JPH0732709B2 - Alkali-tolerant cellulase - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel alkali-tolerant cellulase.

[Conventional technology]

The development of celluloses, which are fibrinolytic enzymes, has hitherto been promoted with the aim of effectively utilizing biomass resources, particularly cellulose resources. Strains that have been isolated as cellulase-producing bacteria are many types, mainly Aspergillus, Penicillium, Trichoderma, Fusarium, Humicola, Acremonium, etc., mainly filamentous fungi, Ciudadmonas, Cellulomonas, Luminococcus, It has also been reported in bacteria such as Bacillus and actinomycetes such as Streptomyces and Thermoactinomyces. However, at present, the use of biomass cellulase on an industrial scale is not large.

On the other hand, as a new industrial use of cellulase, its use as a blending component of a detergent for clothes has been studied and attracted attention (Japanese Patent Publication No. 59-49279 and Japanese Patent Publication No. 23158/1985).
Japanese Patent Publication No. 60-36240). However, most of the cellulases conventionally found in nature are classified as so-called neutral or acid cellulases that show the maximum and stable enzyme activity in the neutral to acidic region, and are detergents for clothes. The fact is that the presence of cellulases that exhibit maximum activity in the alkaline region or that have alkaline acidity, so-called alkaline cellulases and alkali-tolerant cellulases, which are necessary conditions as a compounding ingredient, is extremely small.

The alkaline cellulase referred to here is one whose optimum pH is in the alkaline region, and the alkaline-resistant cellulase is
The optimum pH is in the neutral to acidic range, but it has a sufficient activity and stability even in the alkaline range as compared with the activity at the optimum pH.

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

That is, as a conventional method for producing an alkaline cellulase and an alkaline-tolerant cellulase that can be used as a detergent composition for clothes, a method for collecting cellulase A by culturing an alkalophilic Bacillus bacterium (Japanese Patent Publication No. 28515 / SHO), A method for producing alkaline cellulase 301-A by culturing an alkalophilic bacterium belonging to the genus Cellulomonas (JP-A-58-224686), an alkalophilic Bacillus No. 1139.
To produce carboxymethylcellulase (Fukumori, F., Kudo, T. and Horikoshi, K., J. Gen. Microb
iol., 131, 3339, (1985)) and a method for producing an alkaline cellulase using a Streptomyces genus (JP-A-61-19483). Not suitable for fermentative production.

However, recently, the present inventors have found that Bacillus sp. KSM-635, which is a kind of alkalophilic microorganism.
(FERM P-8872) efficiently produces alkaline cellulase K suitable as a detergent composition for clothing, and further selects culture conditions to obtain alkaline cellulase K in a higher yield, It was found that industrial fermentative production of alkaline cellulase is possible.

[Problems to be solved by the invention]

However, the culture conditions of Bacillus sp. KSM-635 are not necessarily industrially advantageous. That is, the alkalophilic strain needs to keep the pH alkaline during the culture, but so far, the so-called alkaline fermentation method using the alkalophilic strain has a short history,
Compared with normal neutral microorganisms, the knowledge about the physiology and biochemistry of these alkalophilic microorganisms has not been sufficiently accumulated, and the difficulty in operating the medium preparation and culturing method for industrial fermentative production. I was told.

[Means for solving problems]

In order to solve such a problem, the present inventors can maintain an alkaline cellulase having an optimum pH in the alkaline region or a high activity even in the alkaline region as compared with the maximum activity at the appropriate pH. In order to obtain a neutral bacterium that produces an alkali-resistant cellulase, using a strain that grows in a neutral region as a host, cloning the corresponding cellulase gene, in parallel with so-called gene recombination means, continue to search for the bacterium in the natural world. Came.

Then, as a result, it was found that the strain isolated from the soil of Nikko City, Tochigi Prefecture has a sufficient action even on the alkaline side, and retains stability, producing so-called alkali-resistant cellulase, and completed the present invention. .

The strain used in the present invention has the following mycological properties. The media used in the classification and identification below are as follows.

Medium 1. Meat extract, 1.0; Bacto peptone, 1.0; NaCl, 0.5; Bacto agar, 1.5 (pH 7.2) (label is% by weight; the same applies below) Medium 2. Meat extract, 1.0; Bacto peptone, 1.0; NaCl , 0.5 (p
H7.2) Medium 3. Meat extract, 1.0; Bactopeptone, 1.0; NaCl, 0.5; Gelatin, 1.0 (pH7.2) Medium 4. Bactoritomymilk, 10.0 Medium 5. Bactopeptone, 1.0; KNO ₃ , 0.1 Medium 6. Bactopeptone, 1.0; NaNO ₃ , 0.1 medium 7. Bactopeptone, 0.7; NaCl, 0.5; Glucose, 0.5
(PH7.0) Medium 8. Bactopeptone, 1.0 Medium 9. TSI agar (manufactured by Eiken Kagaku); Indicated amount Medium 10. Meat extract, 1.0; Bactopeptone, 1.0; NaCl, 0.5; Soluble starch, 0.2; Agar, 1.5 Medium 11.NaNH ₄ HPO ₄・ 4H ₂ O, 0.15; KH ₂ PO ₄ , 0.1; MgSO ₄・ 7H
₂ O, 0.02; Sodium citrate, 0.25 (pH6.8) medium 12. Christensen medium (Eiken Chemical Co., Ltd.); indicated amount medium 13. Simmons medium (Eiken Chemical Co., Ltd.); indicated amount medium 14. Glucose, 1.0; _{KH 2 PO 4, 0.1; MgSO} 4 · 7H 2 O, 0.05; KC
l, 0.02; Nitrogen source, 0.1 (Nitrogen source was sodium nitrate and ammonium sulfate) (pH7.2) Medium 15. King A medium "Eiken" (Eiken Chemical Co., Ltd.); Indicated amount Medium 16. King B Medium "Eiken" (manufactured by Eiken Chemical); indicated amount Medium 17. Urea medium "Eiken" (manufactured by Eiken Chemical); indicated amount Medium 18. Cytochrome oxidase test paper (Nissui Pharmaceutical) Medium 19.3% excess Hydrogen peroxide water medium 20.OF basal medium (manufactured by Difco); indicated amount medium 21. (NH ₄ ) ₂ HPO ₄ , 0.1; KCl, 0.02; MgSO ₄ .7H ₂ O, 0.02;
Yeast extract, 0.02; Bacto agar, 2.0; BCP (0.2% solution), 0.
4 Medium 2 2. Bact Sabouraud Dextrose Agar (D
ifco); indicated amount medium 23. phenylalanine malonate medium (manufactured by Nissui Pharmaceutical Co., Ltd.); indicated amount medium 24. skim milk, 5.0; Bacto agar, 1.5 (mycological properties) (a) Microscopic observation Results The size of the cells is 0.5-0.8 μm x 1.0-2.0 μm, and the oval endospores (0.5-0.8 μm x
1.0-1.2 μm). It has flagella and is motile. Graph staining is positive. There is no acid resistance.

(B) Growth condition in various media Meat agar plate culture (medium 1) Growth condition is normal. The shape of the settlement is circular, the surface is smooth, and the peripheral edge is smooth or wavy. The color of the village is pale yellow and semi-transparent, and the gloss is a little dull.

Meat broth agar slope culture (medium 1) Growth is normal and its state is spreading, shiny and milky white translucent.

Broth liquid culture (medium 2) Grows. There is surface growth and upper growth is also observed.

Meat broth gelatin puncture culture (medium 3) Liquefaction of gelatin is observed.

Litmus milk medium (medium 4) Milk liquefaction is observed, but there is no clear change in litmus pigment.

(C) Physiological properties Nitrate reduction and denitrification (Medium 5, Medium 6) Nitrate reduction and denitrification are negative.

MR test (medium 7) positive.

VP test (medium 7) positive.

Formation of indole (medium 8) Negative.

Production of hydrogen sulfide (Medium 9) Negative.

Hydrolysis of starch (medium 10) Negative.

Utilization of citric acid (Medium 11, Medium 12, Medium 13) Negative in both Cosa medium and Simmons medium. Positive in Kristensen medium.

Use of inorganic nitrogen source (medium 14) Neither nitrate nor ammonium salt is used.

Pigment formation (medium 15, medium 16) Yellow pigment formation in KING-B medium.

Urease (medium 17) Negative oxidase (medium 18) Positive, not positive.

Catalase (medium 19) positive.

Growth range (medium 2) The growth temperature range is 10 to 50 ° C, and the optimum growth temperature range is 20 to
The pH range for growth at 40 ° C was pH 5-10.

Attitude toward oxygen Aerobic.

OF test (medium 20) Only aerobic growth.

Utilization of sugar (medium 21) (+: used, −: not used) 1. L-arabinose + 2. D-xylose + 3. D-glucose + 4. D-mannose + 5. D-fructose + 6 .D-galactose + 7. Maltose + 8. Sucrose + 9. Lactose -10. Trehalose + 11. D-sorbit -12. D-mannite + 13. Inosit + 14. Glycerin + 15. Starch- In VP medium PH in pH 7 (medium 7) pH 5.0 Growth in salt-containing medium (modified medium 1) Grow at 5%.

Grows at 7%.

Grows at 10%.

Grow at pH 7.7 (Medium 22) Grow.

Deamination of phenylalanine (medium 23) Negative.

Casein decomposition (medium 24) positive.

Regarding the above-mentioned mycological properties, Vergi's Manual
Of Date Bacteriology (Berge
y's Mannual of Determinative Bacteriology) 8th Edition and The Genus Bacillus,
Agriculure Handbook No.427, Agricult by Ruth, E. Gordon
ural Research Service, USDepartment of Agricultr
Based on the result of comparison and search with reference to e.Washingeton DC (1973), it is recognized that the strain used in the present invention is a kind of Bacillus genus, which is a spore bacillus. And this strain is bacteria, Horikoshi and Akiba ("Alkalophilic Microor
ganism ", Japan Scientific Society Press (Tckyo), 19
Published in 1982, the so-called alkalophilicity (Alkaloph)
ilic) microorganisms grow in an alkaline medium with a pH of 8 or more, and cannot grow in a neutral pH range below this,
Since it can grow from a weakly acidic region to an alkaline region (pH 5 to 10), it is clearly different from this alkalophilic microorganism and can be judged to be a general neutral and viable Bacillus microorganism. .

Furthermore, when this strain is compared with other known strains of the genus Bacillus, Bacillus pumilus (Bacillus) is the most closely related species.
pumilis). However, when comparing this strain with known strains of Bacillus pumilus, since the known strain does not produce at least alkali-resistant cellulase, the present inventors determined that this strain is a new strain, and Bacillus sp. KSM-597. It was named and deposited at the Institute of Microbial Technology, Institute of Industrial Technology (FERM P-9573).

In order to obtain the alkali-resistant cellulase and cellulase K-597 using this strain, the medium may be inoculated with the strain and cultured according to a conventional method. It is preferable that an appropriate amount of assimilable carbon source and nitrogen source be contained in the medium. The carbon source and the nitrogen source are not particularly limited, but examples thereof include inorganic ammonium nitrate, ammonium sulfate, ammonium salt as the nitrogen source,
Ammonium phosphate, sodium nitrate, corn gluten meal, soybean flour, corn steep liquor, casamino acid, yeast extract, pharmamedeia, sardine meal, meat extract, peptone, hypro, aji power, corn soybean meal, coffee meal, cottonseed oil cake , Cultivators, amiflex and adipron, zest, azix and the like. Further, as a carbon source, in addition to rice husks, rice flour, paper, general paper, plant fiber such as sawdust, molasses, invert sugar, carboxymethyl cellulose (CMC), avicel, cellulose cotton, xylan, pectin, it can be assimilated Carbon sources such as arabinose, xylose, glucose,
Examples include mannose, fructose, galactose, sucrose, lactose, trehalose, maltose, sorbitol, mannitol, inositol, glycerin, soluble starch, and assimilable organic acids such as acetic acid and citric acid.
In addition, phosphoric acid, Mg ²⁺ , Ca ²⁺ , Mn ²⁺ , Zn ²⁺ , Co ²⁺ , N
Inorganic salts such as a ⁺ and K ⁺ , and if necessary, inorganic and organic micronutrient sources can be appropriately added to the medium.

Collection and purification of the target substance, cellulase K-597, from the thus obtained culture can be carried out according to a general enzyme collection and purification means. That is, the crude enzyme solution can be obtained by removing the bacterial cells from the culture solution by an ordinary means for separating individual liquid such as centrifugation or excess. This crude enzyme solution can be used as it is, but if necessary, a crude enzyme is obtained by a separating means such as a salting-out method, a precipitate, and an ultrafiltration method, and further purified and crystallized by a known method, It is also possible to use it as a purified enzyme.

The alkaline cellulase K-597 of the present invention thus obtained
Has the following enzymatic properties: The enzyme activity is measured according to the following method, and the buffer solution used is as follows.

pH3 ~ 8 Maculbein buffer pH8 ~ 11 Glycine-sodium hydroxide buffer pH12 ~ 13 Potassium chloride-sodium hydroxide buffer Enzyme activity assay: (1) CMCase activity 10mg CMC (A-01L, Sanyo Kokusaku Pulp Co., Ltd.), 0.1 ml of the enzyme solution was added to 0.9 ml of a substrate solution containing 100 μmol of various buffer solutions (McClubein, phosphoric acid, glycine-NaOH, etc.) and reacted at 30 ° C. for 20 minutes. After the reaction, 3,5-dinitro-salicylic acid (3,5-
The reducing sugar was quantified by the dinitro-salicylic acid (DNS) method. That is, 1.0 ml of the DNS reagent was added to 1.0 ml of the reaction solution, color was developed by heating at 100 ° C. for 5 minutes, and after cooling, 4.0 ml of deionized water was added to dilute. Colorimetrically quantified at a wavelength of 535 nm, the enzyme titer was 1 μmol / min under the above conditions.
The amount of enzyme that produces a reducing sugar corresponding to the glucose was set as 1 unit.

(2) p-nitrophenyl cellobioside degrading activity 0.1 μmol p-nitrophenyl cellobioside (Sigma), 100 μmol phosphate buffer solution (pH 7.0) 1.0
After reacting an appropriate amount of enzyme solution in 30 ml at 30 ° C, 1M Na ₂ CO
Add 0.3 ml of ₃ and 1.7 ml of deionized water sequentially to release p-
Nitrophenol was colorimetrically determined at 400 nm. The enzyme titer was defined as 1 unit of the amount of enzyme that liberates 1 μmol of p-nitrophenol in 1 minute under the above conditions.

(3) Avicel, cellulose powder, phosphoric acid swelling cellulose, alkali swelling cellulose, and paper decomposition activity 20 mg Avicel (Merck), 200 μmol phosphate buffer (pH
Add an appropriate amount of enzyme solution to 2.0 m of reaction solution containing 7.0),
It was made to act while shaking at 250 rpm at ℃. After the reaction, carry out cooling centrifugation (5 ℃, 3000rpm, 20 minutes), and the supernatant 1.0
ml to 3,5-dinitro-salicylic acid (3,5-dinitro-salic
The reducing sugar was quantified by the ylic acid (DNS) method. Cellulose powder decomposition activity is cellulose powder (Toyo Paper Co., Ltd.)
The phosphate-swelling cellulose and alkali-swelling cellulose degrading activity was determined by the method of Tomita et al. (Tomita Y et al; J. Fermen.
t.Thechnol., 52 , 235, 1974), the paper decomposition activity of paper (cellulase activity assay paper,
Toyo No. 51-special) was used, and the same procedure as in the case of avicelase activity was performed. The enzyme titer was defined as 1 unit of the amount of enzyme that produced a reducing sugar corresponding to 1 μmol of glucose per minute under the above conditions.

(4) Cellobiase activity 10 mg of cellobiose (Kanto Chemical Co., Inc.) and 50 μmol of phosphate buffer (pH 7.0) in 1.0 ml of a reaction solution containing an appropriate amount of the enzyme solution.
After incubating at 100 ° C for 5 minutes at 100 ° C to inactivate the enzyme, the amount of glucose produced is determined by the mutarotase / GOD method (Gluco
se C-Test, Wako Pure Chemical Industries, Ltd.). The enzyme titer was defined as 1 unit of the amount of enzyme that produced 2 μmol of glucose per minute under the above conditions.

(Enzymatic properties) (1) Action It acts well on cellulose, paper, Avicel, and fibrin such as CMC sugar and dissolves them to form reducing sugar such as glucose.

(2) Substrate specificity In addition to CMC, this enzyme is used for cellulose powder, Avicel,
It had activity on paper and p-nitrophenyl cellobioside.

(3) Working pH and optimum pH The working pH range is extremely wide, from 3 to 13. Optimum pH
Is 7 and has a relative activity of 50% or more of the activity at a suitable pH even in the range of 4.5 to 11.5, which is the most alkaline side of the cellulases studied in the past, and has an action pH range. Is a broad enzyme (Fig. 1).

(4) pH stability The pH stability was examined by measuring the residual activity at pH 6 after holding at 30 ° C for 1 hour at each pH. As a result, it is extremely stable at pH 5 to 11.5 and does not deactivate, and even at pH 4.5 to 12.5, about 50
% Activity was maintained. The enzyme is sufficiently stable even in the high alkaline region (Fig. 2).

(5) Working temperature when activity is measured at optimum temperature pH6 is 15-80
The optimum temperature was 60 ° C over a wide range of ° C.
Even in the range of 45 to 75 ° C, the activity of 50 at the optimum temperature
% Or more (Fig. 3).

(6) Temperature stability After treatment (pH7) for 30 minutes at each temperature, the residual activity was measured at the optimum pH (pH6). As a result, it was stable at 50 ° C and about 50% remained at 55 ° C. It had activity (Fig. 4).

(7) Molecular weight The molecular weight of this enzyme was measured by the gel filtration method using Bio-Gel P-150 (“Bio-Gel P-150”).
A main peak at 0,000 was observed.

(8) Effect of metal ions For this enzyme, various metal ions (Al ³⁺ , Fe ³⁺ , Ba ²⁺ , Ca
²⁺ , Cd ²⁺ , Co ²⁺ , Cu ²⁺ , Fe ²⁺ , Hg ²⁺ , Mn ²⁺ , Mg ²⁺ , Ni ²⁺ , Pb ²⁺ , Zn
²⁺ , K ⁺ , Na ⁺ ) coexisted during the activity measurement, and the effect was examined (the concentration of various metal ions was 1 mM, K ⁺ , Na ⁺ was 50 mM).

As a result, inhibition was observed with Hg ²⁺ and activation was observed with Co ²⁺ .

(9) Effects of surfactants Various surfactants (eg, LAS, AS, ES, AOS, α-SF
The effect of E, SAS, soap, and polyoxyethylene secondary alkyl ether) on the enzyme activity was investigated. The effect of the surfactant was examined by coexisting with 0.05% of the surfactant during the activity measurement. As a result, almost no inhibition was observed with any of the surfactants. Almost no inhibition of the activity was observed even with the strong detergent Zodium dodecyl sulphate.

(10) Protease resistance Detergent proteases such as API-21 (Showa Denko), Maxatase (Gist) and Alcalase (Novo)
When the activity was measured, the coexistence (0.1 mg / ml) was made to exist and the effect was examined, and it was found that they had strong resistance to any protease.

(11) Effects of chelating agents Chelating agents such as EDTA, EGTA, citric acid, zeolite,
Sodium tripolyphosphate was allowed to coexist during the activity measurement and its effect was examined, but almost no inhibition was observed.

〔The invention's effect〕

Although the cellulase K-597 of the present invention has an optimum pH of 7, it has an optimum pH of 60 even at a highly alkaline pH of 11.5.
It has a relative activity of not less than%, and is extremely stable at pH 5 to 11.5. Further, it is hardly affected even by detergent-containing components such as surfactants, proteases and chelating agents. Therefore, the present enzyme can be advantageously used as a blending component of a detergent composition.

Since Bacillus sp. KSM-597 is a strain that grows neutrally, it is possible to industrially produce alcal-resistant cellulase more easily than alkalophilic bacteria.

〔Example〕

 Hereinafter, the present invention will be described in more detail with reference to examples.

Reference Example 1 A spoonful (about 0.5 g) of the soil of Nikko City, Tochigi Prefecture was suspended in sterilized physiological saline and heat-treated at 80 ° C. for 10 minutes. The supernatant of this heat treatment solution was appropriately diluted and applied to a separation agar medium (medium 1). Then, this was cultured at 30 ° C. for 3 days to form a colony. CMCase-producing bacteria were obtained by selecting those that form a transparent zone due to the dissolution of CMC around the settlement. Further, the obtained microorganism was inoculated into a liquid medium of Medium 2 and shake-cultured at 30 ° C. for 3 days. After the culture, the CMCase activity of the supernatant obtained by centrifugation was measured at pH 3 to 13 to screen for alkali-resistant cellulase-producing bacteria.

The Bacillus sp. Used in the present invention is obtained by the above method.
We were able to obtain KSM-597 strain (FERM P-9573).

Medium 1.CMS 2% polypeptone 0.5% yeast extract _{0.05% KH 2 PO 4 0.1%} Na 2 HPO 4 · 12H 2 O 0.25% MgSO 4 · 7H 2 O 0.02% agar 0.75% pH 6.8 medium 2. CMS 1% polypeptone reference 1% yeast extract _{0.5% KH 2 PO 4 0.1%} Na 2 HPO 4 · 12H 2 O 0.25% MgSO 4 · 7H 2 O 0.02% pH6.8 example 1 Bacillus sp KSM-597 obtained in reference example 1 example Liquid medium 2 of No. 1 was inoculated and cultured at 30 ° C. for 3 days with shaking.
After culturing, the bacterial cells were removed by centrifugation to obtain a crude enzyme solution. Ethanol (3) was added to this crude enzyme solution 1 in dry ice-ethanol, and the resulting precipitate was centrifuged and freeze-dried to give cellulase K-597 as a dry powder.
10 g (specific activity 8 units / g; same as below measured value at pH 9)
Got

Example 2 Using liquid medium 2 in which CMC was changed to 1% sucrose and polypeptone was changed from 7% CSC to 7% CSL (corn steep liquor), shaking culture was carried out at 30 ° C. for 3 days according to Example. . The CMCase activity of the supernatant obtained by centrifugation of the obtained culture broth was 300 units /.

[Brief description of drawings]

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-treated pH and relative activity. FIG. 3 is a drawing showing the relationship between enzyme reaction temperature and relative activity. FIG. 4 is a drawing showing the relationship between enzyme treatment temperature and relative activity.

Claims (1)

[Claims] 1. An alkali-tolerant cellulase K-597 having the following enzymatic properties. (1) Action It often acts on fibrin such as carboxymethyl cellulose, cellulose, paper filter, and Avicel, and dissolves them to produce reducing sugar. (2) Substrate specificity In addition to carboxymethyl cellulose, it has activity against phosphoric acid swollen cellulose, cellulose powder, Avicel, paper filter and p-nitrophenyl cellobioside. (3) Working pH and optimum pH The working pH range is 3 to 13. The optimum pH is 7, 4.
It has a relative activity of 50% or more of the activity at the optimum pH even in the range of 5 to 11.5. (4) pH stability It is extremely stable at pH 5 to 11.5 and does not deactivate, and maintains an activity of about 50% or more even at pH 4.5 to 12.5. (5) Optimum temperature The working temperature is in a wide range of 15 to 80 ° C, and the optimum temperature is 60 ° C. Further, it has 50% or more of the activity at the optimum temperature even in the range of 45 to 75 ° C. (6) Molecular weight has a molecular weight peak at about 40,000 (Biogel P-150
By the gel filtration method). (7) Effect of metal ions It is inhibited by Hg ²⁺ and activated by Co ²⁺ . (8) Effect of surfactant LAS, AS, ES, AOS, α-SFE, SAS, soap, and polyoxyethylene secondary alkyl ether hardly inhibit the activity. (9) Protease resistance It has resistance to protease. (10) Effect of chelating agent EDTA, EGTA, sodium tripolyphosphate, zeolite, and citric acid do not inhibit the activity.