JP3310008B2 - Oligosaccharide oxidase, method for producing oligosaccharide oxidase, method for measuring oligosaccharide, method for producing oligosaccharide acid, method for measuring amylase activity, and novel microorganism - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an oligosaccharide oxidase, a method for producing an oligosaccharide oxidase, a method for measuring an oligosaccharide, a method for producing an oligosaccharide acid , and a method for measuring amylase activity using the oligosaccharide oxidase. And novel microorganisms . INDUSTRIAL APPLICABILITY The present invention is widely used in the fields of clinical test drugs, food production, and the like.

[0002]

2. Description of the Related Art Methods for measuring oligosaccharides include (1) a method for measuring the reducing power of a reducing end, such as a dinitrosalicylic acid method and a somogy method, (2) a paper chromatographic method,
(3) High-performance liquid chromatography and the like are known. Also, various methods for measuring the activity of sugar-related enzymes such as amylases and cellulases using these methods have been proposed. As a method for measuring amylase activity in the clinical test drug field and the food field, a blue starch method, an iodostarch reaction method, an enzyme method using an oligosaccharide derivative as a substrate, and the like are used. In particular, a method for measuring amylase in a biological component using a glucose polymer or a cyclic glucose polymer having a reducing terminal modified as a substrate has been reported for clinical examination (JP-B-63-37640).

[0003] Glucose oxidase is known (Japanese Patent Application Laid-Open No. 57-86283), but does not act on oligosaccharides at all. In addition, although the presence of hexose oxidase in red algae is known as an enzyme exhibiting the oxidizing activity of oligosaccharides, its properties have not yet been clarified [“Enzyme Handbook” (published by Asakura Shoten), 68
６９69]. The optimum pH of this enzyme is described as 5.0.

[0004]

It is said that the most preferable method for measuring amylase is a method for quantifying a newly generated reducing group due to amylase activity, such as the Somogyi method, but the operation is very complicated. There is a disadvantage that. In addition, in the blue starch method and the iodine starch reaction, the activity value varies depending on the lot of the substrate, and the former has a problem that a filtration operation is required. Therefore, in order to solve these problems, a method using a substrate having a chromophore bound to an oligosaccharide, for example, modifying the reducing end of the oligosaccharide with p-nitrophenol,
A method of reacting glucoamylase and α-glucosidase after amylase action and colorimetrically determining the released p-nitrophenol (JP-A-60-54395), and further modifying the reducing terminal residue to obtain a maltose dehydrogenase substrate Maltose dehydrogenase and NAD were added to the substrate hydrolyzate produced by the amylase action using a modified reducing terminal glucose polymer modified so as not to cause
(P) was acted on to produce NAD
A method for measuring (P) H (JP-B-63-37640) is known.

However, in these methods, the operation is complicated, and the temperature of the released p-nitrophenol,
There are problems such as stability due to pH, problems such as susceptibility to bilirubin and hemoglobin coexisting in the sample, and difficulty in application to an automatic analyzer due to a large Km value of maltose dehydrogenase. It was not a satisfactory measurement method in terms of measurement accuracy. Therefore, development of a new method for measuring amylase activity has been desired.

On the other hand, regarding the production of oligosaccharide acids, a production method by Pseudomonas graveorens has been reported (Japanese Patent Publication No. 48-20314), but this report plays a central role in the production of oligosaccharide acids. There is no description about the enzyme. The present invention solves the above problems, and provides an oxidase that directly reacts with an oligosaccharide, a method for producing the same, a method for measuring an oligosaccharide that can be carried out easily, inexpensively, and accurately using the oxidase. An object of the present invention is to provide a method for producing a sugar acid, a method for measuring amylase activity, and a novel microorganism .

[0007]

The oligosaccharide oxidase of the first invention has a molecular weight of 5.8 to 64,000 and contains one molecule of FAD (flavin adenine dinucleotide) per one molecule of the oligosaccharide oxidase. , Isoelectric point is 4.2-4.4, optimal p
H is around 10, optimum temperature of 50 ° C. vicinity der Ri, D- Group
Course, D-lactose, D-cellobiose, D-mal
Totriose, D-maltotetraose, D-maltope
Antaose, D-maltohexaose, D-maltohep
Good reaction activity for each substrate of Taose
To FeCl ₂ of HgCl ₂ and 0.5mM concentration of M concentration
And wherein the Rukoto inhibited me. The method for producing the present oxidase is not particularly limited, but is usually the following method of the second invention. The Km value for maltose is usually 2 to 4
It can be about mM.

The method for producing an oligosaccharide oxidase according to the second aspect of the present invention is characterized by the action of producing an acid corresponding to the oligosaccharide and hydrogen peroxide from the oligosaccharide and oxygen (hereinafter referred to as acid / peroxide). Hydrogen oxide generating action).
A microorganism having an ability to produce an oligosaccharide oxidase according to the first aspect of the present invention is cultured in a predetermined medium and collected from the culture. The acid corresponding to this oligosaccharide produces gluconolactone by the action of the present oxidase,
This is caused by hydrolysis. The Acremonium genus microorganism is described in the twelfth invention.
Acremonium Strictham T1 (deposit number: FER
Acremonium Sutorikutamu, including M No. P-11899), etc. (Acremonium strictum)
Other, Acremonium Fujidioidesu (Acrem
onium fusidioides or Acremonium potronii (Acremonium potr)
onii). The production of this oxidase can be carried out by either solid culture or liquid culture. In the case of solid culture, the support is not particularly limited, and generally used bran can be used. In the liquid culture, those generally used for the carbon source, the nitrogen source and the inorganic salts are sufficient, and are not particularly limited. The purification method is not particularly limited. For example, the purification can be performed by a combination of ultrafiltration, salting out, gel filtration, ion exchange chromatography, hydrophobic chromatography and the like.

[0009] The method for measuring an oligosaccharide according to the third invention uses the oligosaccharide oxidase of the first invention having an acid / hydrogen peroxide-producing action to convert an oligosaccharide and oxygen into an acid corresponding to the oligosaccharide. The method is characterized in that hydrogen peroxide is generated, and the consumption amount of the oxygen or the generation amount of the acid or the hydrogen peroxide is determined. The oxidase in the present invention can be applied as long as it is the oligosaccharide oxidase of the first invention having the action of producing acid and hydrogen peroxide, and is not limited to the oligosaccharide oxidase produced by the method of the second invention. These quantification methods include commonly used methods such as a HPLC (high performance liquid chromatography) method, a color development method using peroxidase, and a method using an oxygen electrode or a hydrogen peroxide electrode by immobilizing the enzyme. You can also.

Utilizing this principle, the activity of a saccharide hydrolase can be easily measured as shown in the fifth invention. For example, the activity of amylases can be easily measured by quantifying hydrogen peroxide or the like produced by the action of an enzyme on an oligosaccharide produced by amylase action from a substrate such as starch, dextrin and oligosaccharide. In this case, it is desirable to use the one in which the reducing end of the substrate is crushed in order to suppress the action of the oligosaccharide oxidase on the substrate and increase the measurement accuracy, and for this purpose,
Using a substrate having a oxidized reducing end, which can be prepared by reacting this enzyme with a substrate which is a glucose polymer such as starch, starch hydrolysate, dextrin or oligosaccharide in advance, or using various glucose polymers described above. A glucose polymer having a reducing terminal group modified by esterification, etherification, oxidation or the like can be used as a substrate. Further, a cyclic glucose polymer can be used as a substrate. Similarly, the enzyme activity of cellulases can be measured.

Particularly, the method for measuring amylase activity in a biological body fluid will be described in detail. A glucose polymer or a cyclic glucose polymer can be suitably used as a substrate for amylase. When a glucose polymer is used, its reducing terminal glucose residue is modified by various methods to use a glucose polymer having a modified reducing terminal glucose residue that does not become a substrate for oligosaccharide oxidase. Is preferred.

As a glucose polymer, the degree of polymerization is 4
The above is used. For example, various oligosaccharides, amylose, amylopectin, starch, so-called dextrin such as starch hydrolyzate, and the like can be used. These substrates are available by modifying the reducing terminal glucose residue. Furthermore, cyclic glucose polymers can also be used as substrates. In this case, the degree of polymerization is preferably 5 or more. For example, examples of the cyclic glucose polymer include various cyclodextrins (α-, β-, γ-, δ-, or ξ-).

The method for modifying the reducing terminal glucose residue of the glucose polymer may be any method as long as the substrate can be substantially prevented from reacting with the oligosaccharide oxidase. For example, as described above, a method of oxidizing the reducing terminal using the oligosaccharide oxidase of the present invention, a method of etherification, esterification, oxidation or reduction by a conventional method, and the like are mentioned. Further, more preferably, a sugar alcohol of an oligosaccharide can be used as a substrate. For example, maltote reitol, maltopentitol, maltohexaitol, maltoheptoitol, maltooctaitol and the like are exemplified. The glucose polymer having a modified reducing terminal glucose residue serving as a substrate for these amylases can be used irrespective of its degree of polymerization or a mixture of various degrees of polymerization.

Using a glucose polymer having the above-mentioned modified reducing terminal glucose residue or cyclic glucose as a substrate, a test solution containing amylase is added and reacted. These substrates are hydrolyzed by the amylase action to generate decomposed products such as glucose, maltose and various other oligosaccharides. The oligosaccharide oxidase of the present invention is allowed to act on the generated decomposed product, and the amount of oxygen consumed or the amount of generated hydrogen peroxide is measured, whereby the amylase activity in the sample can be measured.

Known methods can be used for measuring the amount of consumed oxygen and the amount of generated hydrogen peroxide. For example, there is a method using an oxygen electrode or a hydrogen peroxide electrode for measuring a potential difference. Furthermore, in the method of measuring the amount of generated hydrogen peroxide,
For example, a known method of optically measuring using a peroxidase and a hydrogen peroxide detection reagent can be suitably used. Examples of the hydrogen peroxide detection reagent include a combination of a so-called coupler and a Trinder reagent (hydrogen donor) as represented by 4-aminoantipyrine and phenol which have been widely used as clinical diagnostic agents.

Specific examples of the above coupler include 4-aminoantipyrine, 2,6-dibromoaminophenol, 3-methyl-benzothiazolinone hydrazone and the like, and specific examples of a Trinder reagent (hydrogen donor). Is β-chlorophenol, 2,4-
Dichlorophenol, 2,6-dichlorophenol,
N, N-dimethylaniline (DMA), N-ethyl-N
-(2-hydroxy-3-sulfopropyl) -m-anisidine (ADOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -aniline (ALO
S), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS), N-ethyl-N-sulfopropyl) -m-anisidine (ADP)
S), N-ethyl-N-sulfopropylaniline (A
LPS), N-ethyl-N-sulfopropyl-3,5
-Dimethoxyaniline sodium salt (DAPS), N
-Ethyl-N- (2-hydroxy-3-sulfopropyl-3,5-dimethoxyaniline sodium salt (DA
OS), N-sulfopropyl-3,5-dimethoxyaniline (HDAPS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HD
AOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (MA
OS), N-ethyl-N-sulfopropyl-3,5-
Examples include dimethoxyaniline (MAPS), N-ethyl-N-sulfopropyl-m-toluidine (TOPS), N-ethyl-N- (3-methylphenyl) -N'-acetylethylenediamine (EMEA), and the like.

As the test liquid to be measured in the present invention, a biological fluid such as serum, urine or saliva is usually used. When these are used as test liquids, they may be appropriately diluted and used for the reaction. Furthermore, these test liquids often contain a so-called measurement interfering substance that causes measurement interference when carrying out the present invention. The measurement interfering substance is, for example, glucose or maltose. If they coexist, it is desirable to process them beforehand and delete them.

Elimination means conversion to a compound that does not affect the assay of the present invention. For example, in the case of glucose, a method of converting glucose to glucose-6-phosphate by reacting it with hexokinase or glucokinase in the presence of magnesium ion and adenosine triphosphate (hereinafter referred to as ATP) can be mentioned. in addition,
A system in which ATP converted from ATP is supplied again as ATP may be added by allowing phosphoenol pyruvate and pyruvate kinase to be present in the measurement system. Further, mutarotase may be added as needed.

In the case of maltose, the oligosaccharide oxidase of the present invention is allowed to act beforehand, and the generated hydrogen peroxide may be removed with an appropriate trapping agent. As the trapping agent, for example, the coupler or the Trinder reagent described above can be used. Alternatively, maltose is converted to glucose and glucose-1 using maltose phosphorylase.
-Can be converted to phosphoric acid and further eliminated using the glucose elimination system described above.

As a suitable amylase measuring system, a reaction interfering substance in a test solution is treated in advance by the above-described method or the like, and then reacted with the various substrates described above, and further reacted with oligosaccharide oxidase. The resulting hydrogen peroxide is colored with various couplers and a Trinder reagent, and the absorbance is measured with a photometer.

As the reagent composition, for example,
And the composition as described in Example 13. In these, the substrate, the coupler, the Trinder reagent and the like can be used in various combinations, and the type of the buffer and the purity of the enzyme reagents can be appropriately changed as long as they do not affect the measurement. The amylase measurement method of the present invention can be applied to any of a so-called rate assay method and an end point method. The above series of reactions is usually performed at 25 ° C to 40 ° C, pH 6 to
8 is performed, but can be appropriately changed depending on the used reagents and the like.

As described above, the method for producing an oligosaccharide acid according to the fourth aspect of the present invention comprises the first aspect having an acid / hydrogen peroxide generating action.
Using the oligosaccharide oxidase of the present invention, an oligosaccharide acid corresponding to the oligosaccharide is generated from the oligosaccharide and oxygen, and then the oligosaccharide acid is collected. The oligosaccharides include maltooligosaccharides, cellooligosaccharides,
Further, oligosaccharides and the like, for which the oxidase is active, such as lactose, can be used. The oligosaccharide acid obtained by this reaction is obtained by oxidizing the reducing end glucose of the used oligosaccharide to gluconic acid. As a method for producing this oligosaccharide acid, besides a fermentation method using a microorganism that produces this enzyme, an enzymatic method that separates this enzyme can also be used, but in the latter case, catalase is used in combination. It is necessary to let

[0023]

The present invention will be described in detail with reference to the following examples. Example 1 Screening of Oligosaccharide Oxidase Producing Bacteria and Production of Oligosaccharide Oxidase (1) Screening of Producing Bacteria First, a bacterium that produces an oxidase directly acting on an oligosaccharide was converted from a natural microorganism into a bran culture medium (26- (28 ° C.) to screen various bacteria. That is, after adding 1 liter of water to 30 g of bran and extracting by boiling, the bran extract obtained by filtration is:
Glucose 1%, malt extract 0.3%, yeast extract 0.
Medium (pH 7.0) 1 containing 3% and 0.5% peptone
00 ml is added to a 500 ml Sakaguchi flask, which is stoppered with cotton and sterilized at 120 ° C. for 30 minutes. After cooling, the strain of the genus Acremonium was inoculated and cultured with shaking at 26 to 28 ° C. After 5 days, the culture was stopped, and a clear crude enzyme solution was obtained by centrifugation. When the oligosaccharide oxidase activity of this crude enzyme solution was examined,
Acremonium Strictum ATCC-34717,
Acremonium Fujidioides IFO-6813,
4 kinds of Acremonium Acremonium Potoronii IFO-31197 and Acremonium Sutorikutamu T ₁ is found to be suitable as a production microorganism. Of these, Acremonium Strictum ATCC-347
When using 17 and Acremonium Sutorikutamu T ₁ showed especially good results.

The activity was measured by an enzyme-coupling method (phenol 4AA peroxidase method, hereinafter referred to as peroxidase method). In this method, an appropriate amount (10 to 100 μl) of the enzyme diluent is added to maltose 10 mM,
Add 1 ml of Tris-HCl buffer (50 mM, pH 7.8) containing 4 mM of aminoantipyrine, 4 mM of phenol, and 12 to 25 u of peroxidase (enzyme unit) / ml, react at 30 ° C., and follow the change of ΔA ₅₀₀ Is what you do. Incidentally, the activity value is calculated by the following formula,
Compared.

Activity value (u / ml) = (ΔA ₅₀₀ / t) ×
(Vt / Ve) × (1 / 6.8) × n where ΔA ₅₀₀ is the absorbance difference between the reaction value and the blind value at a wavelength of 500 nm, t is the reaction time (min), Vt is the volume of the reaction solution, Ve indicates the volume of the enzyme solution, and n indicates the dilution factor.

The measurement of the enzyme activity was performed by the following method, if necessary. That is, Gilson's oxygra
The measurement was carried out by measuring the amount of oxygen consumed after adding the enzyme solution to a 0.1 mM phosphate buffer (pH 7.5) containing 6 mM maltose using a ph device. One unit of oligosaccharide oxidase was expressed as an enzymatic activity that consumed 1 μmol of oxygen per minute.

[0027] Then, the Acremonium Sutorikutamu T ₁ is, Netherlands · CVS (Centraalbureau Voor
Where was the identification request to Schimmelcultures), recognized as belonging to Acremonium Sutorikutamu, this fungus was Acremonium Sutorikutamu T _1. Then, this bacterium was deposited with the Research Institute for Microbial Industry on December 13, 1990, and the deposit number is FERM P-11899. Below, we were tested using the Acremonium Sutorikutamu T _1.

(2) Production and purification of oligosaccharide oxidase 700 g of bran and water 7 were placed in three 5-liter Erlenmeyer flasks.
After dividing the well-mixed medium into which 00 ml had been added, a cotton plug was placed and sterilization was performed at 120 ° C. for 60 minutes. After cooling down,
It was inoculated Acremonium Sutorikutamu T _1, 26~2
The cells were cultured at 8 ° C for 7 days. Add 4 liters of 50 to this culture
mM Tris-HCl buffer (pH 7.8) was added for extraction and centrifugation to obtain a clear solution. Thereafter, 45-80% ammonium sulfate salting out fractionation was performed. The salted-out precipitate is collected and dissolved, dialyzed, and subjected to column chromatography using "DEAE-Toyopearl 650M". Further, the active fractions are collected and subjected to column chromatography using "phenyltoyopearl 650M". Gel filtration by 100 "and column chromatography by hydroxyapatite were sequentially performed to obtain about 2 mg of a purified oligosaccharide oxidase.

When the purity of this oxidase was examined by electrophoresis of polyacrylamide gel, it showed a single band, indicating that it was a single substance. As shown in FIG. 1A, the molecular weight was about 60,000 from the band of the standard substance B in FIG. The specific activity is 2.2
u / mg, Km value for maltose (Michaelis constant)
Was about 2-4 mM. Note that this Km value caused such an error by the derivation method (extrapolation method) of this value. Furthermore,
When measured by the absorbance method, the oxidase contains one molecule of flavin adenine dinucleotide (FDA) per molecule of the enzyme. In addition, this oxidase oxidizes the reducing end of the oligosaccharide to generate an oligosaccharide acid, as described in “Method for producing oligosaccharide acid” below.

Example 2 Examination of enzymatic properties The following properties were examined using the purified enzyme obtained above. (1) Optimum pH and pH Stability Various kinds of buffer solutions were used to change the pH from 4.5 to 11.5.
Solution H was prepared, and the relationship between this pH and the relative activity of the oxidase was examined. The results for the optimum pH are shown in FIG. 2, and the results for the pH stability are shown in FIG. In addition, acetate buffer at pH 4.5-5.5, phosphate buffer at pH 5.5-8.0, Tris-HCl buffer at pH 7.0-9.0, pH buffer
In 9.0 to 11.5, a carbonate buffer was used, respectively. Further, the relative activity values in FIG.
The remaining activity after one hour was measured. FIG.
According to the above, the optimum pH is about 10, showing good activity at about 8 or more. Also, according to FIG.
The above shows excellent pH stability. In consideration of the above results, it is preferable to use the composition at pH 7 or more.

(2) Optimal Temperature and Temperature Stability FIG. 4 shows the relationship between the temperature (° C.) of the buffer solution and the relative activity of the oxidase.
And FIG. In addition, the case of FIG. 4 was measured with a 50 mM phosphate buffer (pH 7.5), and the case of FIG. 5 was measured with respect to the residual activity after 1 hour in the same phosphate buffer. According to FIG. 4, the optimum temperature is about 50 ° C., and shows good activity at a temperature of 40 to 55 ° C. According to FIG. 5, excellent stability is exhibited below 40 ° C., and above 50 ° C., the residual activity is significantly reduced.
Combining the above results, it is preferable to use at 30 to 50C.

(3) Substrate specificity The oxidase was added to various substrates, and the relative activity of each substrate was evaluated. Here, the relative activity is
It refers to a relative value when the activity on maltose is expressed as 100. According to this test, oxidase is D-Glucouse
(Relative activity value: 59%), D-Lactose (same; 64%), D
-Cellobiose (same; 47%), D-Maltotriose (same; 9%)
4%), D-Maltotetraose (same as above; 74%), D-Maltopen
taose (same; 46%), D-Maltohexaose (same; 66)
%) And D-Maltoheptaose (same as above; 56%). That is, lactose and cellooligosaccharides were also active as oligosaccharides other than maltooligosaccharides, and were also active with monosaccharides such as glucose.

[0033] (4) where on the activity of inhibiting the oxidase to the metal ions, the effects of various metal ions as described below, was examined in terms of the concentration indicated below, the HgCl ₂ and 0.5mM concentration of 1mM concentration Fe
As for Cl ₂ , it was found that the activity was reduced and an inhibitory effect was observed.

Example 3 Production of Oligosaccharide Acid 1 g of maltohexaose was added to a 0.05 M phosphate buffer (p
H7.0), 20 units of oligosaccharide oxidase and 100 units of liver catalase were added, and the reaction was carried out by stirring with a stirrer while introducing air. During the process, the pH was adjusted using a 0.5N NaOH solution because the pH was lowered. After 7 hours, the reaction was stopped, absorption and desorption of the ion exchange resin “Amberlite IRA-410 (manufactured by Organo Co., Ltd.)” were performed, followed by concentration and drying to obtain 0.94 g of a maltopentaose-gluconate compound. The gluconic acid compound was identified by a C ¹³ NMR spectrum and a mass spectrum. For example, the peak positions of the C ¹³ NMR spectrum result are 179.23 (C-1), 73.
33 (C-2), 73.55 (C-3), 83.24
(C-4), 73.56 (C-5), 63.06 (C-
6).

Further, 0.85 g of lactobionic acid was obtained by performing the same operation as above using 1 g of lactose as a substrate instead of maltohexaose. This is also an oligosaccharide acid whose reducing end has been oxidized as described above.

Example 4 Determination of Oligosaccharide 0.1 M containing 1 mM 4-aminoantipyrine, 1.8 mM dimethylaniline and 9 u / ml peroxidase
To 0.7 ml of a phosphate buffer (pH 7.0), 0.1 ml of each concentration of the maltose solution shown in FIG.
After preheating for 0.2 min, 0.2 ml of a 25 u / ml oligosaccharide oxidase solution was added, and reacted at 37 ° C.
As a result of measuring the absorption value of m (the amount of increase in the absorbance), a steady state was obtained after 10 minutes. The result is shown in FIG.

As shown in this figure, the relationship between the concentration of maltose (oligosaccharide) and the amount of increase in absorption was in a good linear relationship in direct proportion, so that the maltose concentration could be accurately measured. In addition, lactose, maltotriose, etc. show the same relationship, so that the concentration can be measured similarly.

Example 5 Measurement of Amylase Activity A dextrin having a reducing end oxidized was prepared according to the method for producing an oligosaccharide acid, and this was used as a substrate for measuring amylase activity. PH 7.0 phosphate buffer (1 mM 4-aminoantipyrine, 1.8 mM dimethylaniline, 9 u /
ml peroxidase and 5 u / ml oligosaccharide oxidase. ) Transfer 0.9 ml into a test tube, preheat at 37 ° C for 5 minutes, add 0.1 ml of each dilution of human saliva, and add
Reacted at ℃. In the blind test, deionized water was used instead of saliva. As a result of reading the absorption value at 550 nm (increase in absorbance) 10 minutes after the start of the reaction, the relationship between the α-amylase activity in saliva and the absorption value at 550 nm showed a good linear relationship in direct proportion. Accordingly, this allows accurate measurement of α-amylase activity.

Example 6 4-Nitrophenyl-α-D
-Measurement of amylase activity using maltopentaoside As reagent A, 0.1 M BES-NaOH containing
Buffer (pH 8.0) 10 mM 4-Nitrophenyl-α-D-maltopentaoside
_{(G 5 pNP) 1.5mM N-} Ethyl-N- (2-hydroxy-3-sulfopropy
l) -m-toluidine (TOOS) 0.1 M BES-NaOH containing 10 mM CaCl ₂ reagent B, including:
Buffer (pH 8.0) 1.5 mM 4-Aminoantipyrine 15 u / ml Peroxidase 2.8 u / ml Oligosaccharide oxidase 10 mM CaCl ₂

660 μl of reagent A was added to 20 μl of saliva amylase of various concentrations, and preliminarily heated at 37 ° C. for 5 minutes.
Thereafter, 360 μl of reagent B was added, and the reaction was performed at 37 ° C. The increase in absorbance at 555 nm per minute was measured. FIG. 7 shows the reaction pattern, and FIG. 8 shows the relationship between salivary amylase activity and absorbance value. As shown in FIG. 7, almost no lag time was observed, and as shown in FIG. 8, the salivary amylase activity and the increase in absorbance per minute showed a good linear relationship in direct proportion. In FIG. 7, indicates a blank,
Are 1/5, 2/5, 3/5, 4/5, 5/5, respectively
The results of the dilution are shown.

Example 7 4-Nitrophenyl-α-D
-Measurement of amylase activity using maltoheptaoside Example 4 was replaced with 4-nitrophenyl-α-D-maltoheptaoside in place of 4-nitrophenyl-α-D-maltopentaoside. In the same manner as in Example 6, an amylase measurement test was performed using an amylase-containing sample derived from saliva and pancreatic juice. As a result, as in Example 6, a good linear relationship was observed between the amylase concentration and the increase in absorbance at 555 nm per minute in all cases.

Example 8 Measurement of Amylase Activity Using Pentaglucosyl-β-D-maltopentaoside Instead of 4-nitrophenyl-α-D-maltopentaoside of Example 6, pentaglucosyl-β-D -An amylase measurement test was performed using fructofuranoside as a substrate and an amylase-containing sample derived from saliva and pancreatic juice in the same manner as in Example 6. As a result, as in Example 6, a good linear relationship was observed between the amylase concentration and the increase in absorbance at 555 nm per minute in all cases.

Example 9 Measurement of Amylase Activity Using Maltohexaitol as Substrate (1) Preparation of Maltohexaitol To 10 mg of maltohexaose was added 10 ml of water, dissolved, 47 mg of NaBH ₄ was added, and the mixture was stirred for 15 hours. Reacted. Thereafter, the mixture was adjusted to pH 8.5 and passed through a strong anion exchange resin “A-111” (manufactured by Kurita Kogyo) and a strong cation exchange resin “L-111” (manufactured by Kurita Kogyo) for the purpose of removing inorganic substances. After adjusting to pH 6.5, the solution was freeze-dried to obtain about 300 mg of maltohexaitol.

(2) Measurement of amylase activity In place of 4-nitrophenyl-α-D-maltopentaoside of Example 6, maltohexaitol was used as a substrate.
As in Example 6, an amylase measurement test was performed using an amylase-containing sample derived from saliva and pancreatic juice. As in Example 6, in each case, as shown in FIG. 9, a good linear relationship was observed between the amylase concentration and the increase in the absorbance at 555 nm per minute.

Example 10 Measurement of Amylase Activity Using Maltopentaose Gluconate as Substrate Oxidation of oligosaccharides from maltohexaose according to Example 3 in place of 4-nitrophenyl-α-D-maltopentaoside of Example 6. A test was carried out in the same manner as in Example 6 using maltopentaose-gluconic acid prepared by the enzyme as a substrate. As a result, as in Example 6, a good linear relationship was observed between the salivary amylase concentration and the increase in absorbance at 555 nm per minute.

Example 11 Measurement of Amylase Activity Using β-Cyclodextrin as Substrate Same as Example 6 except that β-cyclodextrin was used as a substrate instead of 4-nitrophenyl-α-D-maltopentaoside in Example 6. Was measured for salivary amylase. As a result, as shown in FIG. 10, a good linear relationship was observed between the salivary amylase concentration and the increase in absorbance at 555 nm per minute.

Example 12 Elimination of Glucose and Maltose in Test Solution As reagent A, 0.1 M BES-NaOH containing:
Buffer (pH 8.0) 10 mM 4-Nitrophenyl-α-D-maltopentaoside (G
₅ pNP) 1.5 mM N-Ethyl-N- (2-hydroxy-3-sulfopropyl)-
m-toluidine (TOOS) 10 mM CaCl ₂ 1.6 u / ml mutarotase 25.6 u / ml hexokinase 0.85 mM ATP 2.52 mM phosphoenolpyruvate 3.09 u / ml pyruvate kinase 5 u / ml oligosaccharide oxidase 15 mM MgCl _{2 10} u / ml peroxidase As reagent B, 0.1 M BES-NaOH containing:
Buffer (pH 8.0) 1.5 mM 4-Aminoantipyrine 10 mM CaCl ₂

100 mg / dl glucose, 50 mg / dl
20 μl each of 0.8 u / ml salivary amylase solution containing dl maltose and 0.8 u / ml salivary amylase solution
660 μl of reagent A is added thereto, and preliminarily heated at 37 ° C. for 5 minutes to eliminate glucose and maltose. Then, reagent B
Was added and the reaction was carried out at 37 ° C. The increase in absorbance at 555 nm per minute was measured. on the other hand,
The same sample was measured using the reagent used in Example 6. The reaction pattern is shown in FIG. 11 and FIG. still,
11 and 12, the solid line shows the reaction pattern of the test solution containing glucose and maltose, and the dashed line shows the reaction pattern of the test solution containing no glucose and maltose.

According to the above method, it was confirmed that the amylase sample containing glucose and maltose and the sample not containing glucose and maltose showed the same reaction pattern. That is, it was confirmed that the elimination of glucose and maltose was sufficient and the amylase activity could be measured favorably. On the other hand, when the reagent of Example 6 was used without erasure, the α-amylase activity of the sample containing glucose and maltose could not be measured accurately.

Example 13 Elimination of Glucose and Maltose in Sample As a reagent A, a 0.35 M phosphate buffer (p
H6.6) 10 u / ml maltose phosphorylase 10 mM 4-Nitrophenyl-α-D-maltopentaoside (G
₅ pNP) 1.5 mM N-Ethyl-N- (2-hydroxy-3-sulfopropyl)-
m-toluidine (TOOS) 1.6 u / ml mutarotase 25.6 u / ml hexokinase 0.85 mM ATP 2.52 mM phosphoenolpyruvate 3.09 u / ml pyruvate kinase 15 mM MgCl ₂ Reagent B and contains 200 mM H ₃ BO ₃ -50mM NaCl-50mM
Na ₂ B ₄ O ₇ buffer solution (pH 7.5) 1.5 mM 4-Aminoantipyrine 4 mM CaCl ₂ 1.5 u / ml oligo-isooxidase 15 u / ml peroxidase

100 mg / dl glucose, 50 mg / dl
660 μl of reagent A is added to 20 μl each of 0.8 u / ml salivary amylase solution containing dl maltose and 0.8 μl / ml salivary amylase solution, and preheated at 37 ° C. for 5 minutes to eliminate glucose and maltose. Thereafter, 360 μl of reagent B was added, and the reaction was performed at 37 ° C. The increase in absorbance at 555 nm per minute was measured. Example 11
It was confirmed that the measurement could be performed without being affected by glucose and maltose as in the case of. It should be noted that the present invention is not limited to the specific embodiments described above, but can be variously modified within the scope of the present invention according to the purpose and application.

[0052]

The oligosaccharide oxidase of the present invention is an unusual and useful oxidase that produces an oligosaccharide acid or the like having a reduced end oxidized from an oligosaccharide and an enzyme through hydrolysis. And if you use this oxidase,
The oligosaccharide concentration can be measured accurately and inexpensively, and the amylase activity or cellulase activity can be measured simply and accurately. Moreover, since oligosaccharide acids are generated by this enzymatic reaction, oligosaccharide acids can be easily produced.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an explanatory view showing the results of measuring the unity and molecular weight of the oligosaccharide oxidase of Example 1, wherein A is a band relating to the oligosaccharide oxidase, and B is a band relating to the standard protein.

FIG. 2 is a graph showing an optimum pH.

FIG. 3 is a graph showing pH stability.

FIG. 4 is a graph showing an optimum temperature.

FIG. 5 is a graph showing temperature stability.

FIG. 6 is a graph showing the relationship between the maltose concentration and the absorption value at 550 nm in Example 4.

FIG. 7: 4-Nitrophenyl-α-D- of Example 6
It is a graph showing a reaction pattern of the measurement of amylase activity using maltopentaoside, indicates a blank,
Are 1/5, 2/5, 3/5, 4/5, 5/5, respectively
The results of the dilution are shown.

FIG. 8: 4-Nitrophenyl-α-D- of Example 6
It is a graph which shows the relationship between the amylase density | concentration of the amylase activity measurement result using maltopentaoside, and the absorption value at 550 nm.

FIG. 9 shows the results of measurement of salivary amylase activity using maltohexaitol as a substrate in Example 9 and the amylase concentration and 55.
It is a graph which shows the relationship with the absorption value of 0 nm.

FIG. 10 is a graph showing the relationship between the amylase concentration and the absorption value at 550 nm of the results of the amylase activity measurement using β-cyclodextrin as a substrate in Example 11.

FIG. 11 is a graph showing the results of measurement with the reagent composition described in Example 6 in Example 12, wherein a solid line shows a reaction pattern of a test solution containing glucose and maltose, and a broken line shows glucose and maltose. 4 is a graph showing a reaction pattern of a test solution containing no test solution.

FIG. 12 is a graph showing the results of measurement with the reagent composition described in Example 12, in which the solid line represents the reaction pattern of a test solution containing glucose and maltose, and the dashed lines represent glucose and maltose. 4 is a graph showing a reaction pattern of a test solution containing no test solution.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI C12Q 1/54 C12Q 1/54 // (C12N 9/04 (C12N 9/04 C12R 1: 645) C12R 1: 645) (56 References JP-A-2-42980 (JP, A) JP-A-60-114193 (JP, A) JP-B-55-33862 (JP, B1) (58) Fields investigated (Int. Cl. ⁷ , DB Name) C12N 9/04 C12N 1/20 C12P 7/58 C12Q 1/26 C12Q 1/40 C12Q 1/54 BIOSIS (DIALOG) JICST file (JOIS) MEDLINE (STN) WPI (DIALOG)

Claims (12)

(57) [Claims] 1. An oligosaccharide oxidase containing one molecule of FAD (flavin adenine dinucleotide) having a molecular weight of 5.8 to 64,000, and an isoelectric point of 4.2 to 4.4. pH is around 10, optimal temperature is around 50 ° C, and D-
Glucose, D-lactose, D-cellobiose, D-
Maltotriose, D-maltotetraose, D-maltopentaose, D-maltohexaose, and D-maltoheptaose exhibit good reaction activity for each substrate.
1 mM HgCl ₂ and 0.5 mM FeCl
_2. An oligosaccharide oxidase, which is inhibited by ₂ . 2. Acremonium (Acremonium)
2. The microorganism of the genus u) having an ability to produce an oligosaccharide oxidase according to claim 1, which has an action of generating an acid and hydrogen peroxide corresponding to the oligosaccharide from the oligosaccharide and oxygen. The method for producing an oligosaccharide oxidase according to claim 1, which is collected from the culture. 3. claims from the oligosaccharides and oxygen has the effect of generating an acid and hydrogen peroxide corresponding to the oligosaccharides
The acid and hydrogen peroxide corresponding to the oligosaccharide are generated from the oligosaccharide and oxygen using the oligosaccharide oxidase according to Item 1 , and the consumption of the oxygen or the generation of the acid or the hydrogen peroxide is determined. A method for measuring an oligosaccharide, which comprises quantifying the oligosaccharide. 4. claims from the oligosaccharides and oxygen has the effect of generating an acid and hydrogen peroxide corresponding to the oligosaccharides
An oligosaccharide acid corresponding to the oligosaccharide is generated from the oligosaccharide and oxygen using the oligosaccharide oxidase according to item 1 ,
Next, a method for producing an oligosaccharide acid, comprising collecting the oligosaccharide acid. 5. A method for measuring amylase activity using a glucose polymer having a denatured reducing terminal glucose residue as a substrate, wherein the oligosaccharide oxidase according to claim 1 is acted on to measure oxygen consumption or hydrogen peroxide. A method for measuring amylase activity, which comprises measuring the amount of production. 6. The method according to claim 5, wherein the glucose polymer is an oligosaccharide, amylose, amylopectin, starch or a starch hydrolyzate. 7. The method according to claim 5, wherein the modified reducing terminal glucose residue is sorbitol, gluconic acid, 4-nitrophenol, fructose or a derivative thereof. 8. A method for measuring amylase activity using a cyclic glucose polymer as a substrate, wherein the oligosaccharide oxidase according to claim 1 is allowed to act to reduce the amount of consumed oxygen or the amount of generated hydrogen peroxide. A method for measuring amylase activity, characterized in that it is measured. 9. A method for measuring amylase activity of glucose polymers having a modified reducing terminal glucose residue, or cyclic glucose polymer as a substrate, and processing the measurement interfering substances in advance the倹液followed claim 1 A method for measuring amylase activity, which comprises reacting the described oligosaccharide oxidase to measure the amount of consumed oxygen or the amount of generated hydrogen peroxide. 10. A method for measuring amylase activity using a glucose polymer having a modified reducing terminal glucose residue or a cyclic glucose polymer as a substrate, wherein a measurement interfering substance in a liquid to be measured is previously treated with an oligosaccharide oxidase and a kinase. A method for measuring amylase activity, comprising treating and then treating the oligosaccharide oxidase according to claim 1 to measure the amount of oxygen consumed or the amount of hydrogen peroxide produced. 11. A method for measuring amylase activity using a glucose polymer or a cyclic glucose polymer having a modified reducing terminal glucose residue as a substrate, wherein a measurement interfering substance in a liquid to be measured is previously treated with maltose phosphorylase and kinase. Then, the oligosaccharide oxidase according to claim 1 is acted on to measure the amount of consumed oxygen or the amount of generated hydrogen peroxide. 12. Acremonium
Acremonium strictum T1 (deposited in mu) belonging to the genus mu) having an ability to produce an oligosaccharide oxidase having an action of generating an acid and hydrogen peroxide corresponding to the oligosaccharide from the oligosaccharide and oxygen. Number: FERM
P-11899).