JPH0648983B2 - 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 of many types, mainly Aspergillus, Penicillium, Trichoderma, Fusarium, Humicola, Acremonium, etc. , Bacillus, and other 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 novel industrial use of cellulase, its use as a blending component of a detergent for clothing has been studied and attracted attention (Japanese Patent Publication No. 59-49279 and Japanese Patent Publication No. 60-23).
No. 158, 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. In reality, the presence of cellulases that exhibit maximum activity in the alkaline region or that have alkali resistance, so-called alkali cellulases and alkali-tolerant cellulases, which is a necessary condition as a blending component, 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, the neutral means a pH range of 6 to 8, and the alkaline means a higher pH range.

That is, conventionally, as a method for producing an alkaline cellulase and an alkaline-tolerant cellulase that can be used as a detergent composition for clothes, a method of collecting cellulase A by culturing an alkalophilic Bacillus bacterium (Japanese Patent Publication No. 50-28515), A method for culturing an alkaliphilic bacterium belonging to the genus Cellulomonas to produce alkaline cellulase 301-A (Japanese Patent Laid-Open No. 224686/1983), and a method for culturing alkalophilic Bacillus No. 1139 to produce carboxymethylcellulase (Fukumori , F., Kudo, T. and
Horikoshi, K. J. Gen. Microbiol. , 131, 3
339, (1985)) and a method for producing an alkaline cellulase using a Streptomyces sp.
No. 19483) was reported, and none of them was suitable for industrial fermentation production.

However, recently, the present inventors have found that Bacillus sp. KSM-635 (FERM P-88), which is a kind of alkalophilic microorganism.
72) 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 has been found that the fermentative production can be achieved.

[Problems to be solved by the invention]

However, the above Bacillus SP KSM-635
The culturing conditions described above 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, and compared with ordinary neutral microorganisms The knowledge about the physiology and biochemistry of microorganisms has not been sufficiently accumulated, and the medium preparation and culture method for industrial fermentation production have been difficult to operate.

[Means for solving problems]

In order to solve such a problem, the present inventors have maintained an alkaline cellulase having an optimum pH in the alkaline region or a high activity even in the alkaline region even when compared with the maximum activity at the optimum pH. To obtain a neutral bacterium that produces an alkali-resistant cellulase to be obtained, a cell strain that grows in a neutral region is used as a host, and the corresponding cellulase gene is cloned.
In parallel with the so-called gene recombination means, as a result of conducting an intensive search for the above strain in nature, a group of microorganisms belonging to the genus Bacillus produce alkaline-tolerant cellulase despite growing in a neutral medium. I found that.

And, as an example of such an alkali-tolerant cellulase-producing microorganism, the present inventor has isolated Bacillus sp. KSM-534 (FERM) from the soil of Haga-gun, Tochigi prefecture, and depositing it in the Institute of Microbial Technology, Institute of Industrial Science and Technology. P-9010).

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

Medium 1. Meat extract, 1.0: Bactopeptone, 1.0: Nacl,
0.5; Bacto agar, 1.5 (pH7.2) (indicated by weight%; the same applies hereinafter) Medium 2. Meat extract, 1.0: Bactopeptone, 1.0: Nacl,
0.5; (pH 7.2) medium 3. Meat extract, 1.0: Bactopeptone, 1.0: Nacl,
0.5; gelatin, 1.0 (pH 7.2) medium 4. Bactolithomas milk, 10.0 medium 5. Bactopeptone, 1.0: KNO ₃ , 0.1 medium 6. Bactopeptone, 1.0: NaNO ₃ , 1.0 medium 7. Bactopeptone, 0.7: NaCl, 0.5; glucose,
0.5 (pH 7.0) medium 8. Bactopeptone, 1.0 medium 9. TSI agar (manufactured by Eiken Kagaku); indicated amount of medium 10. Meat extract, 1.0: Bactopeptone, 1.0: Nac
l, 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 (pH 6.8) medium 12. Kristensen medium (manufactured by Eiken Chemical); indicated amount of medium 13. _{Glucose, 1.0; KH 2 PO 4,} 0.1; MgSO 4 · 7H
₂ O, 0.05; KCl, 0.02; nitrogen source, 0.1 (sodium nitrate and ammonium sulfate were used as nitrogen sources) (pH 7.2) Medium 14. King A medium "Eiken" (manufactured by Eiken Chemical Co.); indicated amount of medium 15. King B medium "Eiken" (manufactured by Eiken Chemical Co.); indicated amount of medium 16. Urea medium "Eiken" (manufactured by Eiken Chemical Co.); indicated amount of medium 17. Filter paper for cytochrome oxidase test (manufactured by Nissui Pharmaceutical) Medium 18.3% hydrogen peroxide solution Medium 19. OF basal medium (manufactured by Difco); indicated amount of medium 20. _{(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; BC
P (0.2% solution), 0.4 medium 21. Bact Sabouraud Dextrose Agar Medium (manufactured by Difco); indicated amount Medium 22. Meat extract, 0.3: Bactopeptone, 0.5: Yeast extract, 1.0: Glycerin, 2.0 Medium 23. Phenylalanine malonate medium (manufactured by Nissui Pharmaceutical); indicated amount of medium 24. Skim milk, 5.0; Bacto agar, 1.5 medium 25. Meat extract, 0.3; Bactopeptone, 0.5; L-
Tyrosine, 0.5 Bacto agar, 1.5 (Mycological properties) (a) Microscopic observation result The size of the bacterial cell is 0.4 to 0.8 μm × 1.5 to 6.0.
It is a rod-shaped bacterium having a diameter of μm, and columnar or elliptical endospores (0.6 to 0.8 μm × 1.0 to 2.5 μm) are formed in the center of the bacterium. It has periflagellates and is motile. Gram stain is positive.
There is no acid resistance.

(B) Growth condition in various media Meat agar plate culture (medium 1) Growth condition is weak. The shape of the settlement is circular, the surface is smooth and the periphery is also smooth. The color tone of the village is light yellow, semi-transparent and glossy.

Meat broth agar slant culture (medium 1) Growth is weak, the state is spread-like, glossy, and pale yellow and translucent.

Broth liquid culture (medium 2) Grows and becomes cloudy.

Meat broth gelatin stab culture (medium 3) Grows on the surface layer and 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 reactions (medium 5, medium 6) are both negative.

MR test (medium 7) Negative and positive are not attached (pH5.2).

VP test (medium 7) Negative and positive do not adhere (pH5.2).

Formation of indole (medium 8) Negative.

Production of hydrogen sulfide (Medium 9) Negative.

Hydrolysis of starch (medium 10) Positive.

Utilization of citric acid (medium 11, medium 12) Positive in both Cosser medium and Christensen medium.

Use of inorganic nitrogen source (medium 13) Both nitrate and ammonium salts are used.

Dye formation (medium 14, medium 15) Negative.

Urease (medium 16) negative.

Oxidase (medium 17) positive.

Catalase (medium 18) positive.

Growth range (medium 2) Growth temperature range is 15 to 50 ° C, growth optimum temperature range is 25 to 40 ° C Growth pH range is pH 5 to 11, growth optimum pH range is pH 6 to
It was 10.

Attitude toward oxygen Facultative anaerobic.

OF test (medium 19) Both aerobic and anaerobic grow, and gas is generated.

Utilization of sugar (medium 20) (+: used, −: not used) 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-Mannit-13. Inosit + 14. Glycerin + 15. PH in starch + VP medium (medium 7) pH 5.2 Growth in salt-containing medium (modified medium 1) Grow at 5%.

Grows at 7%.

It does not grow at 10%.

Grow at pH 5.7 (Medium 21) Grow.

Production of dihydroxyacetone (medium 22) Negative.

Deamination of phenylalanine (medium 23) Negative.

Decomposition of casein (medium 24) Positive.

Degradation of tyrosine (medium 25) Negative.

This KSM-534 strain is easily recognized as a kind of Bacillus genus, which is a spore-bearing bacterium. And further, regarding the above-mentioned mycological properties, Berges Manual of Definite Native Bacteriology Berg
ey's, Manual of Determinative Bacteriology) 8th
Edition and The Genus Bacillu
s ”, Ruth, E. Gordon“ Agriculture Handbook No. 4
27 ", Agricultural Research Service, US Dep.
artment of Agriculture, Washington D.D. C. (197
When comparing and searching with reference to 3)), this strain was recently found to be
Horikoshi and Akiba (“Alkalophilic Microorganism”, Japan
Scientific Society Press (Tokyo), published in 1982), so-called Alkalophilic microorganisms, that is, grown in an alkaline medium of pH 8 or more,
It does not belong to microorganisms that cannot grow in a neutral pH range below this range, and it is from a weakly acidic range to an alkaline range (pH 5 to 1).
It can be judged as a general neutral and viable Bacillus microorganism capable of growing in 1).

Furthermore, when this strain was compared with other known strains of the genus Bacillus, Bacillus licheniformis (Ba
cillus licheniformis). However,
Comparing the known strain of Bacillus licheniformis with this strain, they differ in the reducing ability of assimilable nitrates of mannitol and xylose. Further, since the strain belonging to Bacillus licheniformis does not produce at least alkali-resistant cellulase, this strain is judged to be a new strain.

In order to obtain the alkali-tolerant cellulase of the present invention using the above strains, the medium may be inoculated with the strains 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 chloride, ammonium phosphate, sodium nitrate as a nitrogen source, corn gluten meal, soybean powder, and corn steep liquor. , Casamino acid,
Yeast extract, pharma media, sardine meal, meat extract, peptone, hypro, ajpower, corn soybean meal, coffee meal, cottonseed oil meal, cultivator, amiflex and adipron, zest, azix and the like can be mentioned. Further, as carbon sources, rice husks, rice, filter paper, general papers, vegetable fibers such as sawdust, molasses, invert sugar, C
In addition to MC, Avicel, cellulose cotton, xylan, pectin, assimilable carbon sources such as arabinose, glucose, fructose, galactose, maltose, sucrose,
Examples thereof include lactose, sorbitol, inositol, glycerin, soluble starch, and assimilable organic acids such as acetic acid and citric acid. In addition, phosphoric acid, Mg ²⁺ , Ca ²⁺ ,
Inorganic salts such as Mn ²⁺ , Zn ²⁺ , Co ²⁺ , Na ²⁺ and K ⁺ , and if necessary, inorganic and organic micronutrient sources can be appropriately added to the medium.

Cellulase, which is a target substance, can be collected and purified from the thus obtained culture in accordance with general means for collecting and purifying enzymes. That is, the crude enzyme solution can be obtained by removing the cells from the culture solution by a usual solid-liquid separation means such as centrifugation or filtration. This crude enzyme solution can be used as it is, but if necessary, salting out,
It is also possible to obtain a crude enzyme by a separation means such as a precipitation method or an ultrafiltration method, further purify and crystallize it by a known method, and use it as a purified enzyme.

The alkali-tolerant cellulase of the present invention thus obtained was designated as cellulase K-534. Hereinafter, the present invention will be further described with reference to this example. The enzyme activity was measured according to the following method, and the following buffer solution was used.

pH3-8 McClubain buffer pH8-11 Glycine-sodium hydroxide buffer pH12-13 Potassium chloride-sodium hydroxide buffer Enzyme activity measurement method: (1) CMCase activity 10 mg CMC (A-01L, Sanyo Kokusaku Pulp Co., Ltd.), 10
0.1 ml of the enzyme solution was added to 0.9 ml of a substrate solution containing 0 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 a (3,5-dinitro-salicylic a
The reducing sugar was quantified by the cid (DNS) method. That is,
Add 1.0 ml of DNS reagent to 1.0 ml of reaction solution, and
Color was developed by heating at 100 ° C, and after cooling, 4.0 ml of deionized water was added to dilute. This was colorimetrically determined at a wavelength of 535 nm. 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.

(2) P-nitrophenyl cellobeoside decomposing activity 0.1 μmol of p-nitrophenyl cellobioside (Sigma) and 100 μmol of phosphate buffer (pH 7.0) in 1.0 ml of a reaction solution containing an appropriate amount of enzyme. After operating at 30 ℃, 1M
Add 0.3 ml of Na ₂ CO ₃ and 1.7 ml of deionized water sequentially,
The liberated p-nitrophenol was colorimetrically determined at 400 nm. The enzyme titer is 1 μmol p / min under the above conditions.
-The amount of enzyme that releases nitrophenol was 1 unit.

(3) Avicel, cellulose powder, and filter paper degrading activity 20 mg Avicel (Merck) and 200 μmol of phosphate buffer (pH 7.0) were added to 2.0 ml of a reaction solution at an appropriate amount of enzyme at 30 ° C. and 250 rpm. It was made to act while shaking. After the reaction, cooling centrifugation (5 ° C, 3000 rpm, 20
Minutes) and 1.0 ml of the supernatant was quantified for reducing sugars by the DNS method. Cellulose powder decomposition activity was measured using cellulose powder (Toyo Roshi Kaisha, Ltd.), and filter paper decomposition activity was measured using filter paper (cellulase activity assay filter paper, Toyo No. 51-special) in the same manner as in the case of avicelase activity. 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 After reacting 1.0 ml of a reaction solution containing 10 mg cellobiose (Kanto Kagaku) and 100 μmol phosphate buffer (pH 7.0) with an appropriate amount of enzyme at 30 ° C., 100 ° C. for 2 minutes After the treatment to deactivate the enzyme, the amount of glucose produced is determined by the amount of mlotase.G.
It was measured by the OD method (Glucose 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 often acts on fibrin such as cellulose, paper, Avicel, CMC and the like to dissolve them and produce reducing sugar such as glucose.

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

(3) Working pH and optimum pH The working pH range is extremely wide ranging from 3 to 12.5.
The optimum pH is about 6.5, and it has a relative activity of 50% or more of the activity at the optimum pH even in the range of 4.5 to 10.5. Among them, it can be said that the enzyme has the widest working pH range from the acidic side to the alkaline side (Fig. 1).

(4) pH stability The pH stability was investigated by measuring the residual activity after holding at 30 ° C for 1 hour at each pH. As a result, it was extremely stable at pH 5 to 11 and did not deactivate, and even at pH 4.5 to 12.5, it was about 5
The activity was maintained at 0% or more. This enzyme is sufficiently stable even in the highly alkaline region (second
Figure).

(5) Optimum temperature The working temperature was in a wide range of 15 to 85 ° C, and the optimum temperature was about 70 ° C. Further, even in the range of 45 to 75 ° C, it had 50% or more of the activity at the optimum temperature (Fig. 3).

(6) Temperature stability After the treatment at each pH for 30 minutes at the optimum pH, the residual activity was measured and found to be stable at about 40 ° C.
It had a residual activity of about 50% even at ℃ (4th
Figure).

(7) Molecular weight When the molecular weight of this enzyme was measured by the gel filtration method using Sephadex G-100 (“Sephadex G-100”), major peaks at about 15,000 and about 30 thousand were observed. .

(8) Effect of metal ions Regarding this enzyme, various metal ions (Al ³⁺ , Fe ³⁺ , Ba ²⁺ ,
Ca ²⁺ , Cd ²⁺ , Co ²⁺ , Cr ²⁺ , Cu ²⁺ , Fe ²⁺ , Hg ²⁺ , Mn ²⁺ , Mo
²⁺ , Ni ²⁺ , Pb ²⁺ , Zn ²⁺ , Li ⁺ , K ⁺ , Na ⁺ ) were made to coexist during the activity measurement, and the effect was examined (various metal ion concentrations were 1 mM, K ⁺ , Na ^+. Is 50 mM).

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

(9) Effect of surfactants Various surfactants (for example, LAS, AS, ES, AO
The effect of S, α-SFE, SAS, soap, polyoxyethylene secondary alkyl ether) on the enzyme activity was investigated. After treatment with a 0.05% solution of a surfactant at 30 ° C. for 15 minutes, the activity was measured. As a result, almost no inhibition was observed with any of the surfactants. No inhibition of activity was observed even with the potent detergent Sodium dodecyl sulphate.

(10) Protease resistance Detergent proteases such as API-21 (Showa Denko), Maxatase (Gist) and Alcalase (Novo) were allowed to coexist (0.1 mg / ml) during activity measurement, and the effect was examined. It was found that they have strong resistance to any protease.

(11) Effect of chelating agent Chelating agents such as EDTA, EGTA, citric acid, zeolite, and sodium tripolyphosphate (STPP) were allowed to coexist during activity measurement, and their effects were examined, but almost no inhibition was observed.

〔The invention's effect〕

The alkali-tolerant cellulase of the present invention is extremely stable at pH 5 to 11 even though it has an optimum pH of 5 to 7. In addition, there is almost no inhibition even by the synthesis of detergents such as surfactants, proteases and chelating agents. Therefore, the present enzyme can be advantageously used as a blending component of a detergent composition.

〔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 in Kai Town, Haga-gun, 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. CMC around the village
Those that form a transparent zone based on the dissolution of
Acquired ase-producing bacteria. Further, the obtained microorganism was inoculated into the liquid medium of the medium 2 and shake-cultured at 30 ° C. for 3 days. After culturing,
The CMCase activity of the centrifuged supernatant was adjusted to pH 3
.About.13 and screened for alkali-resistant cellulase-producing bacteria.

The Bacillus sp. KS of the present invention is produced by the above method.
The M-534 strain (FERM P-9010) could be obtained.

Medium 1. 0.05% CMC 2% polypeptone 0.5% yeast extract _{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. CMC 1% Polypeptone 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% pH 6.8 Example 1 Reference Example Bacillus sp. KSM-534 obtained in 1 was inoculated into the liquid medium 2 of Example 1 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. To this crude enzyme solution 1, ethanol (3) was added in dry ice-ethanol, and the resulting precipitate was centrifuged and freeze-dried to obtain dry powder of cellulase K-534, 10 g (specific activity 3 units / g; The same applies for the measured values at pH 9).

Example 2 A medium in which CMC was replaced with 1% sucrose and polypeptone was replaced with 7% corn steep liquor in the liquid medium 2,
According to Example 2, shake culture was carried out at 30 ° C. for 2 days. The CMCase activity of the supernatant of the obtained culture broth was 50 units / cm.

[Brief description of drawings]

FIG. 1 is a drawing showing the relationship between the enzyme reaction pH and relative activity of alkali-tolerant cellulase K-534. FIG. 2 is a drawing showing the relationship between the treatment pH and the relative activity of the same enzyme. FIG. 3 is a drawing showing the relationship between reaction temperature and relative activity of the same enzyme. FIG. 4 is a drawing showing the relationship between the treatment temperature and the relative activity of the same enzyme.

Claims (1)

[Claims] 1. An alkali-tolerant cellulase K-534 having the following enzymatic properties. (1) Action It acts well on fibrin such as carboxymethyl cellulose (CMC), cellulose, filter paper, and Avicel, and dissolves them to produce reducing sugar such as glucose. (2) Substrate specificity In addition to CMC, cellulose powder, Avicel, filter paper and p
-Has activity against nitrophenyl cellobioside. (3) Working pH and optimum pH The working pH range is 3 to 12.5. The optimum pH is about 6.5, and even in the range of 4.5 to 10.5, the relative activity is 50% or more of the activity at the optimum pH. (4) pH stability It is extremely stable at pH 5 to 11 and does not inactivate, 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 85 ° C, and the optimum temperature is about 70 ° C. Further, it has 50% or more of the activity at the optimum temperature even in the range of 45 to 75 ° C. (6) It has a molecular weight peak at about 15,000 and about 30,000 (by gel filtration method using Sephadex G-100). (7) is activated by the influence Ba ²⁺ metal ions is inhibited by Hg ^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, STPP, zeolite, and citric acid do not inhibit the activity.