptadecaacetate of the formula [II].

The reaction of the obtained [II] chitopentaose heptadecaacetate with p-nitrophenol was conducted under reduced pressure in the range of 180 to 1
Heat for 15 minutes at 90 ℃ to condense. After the reaction, from methanol-chloroform-diethyl ether [III]
Crystals of p-nitrophenyl-hexadecaacetyl-β-chitopentaoside of the formula are obtained.

Next, the compound having the structure represented by the formula [III] is heated to reflux in methanol in the presence of sodium methylate for 10 to 15 hours to carry out a de-O-acetylation reaction, and then the compound represented by the formula [I] of the present invention is expressed. p-nitrophenyl-penta-N-acetyl-β-
Chitopentaoside is obtained.

The compound of the present invention, p-nitrophenyl-penta-N-acetyl-β-chitopentaoside, is an excellent activity measurement substrate for lysozyme.

The present invention provides p-nitrophenyl-penta-N-acetyl-β
P-Nitrophenyl-N- produced by the action of lysozyme using -chitopentaoside as a substrate for lysozyme
It is a method for measuring lysozyme activity by quantifying the amount of p-nitrophenol released by the action of β-N-acetylhexosaminidase on acetyl-β-glucosaminide.

That is, the present invention contains p-nitrophenyl-penta-N-acetyl-β-chitopentaoside as a substrate component,
Further, the present invention relates to a reagent for measuring lysozyme activity, which contains β-N-acetylhexosaminidase.

The β-N-acetylhexosaminidase used in the present invention may be derived from plants, animals, microorganisms, etc., but β-N-acetylhexosaminidase derived from jack bean is p-nitrophenyl. It works well on -N-acetyl-β-glucosaminide, but is preferable because it does not work on glycosides higher than chitobioside.

In addition, the addition of β-N-acetylhexosaminidase produced p-nitrophenyl-N- produced by the action of lysozyme.
Acetyl-β-glucosaminide may be decomposed very quickly, and may be added simultaneously with p-nitrophenyl-penta-N-acetyl-β-chitopentaoside.

The measurement principle of the present invention is shown below.

The activity of lysozyme can be determined by making p-nitrophenol produced in the above reaction yellow in an alkaline solution and optically measuring it.

The novel lysozyme activity measurement substrate of the present invention is used for clinical diagnosis and
In the quality control of pharmaceuticals, the lysozyme activity can be accurately measured in an extremely short time.

Next, the examples of the present invention will be described more specifically.
Of course, the present invention is not limited to the above description.

[Example 1] 6.0 l of 11.5N hydrochloric acid in 800 g of chitosan crushed to 10 mesh
Was added and the mixture was hydrolyzed in a water bath at 80 ° C. for 1 hour and a half.

After adding 6.0 l of water to this hydrolyzate, centrifugation was performed to remove insoluble matter. Activated carbon was added to the obtained supernatant to decolorize it, and then concentrated under reduced pressure until it became a syrup. 3.0 L of methanol was added to this syrup, and the suspension was stirred at room temperature for 1 hour. After removing insolubles by suction, the solution was concentrated under reduced pressure until it became a syrup, acetone was added, and the mixture was left overnight in a refrigerator.

The generated insoluble matter was collected by filtration and dried in a vacuum to obtain 360 g of a chitosan oligosaccharide mixture.

350g of this decomposed product was added to a 5l ion exchange column (Dowex 50w).
And eluted with a linear gradient of 0 to 4 N hydrochloric acid. D-glucosamine and chitosan oligosaccharides were detected by ninhydrin color development and measuring the absorbance at 570 nm.

The chitopentaose hydrochloride fraction as the starting material of the present invention was collected, concentrated and dried to obtain 25 g of chitopentaose hydrochloride.

Chitopentaose hydrochloride 10.0g is gradually added to acetic anhydride solution 120ml containing anhydrous zinc chloride 15g, reaction temperature 55 ~ 60 ℃
The mixture was stirred for 1 hour and a half to be peracetylated.

The reaction mixture was poured into water (1), neutralized by adding excess sodium hydrogen carbonate, and the reaction product was extracted twice with 500 ml of chloroform-methanol (1: 1) and then with 200 ml of chloroform. The extract was dehydrated and the solvent was removed, followed by vacuum drying to obtain 13.5 g (88% of theory) of chitopentaose heptadecaacetate.

Chitopentaose heptadeca acetate obtained here
After mixing 6.0 g and 11.0 g of p-nitrophenol, the mixture was heated under reduced pressure in an oil bath at 180 to 190 ° C for 15 minutes.

After cooling the reaction mixture, 30 ml of methanol was added and left in the refrigerator overnight. After collecting the precipitate and washing it with methanol, the precipitate was mixed with methanol-chloroform (1:
1) It was dissolved in 600 ml and decolorized by adding activated carbon. For decolorizing solution,
25 ml of diethyl ether was added, and the mixture was left overnight in the refrigerator.

The precipitated crystals were collected, washed with diethyl ether, and then dried in vacuum, and p-nitrophenyl-hexadecaacetyl-β-
Obtained 1.30 g (20.7% of theory) of chitopentaoside.

Melting point: 267-270 ℃ (decomposition) 0.200 g of p-nitrophenyl-hexadecaacetyl-β-chitopentaoside was suspended in 50 ml of anhydrous methanol, then about 1.0M
Add 10 ml of sodium methylate, attach a cooling tube, and
Reflux for hours.

The precipitated crystals were collected, washed with methanol, and dried in vacuum to obtain 0.135 g (94.5% of theory) of p-nitrophenyl-penta-N-acetyl-β-chitopentaoside, which was the target substance.

The crystals were decolorized with activated carbon, recrystallized from methanol-water and further purified.

This material showed a single peak in high performance liquid chromatography.

The molecular formula, molecular weight, decomposition point, solubility, measured value of crystal form and observation of this substance were as follows.

Molecular _{weight: C 46 H 70 O 28 N} 6 · 2H 2 O Molecular weight: 1191.14 (theoretical) decomposition point: 204-210 ° C. (starting point) Solubility: Water soluble, insoluble crystals in an organic solvent such as methanol-chloroform Form: tasteless and odorless yellow-white powdery crystal [Example 2] The following solutions were mixed to give a reagent for measuring lysozyme activity.

0.05% p-Nitrophenyl-penta-N-acetyl-β-chitopentaoside 1.00ml 0.5M sodium citrate buffer (pH 5.0, 37 ° C) 0.30ml β-N-acetylhexosaminidase (nama bean, 5 units)
/ ml) 0.04 ml distilled water 0.11 ml 0.05 ml of egg white lysozyme (1 mg / ml) was added to the above measuring reagent, reacted at 37 ° C, and 1.5 ml of 1.0 M sodium carbonate was added, and the absorbance at 405 nm was measured. .

As a control, a system using 0.2% p-nitrophenyl-tetra-N-acetyl-β-chitotetraoside as a substrate and a system not adding β-N-acetylhexosaminidase, which is a conjugated enzyme, were used as controls. I was there. The results are shown in FIG.

The reaction rate is about 20 times higher than that of the system using p-nitrophenyl-tetra-N-acetyl-β-chitotetraoside as the substrate. It can be seen that the absorbance was hardly increased without the addition of β-N-acetylhexosaminidase, which is a conjugated enzyme.

In addition, p-nitrophenyl-tetra-N-acetyl-
β-N-acetylhexosaminidase, which is a conjugated enzyme using β-chitotetraoside, uses p-nitrophenyl-penta-N-acetyl-β-chitopentaoside as a substrate and β-N which is a conjugated enzyme. The reaction rate was only 1/400 as compared with the N-acetylhexosaminidase-added system.

Example 3 Almost the same as in Example 2, except that the lysozyme concentrations were 0.2 mg / ml, 0.4 mg / ml, and 1.0 mg / ml, respectively, and 0.05 ml of these concentrations of lysozyme was added to carry out the reaction. The amounts of lysozyme in the reaction solution are 10 μg, 20 μg, and 50 μg, respectively.

The results are shown in FIG.

A straight line was obtained for each lysozyme concentration.

[Brief description of drawings]

FIG. 1 shows the reaction of lysozyme with respect to the compound of the present invention and a control substrate as measured by the color development intensity of released p-nitrophenol. (1) p-nitrophenyl-penta-N-acetyl-β-chitopentaoside + β-N-acetylhexosaminidase system (2) p-nitrophenyl-tetra-N-acetyl-β-chitotetraoside + β-N-acetylhexyl Sosaminidase system (3) p-nitrophenyl-penta-N-acetyl-β-chitopentaoside system Fig. 2 shows the relationship between the compound of the present invention and the amount of lysozyme. (1) 50 μg of lysozyme in the reaction mixture (2) 20 μg of lysozyme in the reaction mixture (3) 10 μg of lysozyme in the reaction mixture

Claims (3)

[Claims] 1. P-nitrophenyl-penta-N-acetyl-β-chitopentaoside having a structural formula represented by: 2. A p-nitrophenyl-penta- comprising reacting peracetyl-chitopentaose with p-nitrophenol to obtain peracetyl-chitopentaoside with p-nitrophenol as an aglycone and deacetylating it. Process for producing N-acetyl-β-chitopentaoside. 3. p-Nitrophenyl-penta-N-acetyl-
A reagent for measuring lysozyme activity, which contains β-chitopentaoside as a substrate component and further contains β-N-acetylhexosaminidase.