JPH05236985A - Cellulase-inhibiting agent and its productiohn - Google Patents

Abstract

PURPOSE:To provide a new cellulase-inhibiting agent useful in the biomass industry, fiber industry and food industry and useful for controlling injurious pests such as termites because of strongly inhibiting various kinds of the cellulases. CONSTITUTION:A 1-deoxynojirimuycin.cellooligosaccharide of formula I (n is 0 or an integer). The compound is obtained by treating a cellooligosaccharide of formula II (preferably a water-soluble cellooligosaccharide having a polymerization degree of 3-7, such as cellotriol) and 1-deoxynojirimycin with a cellulase (preferably cellulase produced by trichoderma.viride). The reaction is preferably carried out in dimethylsulfoxide. When the cellulase originated from the genus Trichoderma is used as the cellulase, the reaction temperature is preferably about 37 deg.C and the pH is preferably approximately 5.

Description

Detailed Description of the Invention

[0001]

The present invention relates to the following general formula (1)

[0002]

[Chemical 7]

(Where n is 0 or an integer) and the following general formula (2):

[0004]

[Chemical 8]

The present invention relates to a novel cellulase inhibitor represented by (n is 0 or an integer) and a method for producing the inhibitor. More specifically, 1-deoxynojirimycin / cellooligosaccharide in which 1-deoxynojirimycin is β-1,4 linked to cellooligosaccharide (hereinafter collectively referred to as inhibitor group A) and β-1,6 linked 1 -Deoxynojirimycin / cellooligosaccharide (hereinafter collectively referred to as inhibitor B group) and cellulase transglycosylation reaction are used to select the inhibitor A group or inhibitor B group from cellooligosaccharide and 1-deoxynojirimycin. It relates to a method of manufacturing.

The cellulase inhibitor obtained by the present invention strongly inhibits various cellulases and is used for controlling pests such as biomass industry, textile industry, food industry and termites. That is. In the biomass industry, it has been attempted to make cellulase act on natural cellulose to convert it into alcohol or other fuel compounds, chemical industrial fuel, microbial protein, etc., but in the process, use the cellulase inhibitor of the present invention. The cellulase action can be appropriately controlled by. In the textile industry, for example, the treatment strength can be controlled by using the inhibitor of the present invention in a method of making cellulase act on a cellulosic fibrous material to improve its texture. Further, in the food industry, by using the inhibitor of the present invention in a method for producing a food containing fiber by subjecting a food having natural fiber to a cellulase treatment, its action can be controlled, and Food can be produced. In addition, termites are said to be capable of degrading cellulose such as wood by taking in cellulase as a nutrient by cellulase of a protozoan symbiotic in the body, and by applying the inhibitor of the present invention, it is applied to the control of termites It is also possible.

[0007]

2. Description of the Related Art Various substances have been reported as cellulase inhibitors. That is, various phenol compounds such as p-coumaric acid, ferulic acid, vanillin or tannic acid, leucoanthocyanin, N-bromosuccinimide, etc., and glucose, ethanol, etc. are known. They are irreversible inhibitors, and also cellulase inhibitors in Aspergillus terreus wheat bran culture [Can. J. Microbio
L., Vol. 27, No. 12, 1334-1340 (1981)], an inhibitor derived from plant leaves [Siol Biol. Biochem., Vol. 19, 719-725].
(1987)], but they have not been isolated and purified, and their detailed properties are not known.

[0008]

Cellulase is known as an enzyme that hydrolyzes cellulose into glucose,
It is distributed throughout the living world such as protozoa, nematodes, molluscs, bacteria, fungi, and higher plants. Ordinary cellulases are endo-type enzymes (endo-1,4 classified as EC 3.2.1.4)
-Β-D-glucan glucanohydrolase, CM-cellulase, C _X ), exo-type enzyme (exo-1,4-β-D-glucan cellobiohydrolase, avicelase, C ₁ classified as EC 3.2.1.91) , Β-glucosidase (cellobiase classified as EC3.2.1.21) and the like. It is believed that these three enzymes synergize with each other to degrade natural cellulose.

In particular, cellulases produced by microorganisms are industrially used. For example, Tricoderma (Tric
hoderma), Aspergillus, Pellicularia, Penicillium
llium), Acremonium (Acremonium) genus and many of the filamentous fungi. However, the properties of the cellulases produced are diverse, as mentioned above.

The actions of various inhibitors reported so far on such various cellulases have not been very satisfactory. That is, the action of the conventional inhibitor was not uniform due to the origin of the enzyme, and its inhibitory activity was not sufficient.

[0011]

Therefore, the present inventors have replaced conventional inhibitors with low molecular weight substances that bind to the active site of an enzyme-that they are substrate analogs-that have a high inhibitory specificity. We conducted diligent research for the purpose of creating a new inhibitor that combines both points.

That is, the present inventors have the following general formula (1):

[0013]

[Chemical 9]

(Where n is 0 or an integer) and the following general formula (2):

[0015]

[Chemical 10]

The inventors have created an inhibitor group A and an inhibitor group B represented by (wherein n is 0 or an integer), and found that this substance is a cellulase inhibitor satisfying the above-mentioned object. completed.

1-deoxynojirimycin (hereinafter SG
I) is known as a substance having a strong inhibitory effect on α, β-glucosidase, glucoamylase and the like. The present inventor succeeded in site-specifically adding this SGI to cellooligosaccharides using cellulase. That is, the general formula (3)

[0018]

[Chemical 11]

A cellulase having the ability to catalyze a transglycosylation reaction is allowed to act on the cellooligosaccharide represented by (where n represents 0 or an integer) and SGI to give a compound represented by the general formula (1).

[0020]

[Chemical 12]

(Where n is 0 or an integer) or the general formula (2)

[0022]

[Chemical 13]

The inhibitor group A or the inhibitor group B represented by (wherein n is 0 or an integer) can be produced.

The cellulase which can be used in the present invention may be of any origin as long as it has the ability to catalyze a glycosyl transfer reaction, but the cellulase produced by the genus Trichoderma, especially Trichoderma viride (Tric
Cellulase produced by hoderma viride) can be preferably used. Specific examples thereof include cellulase T "Amano" (trade name: manufactured by Amano Pharmaceutical Co., Ltd.) and macerase (trade name: manufactured by Meiji Seika).

The cellooligosaccharide represented by the general formula (3), which is a substrate for the reaction, can be prepared by, for example, the method of Sakamoto et al. [Methods in Car
bohydrate Chemistry, Vol. 3, p. 134 (1963)]. Furthermore, the method described in Japanese Patent Publication No. 1-256394 can be applied.

As the cellooligosaccharides, various cellooligos obtained by the above-mentioned method can be used, and in particular, water-soluble cellooligosaccharides having a degree of polymerization of 3 to 7 such as cellotriose, cellotetraose, cellopentaose, cellohexaose and celloheptaose. Sugar is preferably used. These may be purified so long as they can be used in the present invention, and may be a mixture.

The SGI is, for example, the method of Murao et al. [Agric. B
iol. Chem., 44, 219 (1980)].

The reaction is carried out in various solvents. For example,
In an aqueous solution, in a mixture of various organic solvents and water. More preferably, dimethyl sulfoxide (hereinafter referred to as DMS
It is a method of reacting in 30%.

The reaction temperature or reaction pH may be any temperature that is preferable for the transglycosylation activity of the cellulase used, but if it is derived from the genus Trichoderma, it is around 37 ° C. and around pH 5.0.

The cellulase concentration to be used may be any concentration as long as it exerts its transglycosylation activity, and varies depending on the degree of purification of the cellulase used, but 0.1-10% is used.

The present invention will be described in detail below with reference to experimental examples and examples, but the present invention is not limited thereto.
The reagents and measuring methods used in the following experimental examples and examples are as follows, unless otherwise specified.

As cellulase for assaying inhibitory activity, Aspergillus aculeatu (Aspergillus aculeatu)
s) cellulase (hereinafter referred to as FI-CMCase) produced according to the method of Murao et al. [Method in Enzy
mol., 160 volumes, 274-299 (1988)].

A cellulase solution was prepared by measuring FI-CMCase in 100 mM acetate buffer (pH 5.0) to a concentration of 80 munits / ml. Substrate solution is carboxymethyl cellulose (Wako Pure Chemical Industries, substitution degree
0.65, average degree of polymerization 500), 100 mM acetate buffer (pH 5.0)
The one dissolved in 1.0% (W / V) was used.

0.25 ml of cellulase solution and 0.25 ml of inhibitor solution
After adding ml and letting it work at 37 ℃ for 10 minutes, 0.5 ml of substrate solution
Is added and the enzyme reaction is carried out at 37 ° C for 10 minutes. Then, the reaction is stopped by adding 1 ml of an alkaline copper solution. This is tightly closed, heated in a boiling water bath for 15 minutes, and then cooled in water for 3 minutes. Further, 1 ml of Nelson's reagent is added, color is developed for 20 minutes, and the volume is adjusted to 10 ml, and the absorbance (T) at 500 nm is measured.

As a standard, 0.25 ml of distilled water was added instead of the inhibitor solution, and the same operation was performed to measure the absorbance (S).

As a control, the substrate solution and the alkali copper solution were added in reverse order and the same operation was performed, and then the absorbance (C) was measured.

As a blank, instead of the inhibitor solution,
Distilled water (0.25 ml) is added, and the absorbance (B) is measured in the same manner as the control measurement.

The inhibition rate was determined according to the following formula, and one unit of the inhibitory activity of the inhibitor was the amount of the inhibitor showing a 50% inhibition rate. Inhibition rate (%) = [(SB)-(TC)] / (S-
B) x 100

Cellulase derived from Trichoderma viride (hereinafter referred to as cellulase T) was used as the cellulase used in the enzyme synthesis.

SGI was prepared according to the method of Murao et al.

The cellooligosaccharide mixture was prepared according to the method of Sakamoto et al.

Experimental Example 1 Effect of type of substrate 10% various cellooligosaccharides, 2% SGI, 1% cellulase T were reacted in 100 mM acetate buffer (pH 5.0) containing 50% DMSO at 37 ° C. for 24 hours. Let The reaction solution was heated in a boiling water bath for 15 minutes to stop the reaction, and then the inhibitory activity was examined.

The cellooligosaccharides include cellobiose, cellotriose, cellotetraose, cellopentaose,
Water-soluble cellooligosaccharides and insoluble cellooligosaccharides of cellohexaose and celloheptaose were used. The results are shown in Table 1.

[0044]

[Table 1]

No inhibitory activity was observed when cellobiose or insoluble cellooligosaccharide was used as the substrate. In the case of insoluble cellooligosaccharide, the reaction product may have been removed from the reaction solution as a precipitate. In the case of cellobiose, it is not suitable as a substrate for the enzyme involved in glycosyl transfer, or the product is FI-C
Probably because it does not inhibit MCase.

Experimental Example 2 Effect of Solvent (1) 10% cellooligosaccharide mixture in a mixed solvent prepared by mixing various solvents with 100 mM acetate buffer (pH 5.0) at a ratio of 1: 1 or 3: 7. Add 2% SGI, 1% cellulase T, 37
Perform the reaction for 24 hours at ℃. The inhibitory activity of the produced inhibitor was determined. The inhibitory activity was determined as the number of units of the inhibitor produced from 100 μg of SGI. The results are shown in Table 2.

[0047]

[Table 2]

It was most effective when carried out in an acetate buffer containing 30% DMSO. Then 30% methanol was good. Further, a high inhibitory activity was observed even in the acetate buffer itself containing no organic solvent.

Experimental Example 3 Effect of Solvent (2) From the results of Experimental Example 2, it was decided to use a mixed solvent system containing DMSO as the solvent, and subsequently DM for inhibitor production.
The effect of SO concentration was investigated. 100 mM acetate buffer (pH 5.0)
In DMSO at various mixing ratios,
A 10% cellooligosaccharide mixture, 2% SGI, and 1% cellulase T were added, and the mixture was reacted at 37 ° C. for 48 hours, and the change in inhibitory activity with time was examined. The result is shown in FIG. Inhibitor production was most efficient in solvents containing 30% DMSO throughout the reaction. Moreover, the inhibitory activity is considered to have reached the maximum in about 48 hours.

Experimental Example 4 Effect of Reaction Temperature From the results of Experimental Example 3, 30% DMSO was used as the reaction solvent.
The production amount of the inhibitor at each temperature was examined by setting to use a mixed solvent system containing 100m including 30% DMSO
10% cellooligosaccharide mixture in M acetate buffer (pH 5.0), 2
The reaction was performed using% SGI, 1% cellulase T. The change in inhibitory activity with time was examined. The result is shown in Figure 2.
Shown in. The inhibitory activity was highest at 37 ° C. In the reaction at 50 ° C, it is considered that the hydrolysis reaction is progressing, as inferred from the decrease in the viscosity of the reaction solution.

Experimental Example 5 Effect of pH 10% cellooligosaccharide mixture, 2% SGI and 1% cellulase T were added to buffers of various pH containing 30% DMSO and the reaction was carried out at 37 ° C. The changes with time were examined. The result is shown in FIG. The buffer used is pH
3.0; sodium acetate-HCl buffer, pH 4.0, pH 5.0; sodium acetate-acetate buffer, pH 6.0; sodium phosphate buffer. The maximum amount of inhibitor produced was at pH 5.0.

Experimental Example 6 Effect of enzyme concentration In a reaction at 37 ° C. using a 10% cellooligosaccharide mixture and 2% SGI in an acetate buffer (pH 5.0) containing 30% DMSO,
The enzyme concentration was changed to examine the increase in each inhibitor activity over time. The result is shown in FIG. The amount of inhibitor produced was highest at 2.5% Cellulase T. When the enzyme concentration was increased to 4.0%, it is possible that the hydrolysis proceeded too much and the substrate for the transfer reaction decreased.

As a result of the examination of the reaction conditions of Experimental Examples 1 to 5, the following optimum reaction conditions were set. The inhibitory activity produced under these optimal conditions is 110 units / m
l became the reaction solution.

Cellooligosaccharide mixture 10% SGI 2% Cellulase T 2.5% 30% DMSO-containing 100 mM acetate buffer (pH 5.0), 37 ° C., 48
React on time.

Experimental Example 7 Inhibition obtained under the above optimum conditions
Adsorption of the agent to the resin In order to confirm whether or not the obtained inhibitor is due to the neutral sugar formed by the hydrolysis of cellooligosaccharide, the adsorption to the ion exchange resin was examined. First Dowex 50W X4
When the adsorptivity to (H ⁺ -form) was examined, the inhibitory activity was found in the adsorbed fraction. Then Dowex 1 X8 (OH
^- The inhibitory activity was examined for the adsorption against -form) was found to be present in the non-adsorbed fraction. Further SGI
It does not inhibit cellulase by itself. Therefore, it was determined that this inhibitory substance was a reaction product derived from SGI.

[0056]

Example 1 Production and Purification of Inhibitor Cellooligosaccharide Mixture 5.0 g SGI 1.0 g Cellulase T 1.3 g 100 mM Acetate Buffer (pH 5.0) (containing 30% DMSO) 50 ml Reaction at 37 ° C. for 48 hours After stopping the reaction by boiling for 15 minutes in a boiling water bath, the supernatant was purified as follows.

Column packings for inhibitor purification include Dowex
50W X4, Dowex 1 X8 (Dow Chemical Company), Bio-Gel P-2
(Bio-Rad) using Whatman 3MM chr. Paper (Whatman) for preparative paper chromatography and Kieselgel 60 F254 (Merck) for thin layer chromatography.
Company) was used.

The following reagents were used for detection in paper and thin layer chromatography. 200 ml of acetone is added to 1 ml of an aqueous solution of AgNO ₃ saturated with an alkaline-silver nitrate reagent , and the resulting precipitate is dissolved by adding water (about 5 ml). This solution is sprayed on filter paper or a thin layer to dry it, and sprayed with 0.5N NaOH-ethanol solution. When the spot becomes clear, 5% Na ₂ S is added.
Soak in ₂ O ₃ and wash.

Vanillin-sulfuric acid reagent 0.6 g of vanillin is dissolved in 20 ml of ethanol, and 0.6 ml of concentrated sulfuric acid is further added to this solution. Spray this solution into a thin layer 1
Heat at 00-110 ° C for a few minutes.

Step A Dowex 50W X4 (H ⁺ -form)
Dowex 50W X4 column (H ⁺ -form, 1.5 x 18 cm) in which 50 ml of the reaction supernatant, which is the starting material for the ram chromatography purification, was washed with distilled water beforehand.
It was adsorbed on and washed with 300 ml of distilled water and then eluted with 300 ml of 0.5N ammonia water. The eluate was fractionated into 10 ml and collected.

The inhibitory activity was observed in the adsorbed fraction, and about 200 ml of the active fraction was concentrated with an evaporator to obtain 2.2 g of brown syrup.
Got

Step B Dowex 1 X8 (OH ^-- form) Color
Chromatography Step A The syrup obtained in Step A was dissolved in 10 ml of distilled water, and the Dowex 1 X8 column (OH
^- -form, was added to 1.5 × 10 cm), eluted with distilled water 200 ml. Inhibitory activity was found in non-adsorbed fraction, active fraction 150 ml
Was concentrated with an evaporator to obtain 1.8 g of brown syrup.

Step C Gel filtration with Bio-Gel P-2 Bio-Gel P-2 (2.5%) prepared by dissolving the syrup obtained in Step B in 3 ml of 10% pyridine solution and previously equilibrated with 10% pyridine solution. It was added to a column of × 90 cm) and gel filtration was performed. The same 10% pyridine solution was used as the developing solution, and the eluate was fractionated into 5 ml and collected.

Inhibitory activity is divided into four categories (FI, F-II, F-III
And F-IV). That is, it was found that there are at least four types of inhibitors having different molecular weights. Of these, the activity was low in the FI and F-II fractions, and the TLC was analyzed by n-butanol:
Ethanol: chloroform: 28% ammonia water = 4:
As a result of carrying out with a developing solvent of 5: 2: 8, it was confirmed that the mixture was a small amount and a larger amount of the inhibitor. Therefore, purification of F-III and F-IV was proceeded thereafter.

Each fraction was concentrated using an evaporator to obtain a syrupy substance (41 mg for F-III fraction and 75 mg for F-IV fraction).

Step D According to Whatman 3MM chr. Paper
Preparative Paper Chromatography F-III and F-IV obtained in Step C are each dissolved in a minimum amount of distilled water, and Whatman 3MM chr. Paper (20 × 40 cm)
Preparative paper chromatography was performed. N-Butanol: pyridine: water = 6: 4: 3 was used as the developing solvent, and the solvent was developed by the multiple developing method.

A part of the developed paper was developed with an alkaline-silver nitrate solution, and the corresponding parts were extracted with distilled water. As a result, two types from F-III, F
Since two kinds of active substances were obtained from -IV, the one with the larger Rf value in each paper chromatography was F-II.
The smaller ones were designated as I-1, F-IV-1 and F-III-2, F-IV-2.
Remove the solvent from each using an evaporator and use F-II
I-1 14.7 mg, F-III-2 12.2 mg, F-IV-1 43.3 mg, F-IV
16.4 mg of -2 was obtained.

Step E Dowex 1 X8 (OH ^-- form) Color
The respective fractions obtained in Step D of Chromatography were dissolved in 1 ml of distilled water and added to a Dowex 1 X8 column (OH ^-- form, 1.5 x 5 cm) which had been washed with distilled water in advance, and 40 ml of distilled water was added. It was eluted. The solvent was removed from each using an evaporator, and F-III-1 was 8.6 mg, F-III-2 was 7.0 mg, and F-IV-1 was 32.0 m.
10.4 mg of g and F-IV-2 was obtained.

The homogeneity of the purified inhibitors was investigated by thin layer chromatography (TLC) on silica gel.

Experimental method As a carrier for TLC, commercially available Kieselgel 60F plate (M
erck) was used. As the developing solvent, 2 of the following composition
It was developed at room temperature using two solvent systems. A: Chloroform: Methanol: 18.7% ammonia water =
4: 5: 3 B: n-butanol: ethanol: chloroform: 28
% Ammonia water = 4: 5: 2: 8

As the coloring method, a vanillin-sulfuric acid coloring method and an alkaline-silver nitrate coloring method were used. As a result, it was recognized as a single spot in both solvent systems.

Example 2 Structure Determination of Various Inhibitors Obtained in Example 1 Analysis by Gas Chromatography (GLC) Experimental Method 200 μg of each inhibitor was dissolved in 1 ml of 2N-HCl and hydrolyzed at 100 ° C. for 2 hours. It was disassembled. After removing the solvent by vacuum concentration, the method of Fukuhara et al. [Agric. Biol. Chem., 46, 2021-
2030 (1982)] and trimethylsilylation (TMS
), And sugar and SGI as GL as TMS derivative
Analyzed in C. For TMS conversion, 200 μl of a mixed solution (trimethylchlorosilane: hexamethylsilazane: N, O-bis (trimethylsilyl) acetamide: pyridine = 1: 2: 2: 10) was added to the dried sample and dissolved, followed by vigorous stirring for 30 seconds. After that, it was heated at 80 ° C. for 30 minutes. The solution was used as a sample for GLC. Also, for the purpose of assaying the composition ratio, the molar ratio of glucose and SGI is glucose / SGI = 1,
Samples of 2, 3, and 4 were prepared, and this was converted into TMS to obtain G
Analyzed by LC.

As the GLC, a Hitachi G3000 gas chromatograph equipped with a hydrogen flame ion detector (FID) was used. The column was a stainless steel column having a diameter of 2.2 mm × 220 cm and 4% SE-52 coated Chromosolve W (60- 80 mesh) was used. Column temperature is 140
Starting from ° C, the temperature was raised at a rate of 5 ° C per minute, and finally heated to 190 ° C. Nitrogen gas was used as the carrier gas, and the flow rate was 30 ml / min. The analysis results of each inhibitor in GLC are shown in FIG. 5, FIG. 6, and FIG.
And shown in FIG.

In all the inhibitors, the peaks considered to be glucose and SGI were confirmed from the retention times, and no other peaks were confirmed.

Further, FIG. 9 is a plot of the molar ratio and the peak area ratio of FID for samples prepared with various mixing ratios of glucose and SGI in order to investigate their composition ratio. Table 3 shows the results of obtaining the molar ratio of each inhibitor from this calibration curve.

[0076]

[Table 3]

As described above, it was revealed that all the inhibitors were composed of glucose and SGI, and the molar ratio thereof was 1 to 3.

The reducing terminal residue glucose of the inhibitor was positive for Somogy Nelson's reagent, S
GI is negative. Taking advantage of this difference in properties, we examined what the reducing end residue is. Experimental method 20 μg of each inhibitor was dissolved in 1 ml of distilled water, and the reducing sugar amount was colorimetrically determined by the Somogy Nelson method using glucose as a standard substance.

As a result, all four inhibitors were dominated
Nelson reaction was negative. From this, it is considered that all the inhibitors have SGI at their reducing ends.

For cellulase inhibitor β-glucosidase
The decomposed glucose and SGI have a molar ratio of 2: 1 or 3: 1.
The three inhibitors, F-III-1, F-III-2 and F-IV-2, are decomposed by β-glucosidase, and what kind of decomposition product is produced Attempts were made at regular intervals to identify by TLC.

Experimental Method 200 μg of each inhibitor was dissolved in 50 μl of distilled water, and
50 μl of almond β-glucosidase (manufactured by Sigma) dissolved in 00 mM acetate buffer (pH 5.0) at a concentration of 1 mg / ml.
And at 37 ℃ for 26 hours (F-III -2) or 12 hours (F-III
-1, F-IV-2) reaction was performed. A part of the reaction solution was taken and heat-treated (100 ° C, 15 minutes), and then subjected to TLC analysis.
TLC analysis was performed using Kieselgel 60F254 (Merck) as a carrier.
As a developing solvent, n-butanol: ethanol: chloroform: 28% aqueous ammonia = 4: 5: 2:
8 was used. The color was developed by vanillin sulfate color development. In addition,
Glucose, F-IV-1, F-IV-2 and F-I as standards
II-1 was deployed at the same time.

The results of the decomposition of F-III-2 by β-glucosidase are shown in FIG. Judging from the Rf value 4 hours after the start of the reaction, spots capable of identifying glucose and F-IV-2 were detected. As the reaction proceeded further, some spots were observed in which glucose was further removed from F-IV-2, but this did not match the Rf value with any of the inhibitors isolated this time. The reaction appeared to be almost gone between 14 and 26 hours. This is a small amount of SGI that was released after the unidentified substance was further decomposed.
It is considered that this is because β-glucosidase was inhibited by Since β-glucosidase is an enzyme that releases glucose from the non-reducing end side, these results indicate that F-III-2 has one glucose residue on the non-reducing end side of F-IV-2, β-glucoside. It is considered that they are connected by a bond. In addition, the results of the decomposition of F-IV-2 by β-glucosidase are shown in FIG. Similar to the case of F-III-2, spots of glucose and unidentified substance were detected from F-IV-2 by the decomposition reaction with β-glucosidase. From this, it is considered that F-IV-2 also forms a partial structure of F-III-2.

The results of the decomposition of F-III-1 by β-glucosidase are shown in FIG. Six hours after the start of the reaction, spots of decomposition products of β-glucosidase were detected. The Rf values of glucose and F-IV-1 in the developing solvent used were very close to each other, and it was difficult to say clearly because a good developing solvent that could separate them was not found. Since they are different, this degradation product is considered to be glucose and F-IV-1. Therefore,
F-III-1 is considered to have one glucose residue linked to the non-reducing end of F-IV-1 by a β-glucoside bond.

Structural analysis experimental method by NMR ¹ H and ¹³ C-NMR were analyzed using TSP (Trimethylsily) as an internal standard.
lpropanesulfonic acid) and added to JEOL G in D ₂ O.
It was measured using X-270. The respective signals were assigned by comparison with the chemical shifts of glucose and SGI.

[0085]

[Table 4]

Then, the changes in these chemical shifts were determined by changing the chemical shift values of various glucooligosaccharides [J. Chem.
Soc., 2425-2432 (1973)], F-IV-1 G- (1 → 4) -SGI F-IV-2 G- (1 → 4) -G- (1 → 6) -SGI F-III-1 G- (1 → 4) -G- (1 → 4) -SGI (G: glucose) is considered to be composed of a glucoside bond in a binding mode. In addition, ¹ H- of F-III-1, F-IV-1 and F-IV-2
The chemical shift and the coupling constant of an anomeric proton in an NMR spectrum are shown.

[0087]

[Table 5]

When these values were compared with the binding constants of various glucooligosaccharides [Cabohyd. Res., 33, 105-116 (1974)], the glucoside bonds constituting these inhibitors were all β bonds. It is believed that

The putative structures of the respective inhibitors, which are considered based on the findings obtained so far, are shown in Chemical formulas 14 to 16. F-III-2
Was not analyzed by NMR, but considering that it was decomposed into F-IV-2 and glucose by β-glucosidase and the structure of other inhibitors, SGI was found at the reducing end of cellooligosaccharides of various sizes. Considering that they are added, it is considered that they have the structure shown in Chemical formula 17.

[0090]

[Chemical 14]

[0091]

[Chemical 15]

[0092]

[Chemical 16]

[0093]

[Chemical 17]

Example 3 Inhibitory Effect of Inhibitors on Cellulase The inhibition constant was measured by adding 0.5 ml of the substrate solution to 0.5 ml of the enzyme solution.
The amount of reducing sugar released when the reaction was carried out at 37 ° C. for 10 minutes was measured by the Somogy Nelson method, and glucose was calculated from a standard curve obtained by coloring glucose in the same system. One unit of enzyme activity was defined as the amount of enzyme that released 1 μmol glucose per minute. In addition, the ultraviolet differential absorption spectrum generated by the binding of cellulase and F-III-1 was measured using Hitachi 320 type Spectrophotometer.

The enzyme Carb was reacted with four kinds of inhibitor solutions.
The degree of inhibition of oxymethyl cellulose (hereinafter referred to as CMC) decomposition activity was determined. The enzyme solution is 100 mM acetate buffer (p
It was prepared in H5.0) to a concentration of 80 milliunits / ml. As the inhibitor solution, a solution dissolved in distilled water at a concentration of 4.0 × 10 ⁻⁴ M was used. CMC 100m as a substrate
A solution of 1.0% (W / V) in the same buffer was used. After adding 0.25 ml of the inhibitor solution to 0.25 ml of the enzyme solution and pre-incubating at 37 ° C for 10 minutes, 0.5 ml of the substrate was added and reacted at 37 ° C for 10 minutes to measure the degree of inhibition. The results are shown in Table 6.

[0096]

[Table 6]

The degree of inhibition of FI-CMCase was highest in F-III-1, followed by F-III-2, F-IV-1, and F-IV-2 in that order.
This shows β-1,4 as well as the substrate specificity of cellulase.
It can be seen that it has a high affinity for those having a glucosidic bond. In addition, probably due to the endo-type of this enzyme, it had a high affinity for inhibitors with long glucose chains.

Inhibition constant of F-III-1 against FI-CMCase (K
i) to Dixon Plot [Biochem. J., 55, 170 (195
3)]. As a substrate solution, CMC dissolved in 100 mM acetate buffer at two concentrations of 1.0% and 1.8% was used. The enzyme solution and the measurement conditions were the same as described above. The results are shown in Fig. 13. From this graph, F-II
The inhibition constant of FI-CMCase of I-1 was determined to be 6.9 × 10 ⁻⁶ M.

Example 4 Inhibition Degree of Each Inhibitor against Degradation Activity Using CMC as a Substrate Experimental Method Enzymes (8 kinds) Fungus-derived enzyme a Aspergillus acretas FI-CMCase enzyme b Trichoderma viride Cellulase Ia ₂ enzyme c Cryptococcus・ Flavus CMCase Enzyme d Irpex ・ Lacteus Endo-type Cellul
ase Bacteria-derived enzyme e Bacillus subtilis Cellulase (BSC) enzyme f Bacillus sp. N-4 Cellulase (NK1) enzyme g Clostridium cellulolyticum Endo-β-
1,4-glucanase enzyme h Cellulomonas uda CB4 CMCase

With respect to various cellulases which are said to be CMC saccharified type and further endo type enzymes, the four kinds of the inhibitor solutions obtained in Example 1 were allowed to act to determine the degree of inhibition of CMCase activity of the enzyme. Each enzyme solution was prepared in an appropriate buffer so as to have a concentration of 80 milliunit / ml. As the inhibitor solution, one dissolved in distilled water at a concentration of 4.0 × 10 ⁻⁴ M was used. As a substrate, CMC dissolved in the same buffer solution of 100 mM at 1.0% (W / V) was used. Enzyme solution
After adding 0.25 ml of the inhibitor solution to 0.25 ml and pre-incubating at 37 ° C for 10 minutes, 0.5 ml of the substrate was added and reacted at 37 ° C for 10 minutes to measure the degree of inhibition. The results are shown in Table 7.

[0101]

[Table 7]

F-III-1 showed the strongest inhibitory activity against any cellulase. Inhibition by other inhibitors was different depending on each cellulase, but the enzyme d was F-IV-2.
It was not inhibited by those with β-1,6 bond such as F-III-2 and F-III-2. In addition, this cellulase and enzyme h
Was found to be less susceptible to inhibition by inhibitors than other cellulases.

Example 5 Inhibition Degree of Each Inhibitor against Degradation Activity Using Insoluble Cellooligosaccharide as Substrate Experimental Method Enzymes (Four Kinds ) Mold Derived Enzyme a Aspergillus acretas FI-CMCase Enzyme i Aspergillus acretus FI-Avicelase Enzyme j Aspergillus acretas FIII-Avicelase enzyme k Ilpex lacuteus Exo-type Cellula
se

Preparation of insoluble cellooligosaccharide As the insoluble cellooligosaccharide, the one prepared according to the above-mentioned method of Sakamoto (average degree of polymerization = 18) was used.

Inhibition on insoluble cellooligosaccharide degrading activity
Activity measurement 0.1 ml of each enzyme solution dissolved in an appropriate buffer
Insoluble cellooligosaccharides suspended in the same buffer at 1% concentration
0.1 ml was added and reacted at 37 ° C. for 10 minutes.

After stopping the reaction, 0.8 ml of the same buffer was added to make the total volume 2 ml, and thereafter, the measurement was carried out in the same manner as in the case of CMCase activity.

The enzyme solution was prepared so as to have a concentration of 240 milliunits / ml with each buffer solution. As the inhibitor solution, one dissolved in distilled water at a concentration of 4.0 × 10 ⁻⁴ M was used. In addition, enzyme a was also measured as a comparison with CMC saccharified cellulase. The results are shown in Table 8.

[0108]

[Table 8]

Each inhibitor against FI-CMCase showed a similar inhibitory balance to that of CMCase activity, and the inhibitory activity was higher than that when CMC was used as a substrate although the amount of added enzyme was higher. showed that. In contrast, the inhibitory effect of the exo-type enzyme is considerably different from that of the glycated CMC. Enzyme i and enzyme j are very unlikely to be inhibited by any inhibitor. The enzyme k has β-1,6 bond.
It was strongly inhibited by F-IV-2 and F-III-2. It is also possible that there is a considerable difference in the structure or degradation mechanism of the active site between CMC saccharified cellulase and Avicel saccharified cellulase.

[0110]

The cellulase inhibitor obtained by the present invention strongly inhibits various cellulases and is used for controlling pests such as biomass industry, textile industry, food industry and termites.

[Brief description of drawings]

FIG. 1 is a graph showing the results of Experimental Example 3, in which black circles show 10% concentration, white circles show 20% concentration, black triangles show 30% concentration, and white triangles show 40% concentration.

FIG. 2 is a graph showing the results of Experimental Example 4, in which the black circles indicate the results of 30 ° C. reaction, the white circles indicate the 37 ° C. reaction, and the white triangles indicate the 50 ° C. reaction.

FIG. 3 is a graph showing the results of Experimental Example 5, in which black circles represent pH 3.0, white circles represent pH 4.0, black triangles represent pH 5.0, and white triangles represent pH.
The result of 6.0 is shown.

FIG. 4 is a graph showing the results of Experimental Example 6, in which black circles are 1.0%, white circles are 2.5%, and white triangles are 4.0%.

FIG. 5 shows the results of gas chromatography in which the inhibitor F-III-1 was hydrolyzed and then used as a TMS derivative. In the figure, A indicates glucose and B indicates SGI absorption.

FIG. 6 shows the results of gas chromatography in which the inhibitor F-III-2 was hydrolyzed and then used as a TMS derivative. In the figure, A shows absorption of glucose and B shows absorption of SGI.

FIG. 7 shows the results of gas chromatography in which the inhibitor F-IV-1 was hydrolyzed and then used as a TMS derivative.
Indicates glucose and B indicates SGI absorption.

FIG. 8 shows the results of gas chromatography in which the inhibitor F-IV-2 was hydrolyzed and then used as a TMS derivative.
Indicates glucose and B indicates SGI absorption.

FIG. 9 is a graph in which the molar ratio and the peak area ratio of FID are plotted from the results of gas chromatography of samples prepared with various mixing ratios of glucose and SGI. In the figure, 1) is F-III-1, 2) is F-III-2, and 3) is
F-IV-1, 4) represents F-IV-2.

FIG. 10 shows a TCL pattern of degradation of the inhibitor F-III-2 by β-glucosidase and reaction time. In the figure, S1 is glucose, the upper spot of S2 is F-IV-1, and the lower spot is F-IV.
-2, S3 upper spot glucose, lower spot F-III-
Indicates 1. Moreover, 0, 6, and 12 show reaction time.

FIG. 11 shows a TCL pattern of degradation of the inhibitor F-IV-2 by β-glucosidase and reaction time. In the figure, S1 is glucose, S2 is F-IV-1, the upper spot of S3 is F-IV-1, and the lower spot is F-III-1. Moreover, 0, 6, and 12 show reaction time.

FIG. 12 shows the TCL pattern of the decomposition of the inhibitor F-III-1 by β-glucosidase and the reaction time. In the figure, S1 is glucose, the upper spot of S2 is F-IV-1, and the spot of Sita is F-
IV-2, S3 upper spot glucose, lower spot F-II
Indicates I-1. Moreover, 0, 6, and 12 show reaction time.

FIG. 13 shows the results of Dixon Plot for calculating the inhibition constant of F-III-1 for FI-CMCase. In the figure, the black circles indicate the CMC concentration of 1.0%, and the white circles indicate the CMC concentration of 1.8%.

Claims (4)

[Claims] 1. The following general formula (1): (However, n shows 0 or an integer.) 1-deoxynojirimycin cellooligosaccharide. 2. The following general formula (2): (However, n shows 0 or an integer.) 1-deoxynojirimycin cellooligosaccharide. 3. Cellulase is represented by the following general formula (3): (However, n represents 0 or an integer.) It is reacted with 1-deoxynojirimycin and a cellooligosaccharide represented by the general formula (1) (However, n represents 0 or an integer.) A method for producing 1-deoxynojirimycin cellooligosaccharide. 4. Cellulase is represented by the following general formula (3): (However, n represents 0 or an integer.) The compound is reacted with 1-deoxynojirimycin and a cellooligosaccharide represented by the general formula (2): (However, n represents 0 or an integer.) A method for producing 1-deoxynojirimycin cellooligosaccharide.