JPH01112982A - Alkali-tolerant cellulase and microorganism producing said cellulase - Google Patents

Abstract

NEW MATERIAL:An alkali-tolerant cellulase R-597. Action; well acting and dissolving cellulose, such as CMC, cellulose, etc., to form reducing sugar. Substrate specificity; exhibiting activity for CMC, Avicel(R), etc. Action pH; 3-13. Optimum pH; 7. pH stability; without inactivation at pH5-11.5. Optimum temperature; 15-80 deg.C action temperature and 60 deg.C optimum temperature. Molecular weight; having the molecular weight peak at 40000, etc. USE:A detergent composition for clothes. PREPARATION:A microorganism, belonging to the genus Bacillus and having the ability to produce cellulase K-597, preferably Bacillus sp. KSM-635 (FERM P-8872) is cultivated and the aimed cellulase is isolated and purified from the resultant culture.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel alkali-resistant cellulase and a microorganism that produces the same.

[Conventional technology]

The development of cellulase, a fibrinolytic enzyme, has traditionally been carried out with the primary goal of effectively utilizing biomass resources, particularly cellulose resources. Since nine bacterial strains have been isolated as cellulase-producing bacteria, there are many types, mainly filamentous fungi such as Aspergillus, Penicillium, Trichoderma, Hexathalium, Humicola, and Acremonium, as well as Xutomonas, Cellulomonas, and Ruminococcus. It has also been reported in bacteria such as the genus Streptomyces and genus Bacillus, as well as actinobacteria such as the genus Streptomyces and Thermoactinomyces. However, at present, there are not many uses of cellulase for biomass on an industrial scale.

On the other hand, as a new industrial use of cellulase, its use as a compounding ingredient in laundry detergents is being studied and attracting attention (Japanese Patent Publication No. 59-49279, Japanese Patent Publication No. 60-23
158, Japanese Patent Publication No. 60-36240). However, most of the cellulases previously found in nature are classified as so-called neutral or acidic cellulases, which show maximum and stable enzymatic activity in the neutral to acidic range, and are used in laundry detergents. The reality is that the presence of so-called alkaline cellulases and alkali-resistant cellulases, which are necessary conditions for formulation components, and which exhibit maximum activity in the alkaline region or have alkali resistance, are extremely rare.

The term alkaline cellulase referred to here refers to one whose optimum pH is in the alkaline region, and the term alkaline-tolerant cellulase refers to one whose optimum pH is in the neutral to acidic region, but which has an optimum pH in the alkaline region. It refers to a substance that has sufficient activity and stability compared to the activity of other substances.

Further, neutral refers to a pH range of 6 to 8, and alkaline refers to a pH range higher than that.

That is, conventional methods for producing alkaline cellulase and alkali-resistant cellulase that can be used as laundry detergent compositions include a method in which cellulase A is collected by culturing alkalophilic Bacillus bacteria (Japanese Publication No. 50-28515), Cellulomonas A method for producing alkaline cellulase 301-A by culturing alkaliphilic bacteria belonging to the genus (Japanese Unexamined Patent Publication No. 58-224686), Alkaliphilic Bacillus? Carboxymethyl cell 2- by culturing all139
Method for producing Zee (Fukumori, F., Ku
do, T.

and Horikoshi, K., J. Gen.
Microbiol,, 131°3339, (19
85)) and a method for producing alkaline cellulase using a species of the genus Streptomyces (Japanese Unexamined Patent Publication No. 1983-1948)
No. 3) has been reported, and none of them are suitable for industrial fermentation production.

However, Karichika and the present inventors discovered that Bacillus sp., a type of alkalophilic microorganism,
) KSM-635 (FEBM P-8872) efficiently produces alkaline cellulase K suitable as a laundry detergent composition, and by further selecting the culture conditions, alkaline cellulase K can be produced at a higher yield. was obtained, and it was discovered that industrial fermentation production of alkaline cellulase becomes possible.

[Problem that the invention seeks to solve]

However, the above Bacillus sp. KSM-635
The culture conditions are not necessarily industrially advantageous. In other words, it is necessary for alkaline-loving bacterial strains to keep the pH alkaline during cultivation, but to date, the so-called alkaline fermentation method using alkaline-loving strains has a short history, and compared to normal neutral microorganisms, these alkaline-loving Knowledge about the physiology and biochemistry of microorganisms has not been sufficiently accumulated, and medium preparation and culture methods have been operationally difficult for industrial fermentation production.

[Means for solving problems]

In order to solve this problem, the present inventors have developed an alkaline cellulase that has an optimum pH in the alkaline region, or maintains high activity even in the alkaline region compared to the maximum activity at the optimum pH. In order to obtain a neutral bacterium that produces alkaline-resistant cellulase 2-ase, we cloned the relevant cellulase gene using a bacterial strain that grows in a neutral region as a host, in parallel with so-called genetic recombination. I have continued to explore.

As a result, they discovered that a bacterial strain isolated from soil in Nikko City, Tochigi Prefecture produces so-called alkali-resistant cellulase, which has sufficient activity even in alkaline conditions and maintains stability10.The present invention was completed. .

The bacterial strain used in the present invention has the following mycological properties. In addition, the culture medium used in the following classification identification is as follows.

Medium 1. Meat extract, 1.0: Bactopedetone, LO:
NaCt, 0.5: Pact agar, 1.5 (pI (72)
(Displayed as weight %; same below) Medium Lambda meat extract, 1.0: Pactopezotone, 1.0:
NaCt, 0.5 (pH 7,2) medium Lambda meat extract, i, o; Pactopedetone, 1.0; NaCA, 0
．． 5: Gelatin, 1.0 (pH 7, 2) medium 4. Bactritoma milk, 10.0 medium 5
．． Bactobezoton, l, O: KNOs, 0.
1 medium 6. Baktopezot 7, 1. O;NaNOs
, 0.1 medium7. A) "but7, 0.7;
NaCt, 0.5 Niglucose, α5 (pH 7,0) medium & Bactopedeton, 1.0 medium 9. TSI agar (manufactured by Eiken Chemical); indicated amount medium 10
．． Meat extract, 1.0: Bactopedetone, 1.0: NaC
L, 0.5; Soluble starch, 0.2; Agar, L
5 Medium 11. NaNHaHPOa”4HzO10,15
; KHzPOi +0-1 ; Mg5Oa ” 7
HxO10-02: Sodium citrate, α25 (
ri) I 6.8) Medium 1z Christensen medium (manufactured by Eiken Chemical); indicated amount medium 13. Simmons medium (Eiken Chemical Exhibition); indicated amount medium 1
4. Glucose, 1. O; KHiPO4H0,1:
MgSO4-7H20, 0.05; KCl, 0.0
2; Nitrogen source, 0.1 (nitrogen source is fl- using sodium nitrate and ammonium sulfate) (pH 7,2) Medium 15. King A medium “Eiken’” (manufactured by Eiken Chemical); indicated amount medium 16. King B medium “Eiken” (manufactured by Eiken Chemical Co., Ltd.); indicated amount medium 17. Urea medium “Eiken’” (manufactured by Eiken Chemical Co., Ltd.); indicated amount medium 18. P paper for cytochrome oxidase test (manufactured by Hinaga Pharmaceutical Co., Ltd.) ) Medium 19.3% hydrogen peroxide water medium 20. OF basal medium (Difco If: Indication amount medium 21. (NH4)IHPO4, 0,1; KC
l, 0.02: MgSO4・7HzO, 0.02
; yeast extract.

0,02; Pact agar, ZO; BCP (0.2% solution), 0.4 Medium 2z Pact Sabouraud dextrose agar medium (
Difco); indicated amount medium 23. Phenylalanine malonate medium (manufactured by Hinaga Pharmaceutical Co., Ltd.); indicated amount medium 24. Skim milk, 5.0: Bakuto agar, 1.5
(Mycological properties) ″ (a) Microscopic observation results: The size of the bacterial cells is 0.5 to 0.8 μm x 1.0 to 2
It is a rod with a diameter of 0 μm, and an oval endospore (
0.5~0.8μm x 1.0~1.2)tm)
make. It has flagella and is motile. Durham staining was positive.

It has no anti-acid properties.

(b) Growth status in various media ■ Broth agar plate culture (Medium 1) Growth status is normal. The shape of the colony is circular, the surface is smooth, and the periphery is smooth or wavy. The color tone of the village is pale yellow and semi-transparent with a slightly dull luster.

■Juice agar slant culture (Medium 1) Growth is normal, and the condition is spread-like, glossy, milky white and translucent.

■ Broth liquid culture (medium 2) Grow. There is surface growth, and upper layer growth is also observed.

■Meat juice gelatin puncture culture (medium 3) ° Liquefaction of gelatin is observed.

■ Lidomus Milk Medium (Medium 4) Liquification of milk is observed, but there is no clear change in lidmus pigment.

(C) Physiological properties ■Nitrate reduction and denitrification reactions (Medium 5, Medium 6) Nitrate reduction and denitrification reactions were negative.

■MR test (medium 7) Positive.

■VP test (medium 7) Positive.

■Generation of indole (medium 8) negative.

■Generation of hydrogen sulfide (medium 9) negative.

■Hydrolysis of starch (medium 10) negative.

■Use of citric acid (Medium 11.Medium 12.Medium 13) Negative in both Xhosa medium and Shinanzu medium.

Positive on Christensen's medium.

■Use of inorganic nitrogen source C medium 14) Do not use nitrates or ammonium salts.

■Production of pigment (Medium 15.Medium 16) Yellow pigment production in KING-B medium.

[Phase] Urease (medium 17) negative.

■Oxidase (medium 18) There are no negative or positive results.

@ Catalase (Medium 19.) Positive.

[Phase] Range of Growth (Medium 2) The temperature range for growth was 10 to 50°C, the optimum temperature range for growth was 20 to 40°C, and the pH range for growth was pH 5 to 10.

■Attitude towards oxygen Aerobic.

■O-F test (medium 20) Grows only aerobically.

[Phase] Sugar utilization (medium 21.) (+: Utilize, -
1. L-arabinose + ZD-xylose + 3. 0-gelcose + 4. 0-mannose + 5. D-7 lactose + 6. 0-galactose + L maltose & sucrose 19. Lactose - 10, trehalose + 11, D-sorbitol - 11. D-Mannit + 13, Inosit + 14, Glycerin + 15, De/Den - ■pH in vp medium (Medium 7) pH 5,0 [Phase] Growth in salt-containing medium (Modified medium 1) 5%
Grow in

It grows at 7%.

It grows in 10 fights.

[Phase] Growth at pH 5, 7 (Medium 22) Grows.

@Deamination of phenylalanine (medium 23) negative.

[Phase] Casein decomposition (medium 24) Positive.

Regarding the above dental properties, please refer to Bersey's Manual Fist of Determinative No.
rgey's Manual of Determi
Native Bacteriolo 8th edition and “The Genus Bacillus”
llus ''', Ruth, E-Gordon A
griculture Handbook m427.
Agricultural Research 5erv
ice, U, S, Department of Agr.
iculture, Washington D, C
, (1973) ), the strain of the present invention was found to be a spore-bearing bacterium, Bacillus.
It is recognized as a type of the genus (us). This strain has recently been developed by Horikoshi and Akiha (“Alkalophilic M
icroorganism'', JapanSci
entific 5ociety Press (To
(Kyo), published in 1982), so-called Alkalophilic microorganisms grow in an alkaline medium with a pH of 8 or higher, and cannot grow in a neutral pH range below this, whereas microorganisms with a weakly acidic Since it is possible to grow in the alkaline range (pH 5 to 10), it is clearly different from this alkaliphilic microorganism, and can be judged to be a general Bacillus microorganism that can grow in neutral conditions.

Furthermore, when this strain is compared with other known strains of the genus Bacillus, Bacillus pumilus is the most related species.
lus pumilia). However, when comparing this strain with known strains belonging to Bacillus pumilus, the known strains at least do not produce alkaline-resistant cellulases, so the present inventors determined that this strain is a new strain, and Bacillus sp. KSM- It was named 597 and deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (FE
RM P-9573).

In order to obtain an alkali-resistant cellulase, cellulase -597, using the strain of the present invention, the strain may be inoculated into a medium and cultured according to a conventional method. It is preferable that the medium contains appropriate amounts of assimilable carbon sources and nitrogen sources. There are no particular restrictions on the carbon source and nitrogen source, but examples include inorganic ammonium nitrate, ammonium sulfate,
Ammonium chloride, ammonium phosphate, sotaya nitrate, corn gelten meal, soybean flour, corn steep liquor, casamino acid, yeast extract, Pharmamedia, sardine meal, meat extract, - (Zotone, Hizoro, Azono 9 War, Corn Soy Pean) Examples include meal, coffee grounds, cottonseed oil cake, cultivator, Amiflex and Azozolone, Zest, Azotux, and the like.

In addition, carbon sources include rice husk, iron, filter paper, patterned paper, vegetable fibers such as sawdust, blackstrap molasses, invert sugar, carboxymethyl cellulose (CMC), Avicel, cellulose cotton, xylan, pecten, t, Precious carbon sources such as arabinose, xylose, glucose, mannose, hexalactose, caractose, sucrose, milk sugar, trehalose, maltose, nrubitol, mannitol, inositol, glycerin, soluble starch and assimilable organic acids such as acetic acid, Examples include citric acid. In addition, phosphoric acid, Mg, Ca'', Mn'', Zn
.

Inorganic salts such as Co'', Na'', and K+, and, if necessary, inorganic and organic trace nutrients can also be appropriately added to the medium.

The target substance cellulase -597 can be collected and separated from the culture thus obtained in accordance with conventional methods for collecting and purifying enzymes. That is, the crude enzyme solution can be obtained by removing the bacterial cells from the culture solution using conventional solid-liquid separation means such as centrifugation or filtration. This crude enzyme solution can be used as it is, but if necessary, the crude enzyme can be obtained by separation means such as salting-out method, precipitation method, ultrafiltration method, etc., and further purified and crystallized by a known method. It is also possible to use it as a purified enzyme.

The alkaline cellulase of the present invention thus obtained has -59
7 has the enzymatic properties shown below. .

The enzyme activity was measured according to the following method.
The buffer solution used was as follows.

pH 3-8 McLuvain buffer pH 8-11 Glycine-sodium hydroxide buffer pH 12-13 Potassium chloride-sodium hydroxide buffer Enzyme activity measurement method: (1) CMCase activity 101!9 CMC (A-01L, Yamaba Kokusaku) 9 Rudesha)
.

Add 0 to 0.9 ml of a substrate solution containing 100 μmol of various buffers (Macrupain, phosphoric acid, glycine-NaOH, etc.)
, 1- were added, and the mixture was reacted at 30°C for 20 minutes. After the reaction, 3,5-dinitro-salicylic acid (3°5-di
Nitro-salicylic acid (DN8
)) Reducing sugars were quantified using the method. That is,
1.0 ml of DNS reagent was added to the reaction solution 1.0-ml, heated at 100° C. for 5 minutes to develop color, and after cooling, diluted by adding 4.0 d of deionized water. This was colorimetrically determined at a wavelength of 535 nm. The enzyme titer was 1 μm per minute under the above conditions.
The amount of enzyme that generates reducing sugar corresponding to gelose of ol was defined as 1 unit.

(2) p-nitrophenyl cellobioside degrading activity 0
, 1 μmol p-nitrophenyl cellobioside (Sigma), and 100100p phosphate buffer (pH 7.0) were reacted with an appropriate amount of the enzyme solution at 30°C, and then IM 0.3 m of Na*COs and 1.7 m of deionized water were sequentially added, and the liberated p-nitrophenol was determined colorimetrically at 400 nm. The enzyme titer was defined as the amount of enzyme t-1 unit that releases K1 μmol of p-nitrophenol for 1 minute under the above conditions.

(3) Avicel, cellulose powder, phosphoric acid-swollen cellulose, alkali-swollen cellulose, and filter paper degrading activity 20 ■ Avicel (Merck & Co.), 200 μmol Appropriate amount in 2.0 d of reaction solution containing phosphate buffer (pH 7,0) The enzyme solution was added and allowed to act while shaking at 30°C and 250 rpm. After the reaction, refrigerated centrifugation (5°C, 3000
3.5-dinitro-salicylic acid (3°5- dinit
ro-salicylic acid (DNS)
) method was used to quantify reducing sugars. Cellulose powder decomposition activity was determined using cellulose powder (Toyo Seven Papers), and phosphoric acid-swollen cellulose and alkali-swollen cellulose decomposition activities were determined using the method of Tomita et al.
ment.

Thechenol, 52.235, 1974)
The F paper decomposition activity of the cellulose treated with was carried out in the same manner as the apicelase activity using 7 papers (7 papers for cellulase activity assay, Toyo Magnetic 51-Special). For the enzyme titer, 1 unit was defined as the amount of enzyme that produced reducing sugar equivalent to 1 μmol of gelose per minute under the above conditions.

(4) Cellopiase activity 10q cellobiose (Kanto Kagaku Co., Ltd.), 50 μmol
Reaction solution containing phosphate buffer (pH 7,0>) 1. Oml
After allowing an appropriate amount of enzyme solution to act on the inside at 30℃, it was heated to 100℃
After treatment for 5 minutes to inactivate the enzyme, the amount of gelose produced was measured using the mutarotase-GOD method (Glucose C-Tes
t, Wako Tsumugi Kogyo Co., Ltd.). For the enzyme titer, one unit was defined as the amount of enzyme that produced 2 μmol of gelose per minute under the above conditions.

(Enzymatic properties) (1) Action: Due to cellulose, cellulose, 7 paper, Avicel, CMC, etc.
act to dissolve these and produce reducing sugars such as gelcose.

(2) Substrate specificity In addition to CMC, this enzyme can also be used in cellulose powder, Avicel,
It had activity against filter paper and p-nitrophenyl cellobioside.

(3) Working pH and optimum pH The working pH range is extremely wide, from 3 to 13. The optimal pH is 7, and even in the range of 4.5 to 11.5, the relative activity is more than 50% of the activity at the optimal pH, making it the most cellulase among the cellulases studied in the past. It can be said that it is an enzyme that has a wide action pH range on the alkaline side (Fig. 1).

(4) pH stability The residual activity after being held at 30°C for 1 hour at each pH was calculated as p
The pH stability was investigated by measuring with H6. The result is the pH
Extremely stable and does not deactivate at pH 4.5-115, pH 4.5-12
．． 5 also maintained an activity of about 50% or more.

This enzyme is sufficiently stable even in this highly alkaline region (Figure 2).

(5) When the activity was measured at the optimum temperature, pH 6, the action temperature was 15
The optimum temperature was 60°C over a wide range of ~80°C. Moreover, even in the range of 45 to 75°C, it had more than 50% of the activity at the optimum temperature (Figure 3).

(6) Temperature stability After treatment at each temperature (pH 7) for 30 minutes, the optimum pH (p
As a result of measuring the residual activity of H6), it was stable at 50°C and had about 50% residual activity even at 55°C (Figure 4).

(7) Molecular weight This enzyme was added to Biogel P-150 ('Blo-Ge1P
-150'''), a main peak was observed at about 40,000.

(8) Effect of metal ions Regarding this enzyme, various metal ions (At.

Fe”, Ba, Ca, Cd,
Co”, Cu”2+ 2+
2+Fe, Hg, Mn, M
g, Ni, Pb.

Zn'', K'', Na'') were allowed to coexist during the activity measurement, and their effects were investigated (the concentration of various metal ions was 1).
K+, Na+ is 50mM).

As a result, inhibition was observed with Hg''+ and activation with CO2+.

(9) Effect of surfactant Various surfactants (for example, LASSASS ES).

The effect of AO8, (1-8FE, SAS, soap, polyoxyethylene secondary alkyl ether) on enzyme activity was investigated. 0.05% of a surfactant was present at the time of activity measurement, and its influence was investigated.

As a result, there was almost no inhibition by any of the surfactants. Little inhibition of activity was observed even with dibuium dodecyl sulfate, a potent detersoent.

(10) Protease resistant detergent protease, for example API-21 (Showa Denko)
, Maxatase (Gist), and Alcalase (Noge) were co-present (0,1119/ml) at the time of activity measurement to examine their effects, and it was found that the protein had strong resistance to all proteases.

(11) Effect of chelating agents Chelating agents EDTA, EGTA, citric acid, zeolite, tri? Sodium lyphosphate was allowed to coexist during the activity measurement, and its influence was investigated, but no inhibition was observed.

〔Effect of the invention〕

Although the cellulase of the present invention has an optimum pH of 7, it has a relative activity of 60% or more of the optimum pH even at a highly alkaline pH of 11.5.
．． 5, it is extremely stable. Furthermore, it is hardly inhibited by detergent ingredients such as surfactants, proteases, and chelating agents.

Therefore, this enzyme can be advantageously used as a component of detergent compositions.

In addition, the strain of the present invention, Bacillus sp. KSM-59
Since No. 7 is a strain that grows in neutral conditions, it is easier to industrially produce alkali-tolerant cellulase than with alkaliphilic bacteria.

〔Example〕

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 A spoonful (approximately o, sy) of soil from Nikko City, Tochigi Prefecture was suspended in sterile physiological saline and heat treated at 80°C for 10 minutes. The upper layer of this heat-treated solution was diluted appropriately and applied to a separation agar medium (medium 1). Next, this was heated at 30°C for 3
The cells were cultured for several days to form colonies. Bacteria that formed a pellucid zone around the colony due to CMC dissolution were selected, and CMCase-producing bacteria were obtained. Furthermore, the obtained bacteria were inoculated into a liquid medium of medium 2, and cultured with shaking at 30°C for 3 days. After culturing, the CMCase activity of the centrifuged supernatant was measured at pH 3.
-13 to screen for alkali-resistant cellulase-producing bacteria.

By the method described above, Bacillus sp.
We were able to obtain SM-597 strain (FIRM P-9573).

Medium 1. CMC2chi? Ripezoton 0.5% Yeast extract 0.05% KH2PO40, 1% NazHPO4” 12H200, 25% MgSO4・
7) (,00,02 Chi agar 0.75% pH5,8 Medium 2. CMC1% Bori peptone 1- Yeast extract 0.5% KHzPOa O,1%Na*HPO4
12 Hlo 0.25% pH 6,8 Example 2 Bacillus varnish f-KSM-597 obtained in Example 1 was inoculated into liquid medium 2 of Example 1 and cultured with shaking at 30°C for 3 days. After culturing, The bacterial cells were removed by centrifugation to obtain a crude enzyme solution.To this crude enzyme solution lt, 3L of ethanol was added in dry ice-ethanol, and the resulting precipitate was centrifuged and further freeze-dried to dryness. As a powder, a cellulase with a specific activity of -5971otC 8 units/'; the same as the value measured at pH 9) was obtained.

Example 3 CMC to 1tIb sucrose in liquid medium 2? Using a medium in which lipedeton was replaced with 7% C3L (Corn Steede Liquor), shaking culture was carried out at 30"C for 3 days according to Example 2. The CMCase activity of the centrifuged supernatant of the obtained culture solution When measured, it was 300 units/l.

[Brief explanation of the drawing]

FIG. 1 is a drawing showing the relationship between enzyme reaction pH and relative activity. FIG. 2 is a drawing showing the relationship between enzyme treatment pH and relative activity. FIG. 3 is a drawing showing the relationship between enzyme reaction temperature and relative activity. FIG. 4 is a diagram showing the relationship between enzyme treatment temperature and relative activity. that's all

Claims (1)

[Scope of Claims] 1. Alkaline-resistant cellulase K-597 having the following enzymatic properties. (1) Action It acts well on cellulose such as carboxymethyl cellulose, cellulose, filter paper, and Avicel, and dissolves them to produce reducing sugars. (2) Substrate specificity In addition to carboxymethyl cellulose, it has activity against phosphoric acid-swollen cellulose, cellulose powder, Avicel, filter paper, and p-nitrophenyl cellobioside. (3) Working pH and optimum pH The working pH range is 3-13. The optimum pH is 7, and even in the range of 4.5 to 11.5, the relative activity is 50% or more of the activity at the optimum pH. (4) pH stability Extremely stable at pH 5 to 11.5, does not deactivate, pH 4.5
~12.5, the activity is maintained at about 50% or more. (5) Optimal temperature The working temperature ranges over a wide range from 15 to 80°C, and the optimum temperature is 60°C. In addition, even in the range of 45 to 75°C, the activity at the optimum temperature is 5%.
0% or more. (6) Has a molecular weight peak at approximately 40,000 (Biogel P-
150). (7) Influence of metal ions: inhibited by Hg^2^+ and activated by Co^2^+. (8) Effect of surfactants LAS, AS, ES, AOS, α-SFE, SAS, soap, and polyoxyethylene secondary alkyl ether hardly inhibit the activity. (9) Protease resistance Resistant to proteases. (10) Effect of chelating agents EDTA, EGTA, sodium tripolyphosphate, zeolite, and citric acid do not inhibit the activity. 2. A microorganism belonging to the genus Bacillus and having cellulase K-597 productivity.