JPH0638747A - Cellulase having fiber-disaggregating activity, standard sample of the enzyme and its production - Google Patents

Abstract

PURPOSE:To provide a new cellulase having specific properties, useful for the enzymatic saccharification of cellulosic substances and the enzymatic treatment of paper pulp in a paper pulp-producing process, and having an effect to accelerate the fiber-disaggregating activity. CONSTITUTION:The objective cellulase A has following properties; e.g. (A) action: (i) the production of cellotriose, cellobiose, and glucose from an insoluble cellooligosaccharide, (ii) the production of water-soluble saccharides and water- insoluble saccharides from reduced phosphoric acid-swollen cellulose, and (iii)the acceleration of the fiber-disaggregating activity; (B) substrate specificity: exhibits a strong activity to CM-cellulose, and hydrolyzes the insoluble cellooligosaccharides, the phosphoric acid-swollen cellulose, cellotetraose, and cellopentaose; (C) optimal pH, pH stability: the optimal pH is 5, and the cellulase A is stable at a pH of 2-5, when CM-cellulose is used as a substrate; (D) optimal temperature, thermal stability; the optimal temperature is approximately 70 deg.C, and stable up to 60 deg.C, when the CM-cellulose is used as the substrate; (E) mol.wt.: approximately 53000 by a SDS polyacrylamide gel...slab phoresis method. The cellulase is obtained by culturing Trichoderma SP.SK-1919.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a cellulolytic enzyme. Specifically, it is an enzyme purified and purified from a culture solution of a microorganism capable of producing a defibrated composition with high industrial utility, and recovers the saccharification industry and paper products made from lignocellulose such as wood and straw. The present invention relates to a cellulolytic enzyme used in the paper pulp industry such as used paper recycling treatment and paper pulp production using lignocellulosic materials such as timber and straw as raw materials, and fiber industry such as cotton modification, and a method for producing the same.

[0002]

2. Description of the Related Art Cellulase is a general term for enzymes that hydrolyze cellulose, which is β-1,4-glucan, and is composed of a group of enzymes having various enzyme actions and different protein chemical properties. However, the conventionally known cellulase has a weak saccharification ability, and thus the enzymatic saccharification of a cellulosic substance has not been able to exhibit industrial superiority over the starch saccharification. Further, although the use of cellulase has recently been promoted in the pulp and paper manufacturing industry, in this industry, the decrease in pulp yield hinders the use of cellulase.

[0003]

SUMMARY OF THE INVENTION Therefore, the object of the present invention is industrially useful because it can exert an effective effect on enzymatic saccharification of a cellulosic substance and enzyme treatment in a paper pulp manufacturing process, which has been difficult in the prior art. To provide a novel cellulase.

[0004]

[Means for Solving Problems] In order to break through the present situation, the present inventors have already developed the microorganism Trichoderma sp. S.
Japanese Patent Application No. 1-294469 and Japanese Patent Application No. 294469 show that the culture of the K-1919 strain (Microtech Lab. No. 11054) has a high action of pulping newspaper or copy paper into pieces, that is, defibration activity. -306772. The defibration composition which is this culture has a characteristic that it shows a high defibration activity despite its low CM-cellulase and xylanase activities. Therefore, it is considered that the composition is a completely new composition that can be pulped under the condition that the frequency of hydrolyzing the carbohydrate of the cellulosic material such as pulp is low. Therefore, the SK
As a result of diligent research on the cellulase system of strain -1919, highly purified four kinds of cellulases A, B, C, and D collected from the culture obtained by culturing the strain have the property of promoting defibration activity. It was found that the present invention was completed.

That is, the present invention provides novel cellulases A, B, C and D. The present invention also provides a Trichode
Cellulases A and B obtained from microorganisms belonging to the genus rma
A C, D cellulase enzyme standard is provided. Furthermore, the present invention relates to cellulases A, B belonging to the genus Trichoderma ,
Provided is a method for producing an enzyme, which comprises culturing a microorganism having an enzyme-producing ability of either C or D in a medium, producing the enzyme in the culture solution, and collecting the enzyme from the culture solution. .

[0006]

The present invention provides cellulases A, B, C and D which promote defibration activity. The enzyme of the present invention is an enzyme standard product obtained by purifying and purifying a culture of a defibration composition-producing bacterium, and is used in the fields of paper pulp production using the defibration composition, saccharification of a cellulosic substance, etc. It can be utilized for the treatment with the defibration composition to promote the defibration action, which greatly contributes.

[0007]

DETAILED DESCRIPTION OF THE INVENTION Although the cellulase of the present invention has the properties described below, a method for measuring each property will be described before describing each property.

All cellulase enzyme substrates used were prepared by known methods. That is, insoluble cellooligosaccharides are described in J. Ferment. Technol., Vol. 62, 561.
567 (1984)), cellotriose, cellotetraose, cellopentaose,
Phosphoric acid swollen cellulose is Agric. Biol. Chem., Volume 49,
1283-1290 (1985)). Also, reduced phosphoric acid swollen cellulose is described in Agric. Biol. Chem., 53, 1407-1409.
(1989)).

To investigate the action of cellulase enzymes, J. F.
erment. Technol., Volume 62, 561-567 (198
According to the method described in 4)), the sugar produced from the insoluble cellooligosaccharide was confirmed. That is, 0.04 mg of NaN ₃ was added to 20 mg of insoluble cellooligosaccharide, and
M acetate buffer was added and cellulase A, B, C,
D was added to each substrate as an activity against this substrate to a total amount of 2 ml by adding 0.5 U of enzyme, and the mixture was reacted at 37 ° C. for 18 hours. The reaction solution was sampled at each reaction time, and the produced sugar was confirmed by high performance liquid chromatography.

Water-soluble sugars and water-insoluble sugars produced from reduced phosphoric acid swollen cellulose are described in Agric. Biol. Chem., 53.
Vol., 1407-1409 (1989)). Measurement of defibration activity of copy paper is made in Japanese Patent Application No. 2-
The method was the same as that described in 306772.

In order to examine the substrate specificity, enzyme activity against various substrates was measured. The enzyme activity was measured by the method described in "Cellulase" published by Kodansha Scientific (1987). That is, CM-cellulase activity is 1%
To 0.5 ml of 0.1 M acetate buffer (pH 5) containing CM-cellulose (manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in a concentration, 0.5 ml of the enzyme solution diluted with the same buffer was added, and the mixture was incubated at 37 ° C. for 1
The reaction was allowed for 0 minutes. As a control, after leaving the enzyme solution alone under the same conditions, the copper reagent of the Somogy Nelson method, which is a reaction stop solution, was added first, and then the substrate solution was added. Avicel activity is Avicel SF (made by Asahi Kasei) instead of CM-cellulose
Was used at 37 ° C. for 100
Let react for minutes. After the reaction was completed, the amount of reducing sugar produced was measured by the Somogy Nelson method, and the amount of produced sugar was produced in terms of glucose. The enzymatic activity of 1 U was defined as the amount of enzyme that produces 1 micromol of reducing sugar per minute under the above reaction conditions according to international units.

[0012] As in the case of the CM-cellulase activity, the activity of the substrates other than CM-cellulose and Avicel was measured by increasing the amount of reducing sugars produced by the reaction for 10 minutes at various substrate concentrations of 0.5%. It was measured by the Nelson method. The optimum pH of cellulase A and B was measured in the same manner as the CM-cellulase activity using CM-cellulose as a substrate and buffers having different pHs. Optimal p of cellulase C and D
H uses an insoluble cellooligosaccharide as a substrate and buffers having different pHs. According to the CM-cellulase activity, H is an insoluble cellooligosaccharide instead of CM-cellulose. It was measured by the Somogy Nelson method.

The optimum temperature of cellulases A and B is CM-cellulose as a substrate, and the optimum temperature of cellulases C and D is an insoluble cellooligosaccharide as a substrate.
H5.0), and the reaction was performed under the respective temperature conditions in the same manner as described above to measure the activity.

The pH stability of the four cellulases was treated with a buffer solution of each pH at 25 ° C. for 24 hours, and the residual activity was measured in the same manner as in the optimum temperature measurement.

The thermal stability is 0.1M acetate buffer (pH 5.
0), after enzymatic treatment for 10 minutes under each temperature condition, the enzyme solution was immediately cooled with ice, and the residual activity was measured in the same manner as in the case of measuring the optimum temperature.

The isoelectric point of enzyme protein is O. Vesterberg
And 100 ml of LKB manufactured by H. Svensson
The measurement was performed using a density gradient isoelectric focusing column.

The molecular weight of the cellulase enzyme is SDS as usual.
-Determined by polyacrylamide gel slab electrophoresis. That is, as a final concentration, 2% SDS, 5% mercaptoethanol, 0.0625M Tris-HCl (p
H6.8) and added the enzyme at 100 ° C, 5
After heat treatment for 1 minute, BPB was added to prepare an enzyme sample.
As molecular weight markers, phosphorylase b (molecular weight 94,000), bovine serum albumin (molecular weight 67,000), ovalbumin (molecular weight 43,000), carbonic anhydrase (molecular weight 30,000), soybean trypsin inhibitor (molecular weight 20100) ), Α-lactalbumin (molecular weight 144
An enzyme sample was similarly prepared using the mixing kit of (00). 0.025M Tr using 7.5% separation gel
Electrophoresis was carried out in a buffer solution of is + 0.192M glycine (pH 8.3), stained with CBB and then decolorized, and the molecular weight of the cellulase enzyme was determined from the relative mobility of each protein with respect to BPB.

The properties of each cellulase enzyme of the present invention will be described below.

The first enzyme of the present invention has an action of promoting the defibrating activity of copy paper, which can be obtained by culturing the Trichoderma sp. SK-1919 strain producing the defibrating composition of the prior invention. In addition, it is a novel cellulase A having the following properties. Cellulase A (1) Action It produces cellotriose, cellobiose and glucose from insoluble cellooligosaccharides. In addition to water-soluble sugars, water-insoluble sugars are produced from reduced phosphoric acid swollen cellulose. Promotes defibration activity. (2) Substrate specificity It shows a strong activity on CM-cellulose and hydrolyzes insoluble cellooligosaccharides, phosphate swollen cellulose, cellotetraose and cellopentaose. (3) Optimum pH and pH stability With CM-cellulose as a substrate, the optimum pH is around pH 5, and it is stable at pH 2 to 5. (4) Optimum temperature and thermal stability With CM-cellulose as a substrate, the optimum temperature is around 70 ° C, and it is stable to heat up to 60 ° C. (5) The molecular weight is about 53,000 (by SDS polyacrylamide gel slab electrophoresis).

The second enzyme of the present invention has an action of promoting the defibrating activity of copy paper, which can be obtained by culturing Trichoderma sp. SK-1919 strain producing the defibrating composition of the prior application. In addition, it is a novel cellulase B having the following properties. Cellulase B (1) Action Cellobiose and glucose are produced from insoluble cellooligosaccharides. In addition to water-soluble sugars, water-insoluble sugars are produced from reduced phosphoric acid swollen cellulose. Promotes defibration activity. (2) Substrate specificity It shows a strong activity on CM-cellulose, hydrolyzes insoluble cellooligosaccharides and phosphate swollen cellulose, and acts on cellopentaose, cellotetraose, and cellotriose. (3) Optimum pH and pH stability With CM-cellulose as a substrate, the optimum pH is around pH 5, and it is stable at pH 2 to 5. (4) Optimum temperature and thermal stability With CM-cellulose as a substrate, the optimum temperature is around 60 ° C, and it is stable to heat up to 50 ° C. (5) The isoelectric point is around pH 4.7. (6) The molecular weight is about 61,500 (by SDS polyacrylamide gel slab electrophoresis).

The third enzyme of the present invention is the defibration set of the prior invention.
Produce a productTrichoderma Cultivated sp. SK-1919 strain
Promote the defibration activity of copy paper that can be obtained by feeding
A new cellular system that has the following properties as well as functions
It is Case C. Cellulase C (1) Action Mainly produces cellobiose from insoluble cellooligosaccharides,
At the same time it produces glucose. Producing mainly water-soluble sugars from reduced phosphoric acid swollen cellulose
To do. Promotes defibration activity. (2) Substrate specificity CM-Cellulose-active, insoluble cellooligosaccharide,
Hydrolyzes phosphoric acid swollen cellulose and
It acts on sucrose, cellotetraose and cellotriose. (3) Optimum pH and pH stability Optimum pH around pH 4.5 with insoluble cellooligosaccharide as substrate
It is H and stable at pH 3-5. (4) Optimum temperature and heat stability Optimum temperature around 60 ℃ with insoluble cellooligosaccharide as substrate
It is stable to heat up to 50 ° C. (5) The isoelectric point is around pH 4.6. (6) The molecular weight is about 66,000 (SDS polyacrylic amine
Dogel Slab electrophoresis).

The fourth enzyme of the present invention has an action of promoting the defibrating activity of copy paper, which can be obtained by culturing Trichoderma sp. SK-1919 strain producing the defibrating composition of the prior application. In addition, it is a novel cellulase D having the following properties. Cellulase D (1) Action Mainly produces cellobiose from insoluble cellooligosaccharides,
At the same time, glucose and cellotriose are produced. It produces mainly water-soluble sugars from reduced phosphoric acid swollen cellulose. Promotes defibration activity. (2) Substrate specificity It hydrolyzes insoluble cellooligosaccharides, cellotetraose, cellopentaose and phosphoric acid swollen cellulose, and also acts on CM-cellulose and Avicel. (3) Optimum pH and pH stability Using insoluble cellooligosaccharide as a substrate, the optimum pH is around pH 3.
At the same time, it shows 50% or more of the maximum activity at pH 7. It is stable at pH 3 to 6 and has 80% residual activity even at pH 8. (4) Optimum temperature and heat stability Using insoluble cellooligosaccharide as a substrate, the optimum temperature is around 60 ° C, and it is stable to heat up to 50 ° C. (5) The isoelectric point is around pH 3.9. (6) The molecular weight is about 70,000 (by SDS polyacrylamide gel slab electrophoresis).

As shown in Table 1, the enzyme of the present invention has an action, an optimum pH and a pH as compared with various cellulase purified standard products.
There are differences in stability, optimum temperature, thermal stability, isoelectric point, and molecular weight. Furthermore, even when compared with cellulase derived from other microorganisms, there are differences in action, optimum pH, pH stability, optimum temperature, thermal stability, isoelectric point, and molecular weight. Therefore, the four cellulase enzymes of the present invention Is a novel enzyme with different properties from all known enzymes.

[Table 1]

The four cellulases of the present invention are Trichoderm
inoculated with microorganisms that produce one or any one or more of the four enzymes belonging to a genus to the medium and cultured are those which can be obtained by collecting the cellulase from the culture.

Cultivation of the microorganism for obtaining the enzyme of the present invention is carried out by culturing the microorganism under the medium and culture conditions usually used for culturing the microorganism. The medium contains cellulose or hemicellulose. It is preferable to add a product, for example, lignocellulose or a product produced from lignocellulose such as waste paper or pulp. The content of cellulose or hemicellulose-containing material in the medium is 0.00
It is preferably about 1 to 5% by weight.

In order to collect the enzyme of the present invention, a culture obtained by culturing the microorganism is used for ion separation column chromatography, gel filtration, chromatofocusing, and other usual enzyme separation and purification. Can be carried out by the method described. In some cases, the purified enzyme can be effectively collected by using other enzyme purification methods in combination as necessary.

[0027]

EXAMPLES The contents of the present invention will now be specifically described by showing Examples of the present invention, but the present invention is not limited to the following Examples.

Example 1 Lactose 1%, corn steep liquor 0.2%, ammonium sulfate 0.
_{05%, K 2 HPO 4 0.05} %, MgSO 4 · 7H 2 O 0.005%, 0.01% NaCl,
500m medium (pH 5.5) 100 ml containing FeSO ₄ · 7H ₂ O 0.005%
l It was put in a Sakaguchi flask and sterilized by heating at 120 ° C. for 20 minutes. Trichoderma sp. SK with sufficient spore formation in advance
Sterile distilled water was added to the slant of strain -1919, and 2 ml of the resulting spore suspension was inoculated into the above medium, and precultured for 36 hours under the conditions of 30 ° C. and 120 rpm reciprocal shaking. The main culture medium was LBKP 1% instead of lactose in the culture medium.
Of the medium added with 0.02% of adecanol (pH 5.
5) Put 20l into a 30l jar fermenter and
It was sterilized by heating at 20 ° C. for 20 minutes. Pre-culture liquid 1 in this medium
1 was inoculated, and main culture was carried out for 3 days under the conditions of 30 ° C., aeration rate of 10 l / min, and stirring 250 rpm. Medium pH during culture
The pH was adjusted with aqueous ammonia so that the pH did not become 4 or less.

After completion of the culture, the culture solution collected from the jar fermenter was filtered with a filter cloth to obtain 14 l of culture filtrate. Ammonium sulfate (6.5 kg) was added thereto, and the mixture was allowed to stand in a low temperature room overnight to precipitate the enzyme protein. The precipitate is centrifuged (6000 rpm, 10
Min) and 10 mM acetate buffer (pH 5.0) was added to dissolve the enzyme. The insoluble precipitate was collected by centrifugation.
The operation of dissolution with an acetate buffer and centrifugation was repeated 4 times, and all the obtained supernatants were collected together to obtain 450 ml of an enzyme solution as an ammonium sulfate precipitation fraction.

Next, the enzyme solution was added to PVA-Hollow Fiver.
The dialyser (KL-2-50, A membrane, manufactured by Kuraray Co., Ltd.) was held outside and dialyzed by passing demineralized water into the hollow fiber. The desalting operation was performed until the external dialyzed liquid did not detect ammonia with the Nessler reagent. 1150 ml of the enzyme solution obtained by desalting was used as a crude enzyme preparation.

As a first step for fractionating a crude enzyme preparation into various cellulases, DEAE-Sephadex A-
At 50, it was fractionated into 6 large fractions. That is, 1 ml of crude enzyme solution was added to 25 ml of 1M acetate buffer (pH 5.5) to obtain pH of 5.
DE adjusted to 5 and pre-equilibrated to the same buffer of 0.02M
A column of AE-Sephadex A-50 (5.0 x 50 cm) was loaded. In order to elute the protein which was not adsorbed on this column, the column was washed with 3 l of 0.02 M acetate buffer (pH 5.5). Next, the protein was eluted by changing the salt concentration and pH in 5 steps from pH 5.5 to 3.5 and flowing a buffer solution stepwise. Collect the eluate in 18 ml aliquots and measure the protein content by measuring the absorbance at 280 nm.
CM-cellulase activity and avicelase activity were measured as enzyme activities. The total eluate volume is 12 liters.

D equilibrated in 0.02M acetate buffer (pH 5.5)
The FI fraction eluted without being adsorbed on the column of EAE-Sephadex A-50 (5.0 × 50 cm) was concentrated by an evaporator and pre-equilibrated with distilled water Sephacryl S-200.
It was loaded on a (5.0 × 50 cm) column. An ultrafilter (Amicon Ultrafiltration Molar) that uses a membrane that collects fractions with high CM-cellulase activity and allows passage of substances with a molecular weight of 20,000 or less.
It was concentrated with del 52). Add this concentrated enzyme solution to 0.025M Tris-HC
l HiLord of FPLC device equilibrated in buffer solution (pH 7.0)
Load the column with Q (1.6 × 10 cm) and add 0 to the same buffer.
The enzyme was eluted with a solution containing stepwise 0.25 M NaCl. 70
A sharp activity peak of CM-cellulase activity was obtained near mM NaCl. The obtained active fraction was designated as cellulase A.

Cellulase A obtained by the above separation and purification method showed a single band by polyacrylamide gel slab electrophoresis, and was confirmed to be a uniform enzyme preparation. Its molecular weight was about 53,000. As for cellulase A, 2.4 mg of purified enzyme was obtained from 14 liters of the culture, and the specific activity for CM-cellulose was 85 U / mg. Incidentally. The protein amount was measured by the method of Lowry using bovine serum albumin as a standard.

Example 2 The FIII fraction of the crude enzyme obtained in Example 1 was ammonium sulfate precipitated in the same manner as in Example 1, and the enzyme solution dissolved in distilled water was added to 0.02M.
Bio Gel P-2 (5.0 x 40c) equilibrated in acetate buffer (pH 4.5)
It was desalted by passing it through the column of m). 0.02M of the obtained enzyme solution
DEAE-Toyopearl equilibrated in acetate buffer (pH 4.5)
A 650 M (3.0 x 13 cm) column was loaded. The enzyme was eluted with a solution containing 0 to 0.25M NaCl stepwise in the same buffer, and the 280 nm absorbance and enzyme activity of each fraction were measured. Avicelase activity and C on two protein peaks
The peaks of M-cellulase activity were overlapped and eluted. Collect the active fractions with strong avicelase activity in front of F
The III-Avicelase fraction was used as the FIII-CMCase fraction by collecting the active fractions having a strong CM-cellulase activity.

Then, the FIII-CMCase fraction was subjected to 0.025M Piperazine-HCl using the ultrafilter used in Example 1.
The solution was concentrated while being replaced with a buffer solution (pH 5.5). PBE 94 gel column (1.0 x
(27 cm) for chromatofocusing, and
Elution was performed with lybuffer 74-HCl buffer (pH 4). A peak was obtained in which the protein elution and the CM-cellulase activity coincided with each other. The active fractions were collected and designated as cellulase B. Cellulase B obtained by the above separation and purification method shows a single band on polyacrylamide gel slab electrophoresis,
It was confirmed that the enzyme preparation was uniform. Its molecular weight was about 61500. For cellulase B, 37.1 mg of purified enzyme was obtained from 14 liters of the culture, and the specific activity for CM-cellulose was 36 U / mg.

Example 3 The FIII-Avicelase fraction obtained in Example 2 was used in Example 2
Concentration was performed while substituting with 0.025M Piperazine-HCl buffer solution (pH 5.5) using the ultrafilter used in. PBE94 gel column (1.0 x 27c) in which concentrated enzyme solution was equilibrated with the same buffer solution
m) to perform chromatofocusing and
Eluted with uffer 74-HCl buffer (pH 4). Since the avicelase activity of the eluted protein fraction was weak, 0.05 U of the purified enzyme preparation cellulase B obtained above was added to the reaction system for measuring the enzyme activity, and the synergistic avicelase activity for determining the avicelase activity was measured. A peak was obtained in which the protein elution and synergistic avicelase activity coincided with each other, and the active fractions were collected and designated as cellulase C. Cellulase C obtained by the above separation and purification method showed a single band by polyacrylamide gel slab electrophoresis, and was confirmed to be a uniform enzyme preparation. Its molecular weight was about 66000. Cellulase C yielded 10.6 mg of purified enzyme from 14 liters of culture, and had a specific activity of 0.19 U / mg for Avicel.
Met.

Example 4 The FVI fraction of the crude enzyme obtained in Example 1 was ammonium sulfate precipitated in the same manner as in Example 1, and the enzyme solution dissolved in distilled water was added to 0.02M acetate buffer (pH 4.5). Equilibrated Bio Gel P-2 (5.0 x 40c
It was desalted by passing it through the column of m). 0.02M of the obtained enzyme solution
DEAE-Toyopearl equilibrated in acetate buffer (pH 4.5)
A 650 M (2.5 x 33 cm) column was loaded. The elution of the enzyme was carried out with a solution containing 0 to 0.5 M NaCl stepwise in the same buffer, and the elution fractions of avicelase activity were collected. The obtained enzyme solution was subjected to gel filtration through a column of Sephacryl S-200 (4.0 × 60 cm) equilibrated with 0.05 M acetate buffer (pH 5) containing 0.25 M NaCl.

Further, the obtained avicelase activity fraction was concentrated while substituting it with 0.05 M acetate buffer (pH 5) using the aforementioned ultrafilter. Q-Sepharose column (1.6 × 10 cm) of FPLC device in which concentrated enzyme solution was equilibrated with the same buffer solution.
Loaded on. The enzyme is eluted with 0-0.2M NaCl in the same buffer.
Was carried out stepwise, and the elution fractions of synergistic avicelase activity were collected. A peak was obtained in which the protein elution and synergistic avicelase activity coincided with each other. This fraction was collected and designated as cellulase D. Cellulase D obtained by the above separation and purification methods showed a single band by polyacrylamide gel slab electrophoresis, and was confirmed to be a uniform enzyme preparation. Its molecular weight was about 70,000. For cellulase D, 270 mg of purified enzyme was obtained from 14 liters of the culture, and the specific activity for Avicel was 0.018 U / mg.

Example 5 Cellulases A, B, C and D obtained as described above
Was measured for various characteristics by the methods described above. As a result, the above-mentioned properties of each enzyme were confirmed. Copy paper defibration activity promotion effect of each enzyme, optimum pH, optimum temperature,
The results of examining pH stability and thermal stability are shown in FIGS. 1 to 5, respectively. In addition, in FIG. 1, cellulase A (2 μg addition, Δ), cellulase B (14 μg addition, □), cellulase C (7 μg) were added to the defibration composition (◯) shown in the figure.
It shows the promotion of the defibration activity when the reaction was performed in combination with addition, ▲) and cellulase D (10 μg addition, ●).

[Brief description of drawings]

FIG. 1 is a diagram showing the results of measuring the effect of promoting the defibration activity of copy paper by enzymes A, B, C and D of the present invention.

FIG. 2 is a graph showing the optimum pH of the enzymes A, B, C and D of the present invention.

FIG. 3 is a graph showing optimum temperatures of the enzymes A, B, C and D of the present invention.

FIG. 4 is a graph showing pH stability of enzymes A, B, C and D of the present invention.

FIG. 5 shows the thermostability of enzymes A, B, C and D of the present invention.

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[Procedure amendment]

[Submission date] October 27, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Title of invention

[Correction method] Change

[Correction content]

Title: Cellulase having defibration activity, enzyme standard product and method for producing the same

Claims (8)

[Claims] 1. Cellulase A (1) having the following properties: Action It produces cellotriose, cellobiose and glucose from insoluble cellooligosaccharides. In addition to water-soluble sugars, water-insoluble sugars are produced from reduced phosphoric acid swollen cellulose. Promotes defibration activity. (2) Substrate specificity It shows a strong activity on CM-cellulose and hydrolyzes insoluble cellooligosaccharides, phosphate swollen cellulose, cellotetraose and cellopentaose. (3) Optimum pH and pH stability With CM-cellulose as a substrate, the optimum pH is around pH 5, and it is stable at pH 2 to 5. (4) Optimum temperature and thermal stability With CM-cellulose as a substrate, the optimum temperature is around 70 ° C, and it is stable to heat up to 60 ° C. (5) The molecular weight is about 53,000 (by SDS polyacrylamide gel slab electrophoresis). 2. Cellulase B (1) having the following properties: Action Cellobiose and glucose are produced from insoluble cellooligosaccharides. In addition to water-soluble sugars, water-insoluble sugars are produced from reduced phosphoric acid swollen cellulose. Promotes defibration activity. (2) Substrate specificity It shows a strong activity on CM-cellulose, hydrolyzes insoluble cellooligosaccharides and phosphate swollen cellulose, and acts on cellopentaose, cellotetraose, and cellotriose. (3) Optimum pH and pH stability With CM-cellulose as a substrate, the optimum pH is around pH 5, and it is stable at pH 2 to 5. (4) Optimum temperature and thermal stability With CM-cellulose as a substrate, the optimum temperature is around 60 ° C, and it is stable to heat up to 50 ° C. (5) The isoelectric point is around pH 4.7. (6) The molecular weight is about 61,500 (by SDS polyacrylamide gel slab electrophoresis). 3. Cellulase C (1) action having the following properties: Producing mainly cellobiose from insoluble cellooligosaccharides,
At the same time it produces glucose. It produces mainly water-soluble sugars from reduced phosphoric acid swollen cellulose. Promotes defibration activity. (2) Substrate specificity CM-Cellulose-active, insoluble cellooligosaccharide,
It hydrolyzes phosphoric acid swollen cellulose and acts on cellopentaose, cellotetraose and cellotriose. (3) Optimum pH and pH stability Optimum pH around pH 4.5 with insoluble cellooligosaccharide as substrate
It is H and stable at pH 3-5. (4) Optimum temperature and heat stability Using insoluble cellooligosaccharide as a substrate, the optimum temperature is around 60 ° C, and it is stable to heat up to 50 ° C. (5) The isoelectric point is around pH 4.6. (6) The molecular weight is about 66,000 (by SDS polyacrylamide gel slab electrophoresis). 4. Cellulase D (1) action having the following properties: mainly producing cellobiose from insoluble cellooligosaccharides,
At the same time, glucose and cellotriose are produced. It produces mainly water-soluble sugars from reduced phosphoric acid swollen cellulose. Promotes defibration activity. (2) Substrate specificity It hydrolyzes insoluble cellooligosaccharides, cellotetraose, cellopentaose and phosphoric acid swollen cellulose, and also acts on CM-cellulose and Avicel. (3) Optimum pH and pH stability Using insoluble cellooligosaccharide as a substrate, the optimum pH is around pH 3.
At the same time, it shows 50% or more of the maximum activity at pH 7. It is stable at pH 3 to 6 and has 80% residual activity even at pH 8. (4) Optimum temperature and heat stability Using insoluble cellooligosaccharide as a substrate, the optimum temperature is around 60 ° C, and it is stable to heat up to 50 ° C. (5) The isoelectric point is around pH 3.9. (6) The molecular weight is about 70,000 (by SDS polyacrylamide gel slab electrophoresis). 5. The cellulase enzyme standard product according to any one of claims 1 to 4, which is obtained from a microorganism belonging to the genus Trichoderma . 6. The microorganism belonging to the genus Trichoderma is Tricho
The cellulase enzyme standard product according to claim 5, which is derma sp. SK-1919. 7. A microorganism which belongs to the genus Trichoderma and has the enzyme-producing ability according to any one of claims 1 to 4 is cultivated in a medium, the enzyme is produced in the medium, and the enzyme is produced from the medium. A method for producing a cellulase enzyme, comprising: 8. The microorganism belonging to the genus Trichoderma is Tricho
The method for producing a cellulase enzyme according to claim 7, which is derma sp. SK-1919.