JPS63165401A - 6-maltosylmaltooligosaccharide derivative, production thereof and measurement of alpha-amylase activity using said derivative - Google Patents

Abstract

PURPOSE:To obtain a derivative, capable of suppressing increase of a blank value by alpha-glycosidase which is a coexisting enzyme in measuring alpha-amylase and measurable also by an automatic analyzer, by linking maltose to a maltooligosaccharide through alpha-1,6-bonds. CONSTITUTION:Pullulanase in an amount of normally 125 units based on 1g maltooligosaccharide derivative is added to maltose and the maltooligosaccharide (preferably 2-chloro-4-nitrophenyl beta-maltopentaoside expressed by formula I) and reacted, preferably at 40-60 deg.C and pH 5-7 to afford a 6- maltosylmaltooligosaccharide expressed by formula II (R<1> is nitrophenol residue; m is 0-1; n is 2-5), which is then used as a substrate and brought into contact with a sample, e.g. urine, blood, etc., in the presence of alpha- and/or beta-glucosidase to measure liberated nitrophenol based compounds. Thereby the alpha-amylase activity in the sample is measured.

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a 6-amylase reagent used for measuring the activity of α-amylase in clinical diagnosis, biochemical research, etc.
The present invention relates to a maldocyl malto-oligo II conductor, a method for producing the same, and a method for measuring α-amylase activity using the same. [Prior Art] Measurement of the activity of α-amylase contained in body fluids such as urine or blood is widely practiced in clinical diagnosis. Recently, as a substrate for measuring α-amylase activity in human body fluids, a substrate with a well-defined structure in which a nitrophenol compound is bound to the glucose at the reducing end of celltooligosaccharide has been synthesized.
α-amylase measurement reagents using the following substrates have been proposed. p-Nitrophenylmaltopentaoside [Special Publication 1986
-53079 Publication] p-nitrophenylmaltohexaoside [Japanese Patent Publication No. 53079]
51892] 2.4-dichlorophenylmaltopentaoside [JP-A-56-35998] The following is an example of a method for measuring α-amylase activity using these compounds as substrates. (a) p-nitrophenyl α-maltopentaosa α
-Amylazide p-nitrophenyl α-maltoside + maltotriose (b) p-nitrophenyl α-maltoside lucose 2,4-dichlorophenyl -maltopentaosamer (a) 2.4-dichlorophenyl β-maltopenta α-amyl zeoside 2,4-dichlorophenyl β
-maltoside + maltotriose (b) 2.4-dichlorophenyl β-maltoside β-glucoside +
Glucose (c) 2.4-dichlorophenyl β-glucoside + glucose (d) 2.4-dichlorophenol + 4-aminoanoxidant tibirin → Kino, men pigment

[Problems to be solved by the invention]

However, α-
The amylase measurement system has a problem in that the reagent blank value increases significantly because α-glucosidase used as a coexisting enzyme also acts on the substrate. Furthermore, α−
A common problem with the combination of glucosidase solution and substrate solution is that the stability of the reagent is significantly impaired due to the decomposition of the substrate by α-glucosidase. Therefore, the present inventors conducted extensive research and added maltose to maltooligosaccharide derivatives.
1,6-linked 6-maltosyl maltooligo 1! The inventors realized that these problems could be solved by using derivatives, and thus completed the present invention. [Means for Solving the Problems] The present invention, which has succeeded in achieving the above object, is a maltooligo W derivative in which a nitrophenol compound is α- or β-bonded to the glucose at the reducing end of the maltooligo ring. 6-maltosylmaltooligosaccharide derivative with α-1,6 linkage, the 6-maltosylmaltooligosaccharide derivative is combined with maltose and the maltooligosaccharide derivative with pullulanase (pullulan 6-glucanohydrolase,
EC3, 2, 1, 41) and the production method using the 6-maldocyl malto-oligosaccharide as a substrate
The present invention provides a method for measuring α-amylase activity in a sample by contacting the sample with the sample in the presence of glucosidase and/or β-glucosidase and measuring the liberated nitrophenol compound. The 6-maldocyl maltooligo Ii derivative of the present invention has the following structure. (I) (wherein R1 represents a substituted or unsubstituted nitrophenol residue, m represents an integer of 0 to 1, and n represents an integer of 2 to 5,
) Here, specific examples of P in the above general formula (1) include 2-nitrophenyl group, 4-nitrophenyl group, 2,4-dinitrophenyl group, and aromatic hydrogen of these groups alone or with halogen of it. Examples include those substituted with a group, a sulfonic acid group, or a carboxylic acid group. The 6-maldocyl maltooligo W derivative can be produced by allowing pullulanase to act on maltose and the maltooligosaccharide derivative. Here, the maltooligosaccharide derivative has the structure of the following general formula (I) (wherein R1 represents a substituted or unsubstituted nitrophenol residue, and 1 represents an integer of 2 to 5. ) Furthermore, general formula (I
Specific examples of al in the formula (I) are the same as those in the general formula (I). Among the bending conductors of general formula (II), 2-chloro-4-nitrophenyl β-maltopentaoside represented by the following formula (rn) is particularly suitable. Pullulanase is used to produce a 6-maldocylmaltooligo W-conductor by linking maltose with α-1,6 to a maltooligosaccharide derivative. It can be anything. As for the reaction conditions, pullulanase was added to a solution with a maltose/maltooligosaccharide derivative weight ratio of 2 to 9 and a substrate concentration of 43 to 75%, at a concentration of 25 to 250 units (25 to 250 units) per 1 g of maltooligosaccharide derivative.
One unit of enzyme is the amount of enzyme that acts on pullulan to produce 14 moR of maltotriose per minute), and the reaction temperature is 30 to 80°C and pH 4 to 9. Particularly preferably, 125 units of pullulanase is added per 1 g of maltooligosaccharide derivative to a solution with a maltose/maltooligo 11 derivative weight ratio of about 4 and a substrate concentration of 66.7%, and the reaction temperature is preferably 40 to 60°C and the pH is around 5 to 7. . Produced 6-maltosyl maltooligo 1! The derivative can be quantitatively analyzed by high performance liquid chromatography by measuring absorbance at 280 nm. After the reaction, the desired 6-maldocyl malto-oligo I! is obtained by an appropriate method such as gel filtration chromatography. derivatives can be obtained. Next, a method for measuring α-amylase using a 6-maldocylmaltooligosa conductor will be explained. α used during measurement
- Glucosidases are found in animals and plants. Although it may be from any origin, including microorganisms, α-glucosidase obtained from yeast is particularly desirable in terms of its substrate specificity.In other words, yeast-derived α-glucosidase has a wide range of aglycone specificity, and is furthermore capable of producing glucosides below maltotriside. Although it acts well, it is difficult to act on glucosides higher than maltotetraoside, and it does not act on α-1,6 bonds, which is suitable for the purpose of the present invention. The β-glucosidase may be of any origin, for example, one obtained from almonds can be used. Specifically, α-glucosidase and β-glucosidase can be used by any of the following methods: (a) α-glucosidase (b) β-glucosidase (C) α-glucosidase and β-glucosidase. In the method of the present invention, other additives may be added as necessary. The present invention provides α-
The α-amylase activity in the sample is measured by contacting the sample in the presence of glucosidase and/or β-glucosidase and measuring the liberated nitrophenol compound. As a method for measuring nitrophenol compounds, the nitrophenol compounds liberated from the substrate are p-
In the case of nitrophenol, 2-chloro-4-ditrophenol, etc., it is sufficient to directly measure the absorbance, and if it is not possible to directly measure the change in absorbance, use a coloring reagent such as 4-ditrophenol.
- It is sufficient to carry out oxidative condensation with a compound such as aminoantipyrine and measure the color intensity. According to the present invention, the present reagent, like conventional reagents of this type,
It can be widely used to measure the activity of α-amylase contained in serum, urine, pancreatic fluid, saliva, etc. [Example] Next, the present invention will be explained in more detail with reference to Examples. Example 1 A mixed solution of 16 g of maltose and 4 g of 2-chloro-4-nitrophenyl β-maltopentaoside dissolved at a high concentration (substrate concentration 66.7%) was injected with Bacillus acidoplulyticus (Bacillus acido ullu).
l tlcus) pullulanase per 1 g of 2-chloro-4-nitrophenyl β-maltopentaoside.
When 25 units were added and the reaction was carried out at a temperature of 60°C and pH 5, Q, after 24 hours, 40% of the substrate 2-chloro-4-nitrophenyl β-maltopentaoside was converted into 2
-Chloro-4-nitrophenyl could be converted to β-6-maltosylmaltopentaoside. After the reaction was completed, the reaction solution was divided into several parts and each was loaded onto a gel filtration chromatography column (2.2 x 38 cm).
2-chloro-4-nitrophenyl β-6-maldocylmaltobentaoside was purified by elution with water and then lyophilized to obtain 1.5 g of white powder. Example 2 α-amylase activity in a sample was measured using the following reagents and the following method. Reagents: 50mM glucosidase pH 7.0 α-glucosidase (yeast) 60 U/mI
! β-glucosidase (almond) 70/mi'1st
Substrates shown in the table 2 tag/mR
1 Table measurement method; 3 mA of the above reagent was taken and the change in reagent blank value over time was investigated. (Measurement wavelength 400 nm, reaction temperature 37°C) Sample (
Serum) Add 3ml of reagent to 20ILI, and after addition, add 4ml of reagent.
The change in absorbance (α-amylase activity) after ~6 minutes was measured. (Measurement wavelength: 400 nm, reaction temperature: 37°C) Changes in reagent blank values over time are shown in Table 2, and changes in serum absorbance are shown in Table 3.
Shown in the table. Reagent A of the present invention has a smaller increase in reagent blank value than Reagent B, and the α-amylase activity value is about 173 compared to Reagent B, but the absorbance change per minute is 0.0.
Since a value of 1 or more is sufficient for measurement, the value of 0.016 is a value that is sufficiently measurable for practical purposes. [Effects of the Invention] According to the present invention, when measuring α-amylase, it is possible to suppress the increase in blank value due to α-glucosidase, which is a coexisting enzyme, as described above, and also to suppress the increase in the blank value due to α-glucosidase, which is a coexisting enzyme, as described above. It is extremely useful for measuring the activity of α-amylase because it can be combined with a β-glucosidase solution and can be applied to an automatic analyzer. Patent Applicant: Sapporo Breweries Co., Ltd.
Method) % formula % 1. Indication of the case Patent application 1986-312847 2. Name of the invention 6-Maldosyl Maltooligo I! Derivative, its manufacturing method and α-amylase activity measurement method using the same 3, Relationship with the amended person's case Patent applicant (219) Sapporo Breweries Ltd. 4, Agent ■ 104 1-10 Kyobashi, Chuo-ku, Tokyo No. 5. Date of amendment order March 4, 1988 March 31, 1988 (Shipping date) 6. Specification subject to amendment 7. Contents of amendment As per (no change in content) (or more)

Claims (5)

[Claims] (1) General formula ▲ There are mathematical formulas, chemical formulas, tables, etc. 6-maltosylmalto-oligosaccharide derivative represented by ). (2) A general formula characterized by the action of pullulanase on maltose and malto-oligosaccharide derivatives ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (In the formula, R^1 represents a substituted or unsubstituted nitrophenol residue, m is an integer of 0 to 1, n is an integer of 2 to 5). (3) The method according to claim 2, wherein the maltooligosaccharide derivative has the following structure. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R^1 represents a substituted or unsubstituted nitrophenol residue, and l represents an integer from 2 to 5.) (4) The method according to claim 2, wherein the maltooligosaccharide derivative has the following structure. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (5) General formula ▲ There are mathematical formulas, chemical formulas, tables, etc. α-glucosidase and/or β-glucosidase using a 6-maltosylmalto-oligosaccharide derivative represented by
1. A method for measuring α-amylase activity, which comprises contacting the sample in the presence of glucosidase and measuring liberated nitrophenol-based compounds to measure α-amylase activity in the sample.