JPH09297A - Determination of activity of cellulose hydrolase - Google Patents

Abstract

PURPOSE: To determine the activity of cellulose hydrolase in high accuracy and efficiency. CONSTITUTION: The activity of cellulose hydrolase is determined by the following manner. The reducing terminal group produced from a cellulose substrate (e.g. water-soluble cellulose such as cellooligosaccharide) by the action of cellulose hydrolase is subjected to an oxidation-reduction reaction with cellobiose dehydrogenase and the change caused by the reaction is detected by a spectroscopic means or electro-chemical means. The change caused by the oxidation- reduction reaction can be detected as the spectroscopic or electro-chemical change of an electron acceptor existing in the reaction system. The cellulose substrate may be a reduced cellooligosaccharide, etc., and a compound containing heme iron, etc., can be used as the electron receptor.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a cellulose hydrolase (cellulase) which decomposes cellulose or its derivatives.
The present invention relates to a method for measuring the activity of Escherichia coli, and an activity measuring kit useful for this method.

[0002]

Cellulase is an enzyme that hydrolyzes the β-1,4-glucopyranosyl bond of β-1,4-glucan (cellulose) or its derivatives. It is a microorganism such as higher plants, fungi, bacteria, or molluscs. It is widely distributed in Japan. Cellulase is a so-called endoglucanase (CMCase) that hydrolyzes a β-1,4-glucopyranosyl bond of a cellulose main chain to an endo type, and mainly cuts out a cellobiose residue from the non-reducing end side of the cellulose main chain. , So-called exoglucanase (avicellase)
It is known to be roughly divided into. It is said that these hydrolases act cooperatively on cellulose or a derivative thereof to lower the molecular weight of the cellulose substrate, and further involve β-glucosidase to decompose the cellulose substrate.

There has been a strong interest in the method of converting cellulose to glucose by utilizing cellulase. More recently, the use of cellulase has been studied in a very multifaceted manner, such as addition to household detergents, modification by surface treatment of cellulosic polymeric materials such as fibers, deinking treatment from waste paper, and food processing. ing. in this way,
Cellulose hydrolase is one of the important enzymes in the chemical industry regarding cellulose or its derivatives.

Regarding the method for measuring the activity of cellulose hydrolase, for example, a method utilizing the reduction of the turbidity of a suspension of cellulose powder and a reduction of the viscosity of a highly viscous aqueous solution of carboxymethyl cellulose (CMC) are utilized. A method, an activity measuring method using a reducing terminal group generated from cellulose, etc. have been proposed.

Among these measuring methods, a method of measuring a change in the amount of reducing end groups, that is, a soluble cellooligosaccharide is obtained by treating a cellulose substrate with cellulase for a certain period of time (usually several tens of minutes to several hours). A method for evaluating the enzymatic activity of cellulase by producing it and chemically quantifying the increased amount of the reducing end group accompanying the production of cellooligosaccharide is widely used. For example, in the generally performed Nelson-Somogyi method, cellulose or the like is hydrolyzed by a cellulase for a certain period of time, and heated in an alkaline aqueous solution, so that the reducing end group of the product causes divalent Cu. ²⁺ is reduced to copper (I) oxide
Of Nelson's reagent (ammonium molybdate, sodium arsenic, sulfuric acid, water) is added and dissolved, and ammonium molybdate is reduced by Cu ⁺ to give a blue color of molybdenum blue. Then, by measuring the change in absorbance with coloration with a spectrophotometer, the amount of reducing terminal groups produced, and further the activity of the cellulose hydrolase is evaluated.

However, the measurement limit by the Nelson-Somogi method is about 100 μM glucose equivalent, and it is said that the so-called DNS method of treating with dinitrosalicylic acid has a measurement limit of about 500 μM glucose equivalent. Therefore, in these measurement methods, the activity of cellulose hydrolase is detected with high sensitivity,
It is difficult to evaluate with high accuracy. In addition, the conventional method for measuring the amount of reducing terminal groups produced by hydrolysis involves a large number of steps, so the operation is complicated and the measurement efficiency is low.

Furthermore, since the enzyme reaction cannot be continuously tracked, it is difficult to analyze the reaction rate in the initial stage of the reaction, which is important information for knowing the reaction characteristic of the enzyme, and the characteristic of the enzyme can be accurately evaluated. I can't.

On the other hand, since the substrate specificity varies depending on the type of cellulase, the cellulase activity may not be measured depending on the type of cellulose substrate. In general, exoglucanase has high substrate specificity, and it is difficult to use carboxymethylcellulose, which is a water-soluble cellulose derivative, as a substrate. On the other hand, since endoglucanase has a very low activity on the crystalline region of cellulose, for example, “Avicel” which is highly crystalline cellulose cannot be used as a substrate. Although exoglucanase is said to be able to use "Avicel" as a substrate, its activity for "Avicel" is low, and therefore it may take several hours to measure the activity. Therefore, the enzyme activity cannot be measured efficiently.

As described above, it is difficult for the conventional methods to detect the enzymatic activity of cellulase with high sensitivity or to continuously analyze the enzymatic reaction by cellulase to analyze the reaction characteristics of the enzyme. Therefore, even if the surface of cellulose or its derivative is modified with cellulase, there is a case where the correlation between the effect of the enzyme treatment and the measured enzyme activity is not always recognized. Furthermore, a weak enzyme activity that has been overlooked in the past may have a more important function.

[0010]

SUMMARY OF THE INVENTION It is, therefore, an object of the present invention to provide a method capable of measuring the activity of a cellulose hydrolase with high accuracy even if the amount is minute, and a measuring kit therefor. Another object of the present invention is to provide a method capable of reducing the substrate specificity of cellulase and efficiently measuring the activity of cellulose hydrolase, and a measurement kit therefor. Still another object of the present invention is to provide
It is an object of the present invention to provide a method capable of measuring the activity of cellulose hydrolase with high sensitivity and a measurement kit therefor.
Another object of the present invention is to provide a method capable of continuously tracking a reaction process by a cellulose hydrolase and a measurement kit therefor.

[0011]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to achieve the above-mentioned object, and as a result, when the redox reaction of cellobiose dehydrogenase was used, the cellulose substrate (cellulose or its derivative) was produced by the action of cellulase. The present inventors have found that the amount of newly produced reducing end group can be detected and quantified with high sensitivity as the enzyme activity of cellulase, and the present invention has been completed.

That is, in the method of the present invention, by detecting the change caused by the reaction between the reducing group produced from the cellulose substrate by the action of the cellulose hydrolase and the cellobiose dehydrogenase, the activity of the cellulose hydrolase is detected. Measure activity. In this method, the change that accompanies the redox reaction between the cellooligosaccharide produced by the cellulase reaction and cellobiose dehydrogenase may be detected as a spectroscopic change in the electron acceptor present in the reaction system. The measurement data detected by the above method can be used as an index of cellulase activity. The present invention also provides a kit for activity measurement, which comprises the cellulose substrate, cellobiose dehydrogenase and an electron acceptor. Hereinafter, the present invention will be described in detail.

As the cellulose substrate, glucose is β
Various compounds having -1,4-glucoside bonds, for example,
Examples thereof include polysaccharides such as cellulose (β-1,4-glucan), small sugars and their derivatives. The average molecular weight of the small sugar is usually 10,000 or less. The derivatives include, for example, esters in which part or all of the hydroxyl groups of cellulose or glucose units are esterified, ethers in which part or all of the hydroxyl groups of cellulose or glucose units are etherified, and the like.

Examples of cellulose esters include, for example,
Examples thereof include esters with C _1-4 organic acids such as cellulose acetate, cellulose propionate and cellulose butyrate, inorganic acid esters such as nitrocellulose and cellulose sulfate, mixed acid esters such as cellulose acetate butyrate and cellulose nitrate acetate. The esters of small sugars include esters corresponding to the above cellulose esters.

Examples of cellulose ethers include:
Alkyl ethers such as methyl cellulose and ethyl cellulose, aralkyl ethers such as benzyl cellulose, phenethyl cellulose and trityl cellulose,
Examples thereof include cyanoethyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, aminoethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, and other alkyl ethers that may have a substituent (cyano group, carboxyl group, amino group, hydroxyl group, etc.). The ethers of small sugars include ethers corresponding to the above cellulose ethers.

Of these cellulose substrates, water-soluble cellulose or its derivatives (for example, the above-mentioned cellulose ethers), especially water-soluble oligosaccharides or its derivatives are used in order to continuously measure and improve the measurement accuracy. It is advantageous to use. Small sugars include, for example, low molecular weight cellulose having a glucose unit number (degree of polymerization) of 3 to 50, preferably about 3 to 25, and particularly having a degree of polymerization of 3 to 20 (preferably 53 to 15, more preferably 5). ~ 9) cellooligosaccharides (eg, cellotriose, cellotetraose, cellopentaose, cellohexaose, celloheptaose, cellooctaose, etc.) are preferable. As the cellooligosaccharide, a water-soluble cellooligosaccharide having a high monodispersity is preferably used, and examples of such cellooligosaccharide include the method of Isogai and Usuda (Mokuzai Gakkaishi, 37 , 339-3).
44, (published in 1991)), a cellooligosaccharide having an average degree of polymerization of about 7 obtained by partial hydrolysis of cellulose with phosphoric acid is included. When a water-soluble small saccharide, particularly a water-soluble cellooligosaccharide, is used, the substrate specificity by cellulase can be reduced and it can be hydrolyzed according to the activity of cellulase.
The measurement accuracy can be improved.

Further, depending on the type of cellulosic substrate such as cellooligosaccharide, it may have reactivity with cellobiose dehydrogenase. When such a cellulose substrate is used, the reducing group having reactivity with cellobiose dehydrogenase is detected as noise, and the reducing end group generated by the action of cellulase cannot be accurately detected / measured. Therefore, in order to improve the measurement accuracy by detecting only the change associated with the reaction between the reducing terminal group generated by the action of cellulase and cellobiose dehydrogenase, in order to improve the measurement accuracy, a cellulose substrate that has been inactivated by reduction treatment as a cellulose substrate is used. It is preferably used.

As a reducing agent for a cellulosic substrate, a conventional metal hydride such as sodium borohydride, lithium borohydride, lithium aluminum hydride, sodium cyanoborohydride, lithium cyanoborohydride, lithium aluminum hydride is used. Etc. The amount of the reducing agent used with respect to the cellulose substrate can be appropriately selected from the range of about 0.01 to 50 parts by weight of the reducing agent with respect to 100 parts by weight of the cellulose substrate. The reduction treatment of the cellulose substrate is performed, for example, in an appropriate solvent (for example, water, alcohols such as methanol and ethanol, ketones such as acetone, ethers, etc.) depending on the type of the substrate,
It can be carried out at an appropriate temperature (for example, about 0 to 70 ° C.).

The origin of the cellulase for hydrolyzing the cellulose substrate is not particularly limited, and may be derived from any of animals such as snails, plants, fungi such as Koji fungi, bacteria and protozoa. The cellulase may be either exoglucanase or endoglucanase. When cellulase is allowed to act on a cellulose substrate, a cellulose hydrolyzate having a reducing end group is produced depending on the activity of cellulase. The amount of reducing end groups produced also changes depending on the amount of cellulase, and the amount of reducing end groups produced increases as the amount of cellulase increases.

In the present invention, the amount of reducing terminal groups produced from a cellulose substrate by the action of cellulase is measured as a change caused by the redox reaction with cellobiose dehydrogenase. That is, cellobiose dehydrogenase is
It is an enzyme that catalyzes a reaction that oxidizes the reducing end group of cellobiose, soluble cellooligosaccharides and cellulose produced by hydrolysis of a cellulose substrate, and converts this into a lactone.In the above redox reaction, cellobiose dehydrogenase and reduction are used. Electrons are exchanged with the sex terminal group. That is, in the redox reaction, cellobiose dehydrogenase receives two electrons due to the oxidation of one reducing end group. Therefore, the activity of cellulase can be measured by detecting a change caused by electron transfer between a reducing group generated by hydrolysis of a cellulose substrate and cellobiose dehydrogenase.

The cellobiose dehydrogenase has only to have a function of converting a reducing group into a lactone as described above, and the origin of the dehydrogenase is not particularly limited. Examples of cellobiose dehydrogenase include, for example, Phaneroquite ( Phan
erochaete ), Sporotrichume ( Sporotrichume )
Examples thereof include cellobiose dehydrogenase produced by microorganisms belonging to the genera, Monilia genus and Fomes genus. These dehydrogenases may be used alone or in combination of two or more.

Preferred cellobiose dehydrogenases include microorganisms belonging to the genus Phanerochaete , especially the white-rot fungus Phanerochaete chrysosporium ( Ph
Anerochaete chrysosporium ) includes cellobiose dehydrogenase produced by microorganisms belonging to anerochaete chrysosporium . The preparation method and properties of this cellobiose dehydrogenase have been studied in detail, and it is possible to stably produce it from Faneroquiete chrysosporium by shaking culture with cellulose as a carbon source. Cellobiose dehydrogenase can be easily separated and purified by a conventional method such as centrifugation, fractionation by salting out, or column chromatography of an enzyme produced by a microorganism. The obtained cellobiose dehydrogenase is relatively stable even at room temperature, can be stored at 4 ° C. for more than half a year, and can be stored for a longer period if frozen.

In the measuring method of the present invention, the enzymatic reaction between the cellulase and the cellulose substrate may be followed by the reaction with cellobiose dehydrogenase. However, in order to improve the measuring efficiency or to measure continuously, In many cases, the enzymatic reaction between cellulase and a cellulose substrate is carried out in the presence of cellobiose dehydrogenase.

The change caused by the redox reaction is
It is also possible to directly detect and measure by various analysis means described later. In order to accurately detect and measure the change caused by the redox reaction with higher sensitivity, it is advantageous to detect it as a spectroscopic or electrochemical change of the electron acceptor present in the reaction system. That is, when the reaction is performed in the presence of the electron acceptor, the electron acceptor receives an electron from cellobiose dehydrogenase. Therefore, when a reagent that causes a spectroscopic change or an electrochemical change by a redox reaction is used as the electron acceptor, the change that accompanies the redox reaction can be detected and measured as a change in the electron acceptor, and the enzyme of cellulase can be used. The activity can be evaluated.

With respect to the electron acceptor, a change accompanying electron transfer between the cellobiose dehydrogenase and the cellobiose dehydrogenase is determined according to the difference between the redox potential of the cellobiose dehydrogenase and the redox potential of the acceptor. The compound is not particularly limited as long as it is a detectable compound, but a compound useful for increasing detection sensitivity,
For example, redox indicators, trivalent iron compounds (cyano complex salts, etc.), quinones (hydroquinone, benzoquinone, etc.),
Radicals and molecular oxygen can be used. As these electron acceptors, compounds having a higher redox potential than heme contained in cellobiose dehydrogenase are preferable. Among these electron acceptors, it does not adversely affect the activity of cellobiose dehydrogenase and can enhance the detection sensitivity. 3
Valuable iron compounds, especially compounds containing heme iron (hematin) are preferably used. Compounds containing heme iron include
For example, in addition to protoheme, heme c, heme a, etc., heme proteins (eg, hemoglobin, myoglobin, cytochrome, catalase, peroxidase, etc.) are included, and among these, there is a characteristic strong absorption band depending on the type. Then, cytochrome is preferably used.

Cytochromes are classified according to the position of the absorption band, etc. Cytochromes a, a1, a2, ..., B, b
1, b2, ..., C, c1, c2, ..., f, h, etc., which are classified according to the structure of the heme of the prosthetic group A, A-2, B, C, D You may belong to either. Preferred cytochromes include cytochromes whose absorbance changes remarkably in a specific wavelength range (for example, about 540 to 560 nm), for example, cytochromes c and c1 to c5 belonging to group C. When such a cytochrome is used, when Fe ^{3+ of} cytochrome which has received an electron from cellobiose dehydrogenase is reduced to Fe ²⁺ , the absorbance in a characteristic absorption band increases depending on the degree of reduction. For example, as the electron acceptor, cytochrome c
When Fe ³⁺ is reduced to Fe ²⁺ , the absorbance at a wavelength of 550 nm remarkably increases depending on the degree of reduction. Therefore, the change in absorbance can be accurately measured with a spectrophotometer. In addition, by continuously measuring the change in absorbance with a spectrophotometer, it is possible to observe the increase and fluctuation of the reducing end groups produced from the substrate by the enzymatic reaction with cellulase. At that time, since the molar extinction coefficient in the absorption region due to the reduction reaction of cytochrome is large,
The reducing terminal group can be detected with high accuracy and high sensitivity. For example, since the molar absorption coefficient at 550 nm due to the reduction reaction of cytochrome c is 23.2 mM ⁻¹ cm ⁻¹ , 1 μM
It is possible to measure up to glucose equivalent (0.12 μg / ml). This detection sensitivity corresponds to about 100 times that of the Nelson-Somogi method and about 500 times that of the DNS method.

Further, in the method of the present invention, it is possible to quantify the reducing end group even in an extremely short period of time, for example, at least about 30 seconds. The detection sensitivity may be increased by increasing the amount of cellulase added to the cellulose substrate and increasing the amount of reducing terminal groups produced. The means for detecting or measuring the change caused by the redox reaction may be any means capable of measuring the change caused by electron transfer as described above, and the absorbance associated with the redox reaction, spectroscopy such as color development Means for detecting chemical changes (for example, infrared absorption method, ultraviolet-visible absorption method, spectroscopic analysis method such as fluorescence analysis method), means for detecting electrochemical changes such as redox potential (for example, potentiometric titration, etc.) Electrochemical analysis means), means for detecting the residual rate of radicals (for example, electromagnetic analysis means such as nuclear magnetic resonance method (NMR), electron spin resonance method (ESR) and electron paramagnetic resonance method (EPR)) And so on. As a preferable detection means, a spectroscopic or electrochemical analysis means having high measurement accuracy and high measurement efficiency is often used. When the above change is detected and measured electrochemically, an enzyme electrode carrying cellobiose dehydrogenase can be used.

When measured in this manner, reducing end groups are produced depending on the degree of hydrolysis of the substrate by cellulase. Therefore, the detection or measurement data of the amount of reducing end groups produced is used as an index of cellulase activity. can do. In the evaluation of the activity of cellulase, the value detected or measured after carrying out the enzymatic reaction of the cellulase under constant conditions may be used as an index of the activity of cellulase, and it may be intermittent or continuous in the enzymatic reaction process of cellulase. The data (for example, reaction rate) obtained on the basis of the values measured in the above may be used as an index of cellulase activity.

The pH of the enzyme reaction system in the measuring method of the present invention can be appropriately selected from the range of about pH 3 to 6 depending on the optimum pH of cellulase and cellobiose dehydrogenase. The reaction between the cellulase and the cellulose substrate has an optimum pH depending on the type of cellulase, for example, a pH of about 5.2 to 5.4 for snail-derived cellulase,
It can be carried out at a pH of about 4.5 for cellulase derived from Koji, and at about 7.0 for cellulase derived from bacteria. The pH is often adjusted using a buffer solution. Further, the reaction temperature is in a range that does not adversely affect the enzyme activity,
For example, it can be selected within the range of 10 to 50 ° C., preferably 25 to 37 ° C.

As is apparent from the above, the kit for measuring the activity of the cellulose hydrolase of the present invention comprises a cellulose substrate, cellobiose dehydrogenase and an electron acceptor. The kit may include other reagents such as a buffer solution. The kit may be composed of each component contained in a separate container, as long as the components are non-reactive with each other (for example, a combination of a cellulose substrate and an electron acceptor, a cellulose substrate and a buffer solution, etc.). You may store in one container. The enzyme may be contained in a container as a mixed solution with a buffer solution.

According to the method of the present invention, since the activity of cellulase can be measured with high sensitivity and accuracy, the amount and method of use of cellulase for detergents, printed waste paper, etc. can be accurately controlled. In addition, cellulase can be effectively used also in the surface treatment of cellulosic polymers such as cellulosic fibers and the processing of foods based on the relationship between the enzyme activity and the degree of treatment.

[0032]

INDUSTRIAL APPLICABILITY Since the measuring method of the present invention utilizes the redox reaction of cellobiose dehydrogenase, cellulase activity can be measured with high accuracy even if the amount is very small. Further, by using cellooligosaccharides as the cellulose substrate, the substrate specificity of cellulase can be reduced, and cellulase activity can be efficiently measured. In addition, cellulase activity can be measured more efficiently by using a water-soluble cellulose substrate. Furthermore, by using an electron acceptor, the activity of cellulase can be measured with high sensitivity. Furthermore, since the reaction process by cellulase can be continuously traced, the enzyme characteristics of cellulase can be effectively and accurately evaluated. The measurement kit of the present invention is useful for a measurement method having the above advantages.

[0033]

EXAMPLES The present invention will be described below in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Preparation of cellobiose dehydrogenase Cellobiose dehydrogenase was obtained from Faneroquiete chrysosporium K-3 strain by the following procedure. That is, Wood et al. (Biochem. Biophys. Act)
a., 1119 , 90-96, 1992), yeast extract (0.5 g / L) and polypeptone (0.5 g / L) were added to the basal medium described above, and about 1 × 10 ⁶ cells / L spores were inoculated and cultured with shaking at 27 ° C for 5 days. After cultivation,
The culture solution was separated from the bacterial cells and the substrate by filtration or centrifugation to obtain a culture solution containing cellobiose dehydrogenase as a crude enzyme solution.

The crude enzyme solution was subjected to precipitation fractionation with 70% saturated ammonium sulfate water, the resulting precipitate was dissolved in a 20 mM acetate buffer solution (pH 6.0), and dialyzed with this buffer solution. Column equilibrated with liquid (QAE-Toyopearl, manufactured by Tosoh Corp.), 44
mmφ × 170 mm). The cellobiose dehydrogenase activity fraction is a linear concentration gradient (20 mM →
Eluted with 1M). The obtained enzyme active fraction was concentrated and 20 mM sodium phosphate buffer solution (pH 7.8)
Column that has been equilibrated with the same buffer solution in advance (DEAD-Toyopear, manufactured by Tosoh Corporation)
l), 44 mmφ × 145 mm), and the cellobiose dehydrogenase active fraction was eluted with a linear concentration gradient (0 → 44 mM) of potassium chloride. After concentrating the obtained fractions, the column was equilibrated with 50 mM sodium phosphate buffer (pH 7.0) containing 0.7 M potassium chloride and preliminarily equilibrated with the same 0.8 M buffer (Tosoh Corp. ), Phenyl-Toyopearl, 32mmφ ×
130 mm) and the target enzyme active fraction was eluted with an inverse gradient of potassium chloride (0.8 → 0 M) to obtain cellobiose dehydrogenase. The Rz value (A ₄₂₁ / A ₂₈₀ ) of the purified enzyme was 0.62. In addition, the literature (Eu
r. J. Biochem. , 207 , 103-107, 1992) had an activity of 15.3 units / mg (U / mg). This cellobiose dehydrogenase was used for the activity measurement of the cellulose hydrolase described below.

Measurement of cellulase activity 500 mM sodium acetate buffer (pH 4.2), 50
500 μl of purified water was added to an aqueous solution containing 100 μl each of a 0 μM cytochrome c aqueous solution, 500 mU / ml cellobiose dehydrogenase aqueous solution, and a 0.25% aqueous solution of reduced cellooligosaccharide, and the concentration of the resulting mixed solution was 0.4 mg / 100 μl of cellulose hydrolase (Cellulase Onozuka R-10, Yakult Co., Ltd.) was added, and the enzyme reaction was started at a temperature of 30 ° C. After the reaction is started, the absorbance at the wavelength of 550 nm (optical path length 1c
When m) was continuously measured, the results shown in FIG. 1 were obtained.

As shown in FIG. 1, a clear positive correlation was observed between the reaction time and the absorbance, and the absorbance increased by 0.26 in the reaction for 1 minute from the start of the reaction. Since the molar extinction coefficient of cytochrome c was 23.2 mM ^-1 cm ^-1, it was confirmed that the reaction for 1 minute produced 5.6 μM of reducing end groups and 140 μM of reducing end groups per mg of cellulase. become. Further, assuming that the reliable detection sensitivity as a change in absorbance is 0.05, if the reducing terminal group of 1.1 μM is produced in a reaction time of 1 minute, it can be detected. Therefore, the reaction time is 10
In the case of minutes, it is possible to measure the production amount of the reducing terminal group of about 0.1 μM.

Example 2 500 mM sodium acetate buffer (pH 4.2) 200
μl, 0.5% carboxymethylcellulose (substitution degree 0.63) aqueous solution 200 μl, 820 mU / ml cellobiose dehydrogenase 100 μl, 500 μM cytochrome c aqueous solution 100 μl, and purified water 3
00 μl was added, and the resulting mixed solution had a concentration of 0.5 mg / m 2.
l Cellulose hydrolase (Cellulase Onozuka R-
10, Yakult Co., Ltd.) 100 μl was added, and the temperature was adjusted to 3
The enzymatic reaction was carried out at 0 ° C. for 1 minute. The amount of reducing end groups produced was 5.6 μM, which was determined from the change in absorbance (optical path length 1 cm) at a wavelength of 550 nm, which means that 116 μM reducing end groups were produced per mg of cellulase. Example 3 500 mM sodium acetate buffer (pH 4.2) 100
μl, 500 μM Cytochrome c aqueous solution 100 μl, 2
50mU / ml cellobiose dehydrogenase aqueous solution 100μ
1 and an aqueous solution containing 100 μl of a 0.25% aqueous solution of reduced cellooligosaccharide, a cellulose hydrolase (cellulase Onozuka R-1) having a concentration of 0.4 mg / ml was added.
0, a predetermined amount of Yakult Co., Ltd.) was added, and purified water was added to 1
The enzyme reaction was started at a temperature of 30 ° C. with a volume of 00 μl. In addition, the addition amount of the cellulose hydrolase having the above concentration is 0 to
The range was 125 μl. And after starting the reaction,
The absorbance at a wavelength of 550 nm (optical path length 1 cm) was continuously measured, and the relationship between the reaction time (seconds) and the absorbance was determined. The results shown in FIG. 2 were obtained.

As shown in FIG. 2, a clear positive correlation is observed between the reaction time and the absorbance, and
As the amount of cellulose hydrolase added increases,
The absorbance also increases. Therefore, the produced reducing terminal group can be accurately quantified by adjusting the addition amount of the cellulose hydrolase.

Comparative Example 1 700 μl of purified water was added to an aqueous solution containing 100 μl each of a 500 mM sodium acetate buffer solution (pH 4.2) and a 0.25% aqueous solution of a reduced cellooligosaccharide, and the resulting mixed solution had a concentration of 0. 100 μl of 4 mg / ml cellulose hydrolase (Cellulase Onozuka R-10, manufactured by Yakult Co., Ltd.) was added, and the enzyme reaction was performed at a temperature of 30 ° C. for 1 minute. After completion of the reaction, the amount of reducing end group produced was measured by the Nelson-Somogyi method, and the detection sensitivity was low.Therefore, no difference from the blank containing no cellulase was observed, and the activity of cellulase can be quantified. could not.

Comparative Example 2 500 mM sodium acetate buffer (pH 4.2) 100
μl and 1% carboxymethylcellulose (degree of substitution 0.63) aqueous solution of 500 μl, 0.5 mg /
ml of cellulose hydrolase (Cellulase Onozuka R
-10, 400 μl of Yakult Co., Ltd. was added, and the enzyme reaction was carried out at a reaction temperature of 30 ° C. for 10 minutes. After the reaction was completed, the amount of reducing end groups produced was measured by the Nelson-Somogyi method, and the amount of reducing end groups produced was 1
It was calculated to be 99 μM per minute, and the amount produced per 1 mg of cellulase was 495 μM. At the measurement wavelength of 660 nm of the absorbance, the change per absorbance of 0.05 corresponded to the production amount of reducing end groups of 170 μM. Therefore, the sensitivity (reducing end group production amount 17
0 μM / absorbance 0.05) is the sensitivity in the method of Example 1 (reducing end group production amount 1.1 μM / absorbance 0.0
It is about 1/100 of that of 5).

Comparative Example 3 500 mM sodium acetate buffer (pH 4.2) 100
μl and 500 μl of 1% Avicel suspension in water,
400 μl of 0.5 mg / ml cellulose hydrolase (Cellulase Onozuka R-10, manufactured by Yakult Co., Ltd.) was added, and the enzyme reaction was carried out at a reaction temperature of 30 ° C. for 60 minutes. When the amount of reducing end groups produced was quantified by the Nelson-Somogi method, the amount of reducing end groups produced was calculated to be 5.5 μM per minute, and 27.5 per mg of cellulase.
μM reducing end groups were generated.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the absorbance at a wavelength of 550 nm and the reaction time in Example 1.

FIG. 2 is a graph showing the relationship between the absorbance at a wavelength of 550 nm and the reaction time in Example 3.

Claims (11)

[Claims] 1. A method for measuring the activity of a cellulose hydrolase, which detects changes caused by the reaction between a reducing group produced from a cellulose substrate by the action of a cellulose hydrolase and cellobiose dehydrogenase. 2. A change caused by a redox reaction between a reducing group generated from a cellulose substrate and cellobiose dehydrogenase is detected as a spectroscopic or electrochemical change of an electron acceptor existing in the reaction system. The activity measuring method according to claim 1. 3. The activity measuring method according to claim 1, wherein the cellulose substrate is water-soluble cellulose or a derivative thereof. 4. The activity measuring method according to claim 1, wherein the cellulose substrate is cellooligosaccharide or a derivative thereof. 5. The activity measuring method according to claim 1, wherein a cellulose substrate having a reducing terminal group subjected to a reduction treatment is used as the cellulose substrate. 6. The water-soluble cellooligosaccharide whose reducing end group is subjected to reduction treatment is used as the cellulose substrate.
The method for measuring activity described. 7. cellobiose dehydrogenase, Fanerokite (Phanerochaete) genus, sports Lot requeue arm (Sporo
Trichume) genus Monilia (Monilia) genus and form scan (Fomes) according to claim 1 or 2 activity measurement method described kind of microorganisms at least member selected from the genus is cellobiose dehydrogenase produced. 8. The cellobiose dehydrogenase is Phanerochaete chrysospor.
3. The activity measuring method according to claim 1 or 2, which is a cellobiose dehydrogenase produced by ( ium ). 9. The activity measuring method according to claim 2, wherein the electron acceptor is a trivalent iron compound. 10. A component produced by a redox reaction between a water-soluble cellooligosaccharide reduced in the presence of a compound containing heme iron and a cellobiose dehydrogenase produced by Phanerochaete chrysosporium. A method in which the optical change is measured and used as an index of the activity of cellulose hydrolase. 11. A kit for measuring the activity of a cellulose hydrolase, which comprises a cellulose substrate, cellobiose dehydrogenase and an electron acceptor.