JPH06339369A - Microorganism producing alkali-resistant cellulase - Google Patents

Abstract

PURPOSE:To obtain an alkali-resistant cellulase capable of advantageous blending as a mixture component of a detergent composition by growing a specified microorganism producing an alkali-resistant cellulase in a neutral medium. CONSTITUTION:Bacillus sp. KSM-534 stipulated as Bikoken stipulation no. 9010 is cultured. This microorganism grows within a neutral pH range and the optimum pH of the produced alkali-resistant cellulase is 5 to 7. This cellulase is stable even at pH 5 to 11 and is scarcely deactivated by a surfactant, protease a chelating agent, etc.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a microorganism which produces a novel alkali-tolerant cellulase.

[0002]

2. Description of the Related Art Development of cellulase, which is a fibrinolytic enzyme, 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 and other filamentous fungi, Pseudomonas, Cellulomonas, Luminococcus. , 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 clothes has been studied and attracted attention (Japanese Patent Publication No. 59-49279, Japanese Patent Publication No. 23158/60). Kokoku 60-36240). However, most of the cellulases hitherto found in nature are classified into so-called neutral or acid cellulases that show the maximum and stable enzyme activity in the neutral or 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 optimum
Alkali-tolerant cellulase has a pH in the alkaline region, and the optimum pH is in the neutral to acidic region, but even in the alkaline region, it is sufficiently active as compared with the activity at the optimum pH. Those that have and retain stability.

Neutral means a pH range of 6 to 8, and alkaline means a higher pH range. That is, conventionally
As a method for producing an alkaline cellulase and an alkaline-tolerant cellulase which 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) and belonging to Cellulomonas genus Method for producing alkaline cellulase 301-A by culturing alkalophilic bacterium (JP-A-58-58)
No. 224686), alkaliphilic Bacillus No. 1
Method for producing carboxymethylcellulase by culturing 139 (Fukumori, F., Kudo, T. an
d Horikoshi, K .; J. Gen. Micr
obiol. , 131, 3339, (1985)) and a method of producing an alkaline cellulase using one of the genus Streptomyces (Japanese Patent Laid-Open No. 61-19483), and all of them are industrial fermentations. It was not suitable for production.

However, recently, the present inventors have found that Bacillus sp.
lus sp. ) KSM-635 (FERM P-88
72) efficiently produces alkaline cellulase K suitable as a detergent composition for clothes, 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.

[0007]

However, the culture conditions for Bacillus sp. KSM-635 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 methods for industrial fermentation production have become operational difficulties.

[0008]

In order to solve such problems, the present inventors have found that alkaline cellulase having an optimum pH in the alkaline region or even in the alkaline region
In order to obtain a neutral bacterium that produces an alkali-tolerant cellulase that can maintain a high activity even when compared to the highest activity at pH, a strain that grows in a neutral region is used as a host, and the corresponding cellulase gene is cloned. As a result of conducting an intensive search for the above strains in the natural world in parallel with the means of gene recombination, microorganisms belonging to the genus Bacillus isolated from the soil of Haga-gun, Tochigi Prefecture, grew in neutral medium The present invention was completed by discovering that it produces resistant cellulase and depositing it at the Institute for Microbial Technology, Institute of Industrial Science and Technology.

That is, the present invention has the ability to produce alkali-tolerant cellulase K-534,
Bacillus SP deposited as No. 10
illus sp. ) KSM-534 is provided.

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
(PH 7.2) (label is% 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. Bactolithos 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; 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 (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; M
_{gSO 4 · 7H 2 O, 0.05} ; KCl, 0.02; nitrogen source, 0.1 (nitrogen source, using sodium nitrate and ammonium sulfate) (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; BCP (0.2% solution), 0.4

Medium 21. Bacto 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; Bect 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 results The size of the cells 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 (1) 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.

(2) Meat broth agar slant culture (medium 1) Growth is weak, and its state is spreading, glossy, and pale yellow and translucent.

(3) Broth liquid culture (medium 2) It grows and becomes cloudy. (4) Meat broth gelatin stab culture (medium 3) Grows on the surface layer and liquefaction of gelatin is observed. (5) Litmus milk medium (medium 4) Although liquefaction of milk is observed, there is no clear change in litmus pigment.

(C) Physiological properties (1) Nitrate reduction and denitrification reactions (medium 5 and medium 6) are both negative. (2) MR test (medium 7) Negative and positive are not clear (pH 5.2). (3) VP test (medium 7) Negative and positive are not clear (pH 5.2). (4) Formation of indole (medium 8) Negative. (5) Generation of hydrogen sulfide (medium 9) Negative.

(6) Starch hydrolysis (medium 10) Positive. (7) Utilization of citric acid (medium 11 and medium 12) Both were positive in Qosa medium and Christensen medium. (8) Use of inorganic nitrogen source (medium 13) Both nitrate and ammonium salt are used. (9) Formation of pigment (medium 14, medium 15) Negative. (10) Urease (medium 16) Negative.

(11) Oxidase (medium 17) positive. (12) Catalase (medium 18) positive. (13) Range of growth (medium 2) The temperature range of growth is 15 to 50 ° C., the optimum temperature range of growth is 25 to 40 ° C. The pH range of growth is pH 5 to 11, and the optimum pH range of growth is pH 6 to
It was 10. (14) Attitude toward enzymes Facultative anaerobic. (15) OF test (medium 19) Both aerobic and anaerobic grow, and gas is generated.

(16) 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. Starch +

(17) pH in VP medium (medium 7) pH 5.2 (18) Growth in salt-containing medium (modified medium 1) Grow at 5%. Grows at 7%. It does not grow at 10%.

(19) Growth at pH 5.7 (medium 2
1) Grow. (20) Production of dihydroxyacetone (medium 22) Negative. (21) Deamination of phenylalanine (medium 23) Negative. (22) Casein degradation (medium 24) Positive. (23) Degradation of tyrosine (medium 25) Negative.

It is easily recognized that the KSM-534 strain is a kind of Bacillus genus which is a spore bacillus. And further, regarding the above-mentioned mycological properties,
Vergie's Manual of Definite Native
Bacteriology (Bergey's Manual)
of Determinative Bacteri
8th edition and The Genus Bacillus ("T
he Genus Bacillus ”, Ruth,
E. Gordon “Agricultural Hand
book No. 427 ", Agricultural
Research Service, U.S.A. S. Dep
art of of Agricultural, Wa
shinton D. C. (1973)) and comparing and searching, this strain was recently found to be Horikoshi and Akiba (“A
lkalophilic Microorganis
m ”, Japan Scientific Society
tyPress (Tokyo), published in 1982, claims so-called alkalophili.
c) Microorganisms, that is, microorganisms that grow in an alkaline medium having a pH of 8 or more and cannot grow in a neutral pH range below this, and grow in a weakly acidic range to an alkaline range (pH 5 to 11). It is possible to judge that it is a general neutral and viable Bacillus microorganism.

Further, when this strain is compared with other known strains of the genus Bacillus, Bacillus licheniformis (Bacillus lichenformi) is the most closely related species.
s). However, when the strains belonging to the known Bacillus licheniformis and this strain are compared, they differ in the reducing ability of nitrite and assimilating nitrates of 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.

Using the strain of the present invention as described above,
To obtain potash-resistant cellulase, inoculate the strain with the medium,
Culture may be performed according to a conventional method. Can be assimilated into the medium
It is preferable to include an appropriate amount of carbon source and nitrogen source.
Good There are no particular restrictions on this carbon source and nitrogen source.
However, as an example, as a nitrogen source, inorganic ammonium nitrate or sulfur is used.
Ammonium, ammonium salt, ammonium phosphate, sodium nitrate, corn
Gluten meal, soy flour, corn steep liquor,
Casamino acid, yeast extract, pharma media, sardines
, Meat extract, peptone, high pro, ajpower, ko
Bean soybean meal, coffee meal, cottonseed oil meal, cult
Beta, Amiflex and Azipron, Zest, Aji
Such as kusu. Moreover, as a carbon source, rice husks,
Malt, filter paper, general papers, plant fiber such as sawdust, waste sugar
Honey, invert sugar, CMC, Avicel, cellulose cotton, xyla
And pectin, as well as carbon sources that can be assimilated, such as ara
Binose, glucose, fructose, galactose,
Maltose, sucrose, lactose, sorbitol, inositol, sugar
Lyserine, soluble starch and assimilable organic acids such as
Examples thereof include acetic acid and citric acid. Also, other, phosphorus
Acid, Mg²⁺, Ca²⁺, Mn ²⁺, Zn²⁺, Co²⁺, N
a⁺, K⁺Inorganic salts such as
A quantitative nutrient source can be appropriately added to the medium.

Collection and purification of the target substance, cellulase, from the thus obtained culture can be carried out 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, a crude enzyme is obtained by a separation means such as a salting-out method, a precipitation method, an ultrafiltration method, and further purified and crystallized by a known method, It is also possible to use it as a purified enzyme. The alkali-resistant cellulase thus obtained was used as cellulase K-534.
I named it. This will be further described below. The enzyme activity was measured according to the following method, and the following buffer solution was used.

PH 3-8 McClubain buffer solution pH 8-11 glycine-sodium hydroxide buffer solution pH 12-13 potassium chloride-sodium hydroxide buffer solution

Enzyme activity assay 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 acid (3,5-dinitro-
The reducing sugar was quantified by the salicylic acid (DNS) method. In other words, 1.0 ml of the reaction solution is DNS
1.0 ml of the reagent was added, color was developed by heating at 100 ° C. for 5 minutes, 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 cellobioside degrading activity 0.1 μmol p-nitrophenyl cellobioside (Sigma) and 100 μmol phosphate buffer (pH 7.0) in an appropriate amount in a reaction solution of 1.0 ml. After the enzyme was reacted at 30 ° C., 0.3 ml of 1M Na ₂ CO ₃ and 1.7 of deionized water were added.
ml sequentially added, and released p-nitrophenol at 400 nm
Colorimetrically determined by. 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 decomposing activity, and filter paper decomposing activity An appropriate amount of enzyme was added to 2.0 ml of a reaction solution containing 20 mg Avicel (Merck) and 200 μmol phosphate buffer (pH 7.0), and 30 It was made to act while shaking at 250 ° C. and 250 rpm. After the reaction, cooling centrifugation (5 ° C, 3000 rpm, 20
Min) 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 for 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 A suitable amount of enzyme was allowed to act at 30 ° C in 1.0 ml of a reaction solution containing 10 mg cellobiose (Kanto Chemical Co., Inc.) and 100 µmol phosphate buffer (pH 7.0), and then 100 ° C. After deactivating the enzyme by treating for 2 minutes, the amount of glucose produced is determined by
It was measured by the GOD 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 acts well on fibrin such as cellulose, filter paper, Avicel, CMC and the like to dissolve 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 filter paper and p-nitrophenyl cellobioside.

(3) Working pH and optimum pH The working pH range is extremely wide, 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 the optimum pH for 30 minutes at each temperature, the residual activity was measured. As a result, it was stable at about 40 ° C and about 65 ° C.
It had a residual activity of about 50% even at ℃ (4th
Figure).

(7) Molecular Weight This enzyme was prepared by using Sephadex G-100 (“Sephade”).
x G-100 ″), the molecular weight was measured based on the gel filtration method. Main peaks were observed at about 15,000 and about 30 thousand.

(8) Effect of Metal Ions For this enzyme, various metal ions (Al ³⁺ , Fe ³⁺ , B
a ²⁺ , Ca ²⁺ , Cd ²⁺ , Co ²⁺ , Cr ²⁺ , Cu ²⁺ , Fe
²⁺ , Hg ²⁺ , Mn ²⁺ , Mo ²⁺ , Ni ²⁺ , Pd ²⁺ , Z
(n ²⁺ , Li ⁺ , K ⁺ , Na ⁺ ) were made to coexist during the activity measurement, and the effect was examined (the concentration of each metal ion was 1 mM,
K ⁺ and Na ⁺ are 50 mM). As a result, inhibition was observed with Hg ²⁺ and activation was observed with Ba ²⁺ .

(9) Effects of surfactants Various surfactants (eg 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, it was hardly inhibited by any of the surfactants. No inhibition of activity was observed with the powerful detergent Sodium dodecyl sulfate.

(10) Protease resistance Detergent proteases such as API-21 (Showa Denko), Maxatase (Gist Co.) and Alcalase (Novo Co.) are allowed to coexist (0.1 mg / ml) at the time of activity measurement, and their effects are affected. As a result of examination, it was found to 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 the activity measurement, and their effects were examined, but almost no inhibition was observed. .

[0043]

The alkali-tolerant cellulase obtained from the microorganism of the present invention has an optimum pH of 5 to 7,
It is extremely stable at pH 5-11. Further, it is hardly affected 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. Moreover, since the microorganism of the present invention grows neutrally, it is possible to industrially produce an alkali-resistant cellulase more easily than an alkaliphilic bacterium.

[0044]

EXAMPLES The present invention will be described in more detail below with reference to examples.

Example 1 One 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
The measurement was performed at ~ 13 to screen for alkali-resistant cellulase-producing bacteria.

The Bacillus sp. KSM-534 strain (FERMP-9010) of the present invention could be obtained by the above-mentioned method.

[0047]

[Table 1] Medium 1. CMC 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

[0048]

[Table 2] 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% pH6.8

Reference Example 1 Bacillus sp. KSM-534 obtained in Example 1 was inoculated into the liquid medium 2 of Example 1 and cultivated with shaking at 30 ° C. for 3 days. After culturing, the bacterial cells were removed by centrifugation to obtain a crude enzyme solution. To 1 liter of this crude enzyme solution, 3 liters of ethanol was added in dry ice-ethanol, and the resulting precipitate was centrifuged and freeze-dried to give cellulase as a dry powder.
K-534, 10g (specific activity 3 units / g; measured value at pH 9 and below is the same) was obtained.

Reference Example 2 In liquid medium 2, CMC was replaced with 1% sucrose, and polypeptone was replaced with 7% corn steep liquor, and a medium was used. The CMCase activity of the supernatant of the obtained culture broth was 50 units / l.

[Brief description of drawings]

FIG. 1 is a drawing showing the relationship between 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 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.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Aki Kimioka 1-229-8 Nanachiza, Koshigaya City, Saitama Prefecture (72) Kei Mori 396-12 Minemachi, Utsunomiya City, Tochigi Prefecture

Claims (1)

[Claims] 1. Bacillus sp., Which has the ability to produce alkali-tolerant cellulase K-534 and has been deposited as Micromachine Research Institute No. 9010.
p. ) KSM-534.