JP2509533B2 - Microorganisms producing alkali-tolerant cellulase - Google Patents

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 a wide variety, mainly Algalilus genus, Penicillium genus, Trichoderma genus, Fusarium genus, Humicola genus, Acremonium and other filamentous fungi, Pseudomonas sp., Cellulomonas sp., Luminococcus sp., It has also been reported in bacteria such as Bacillus and actinomycetes such as Streptomyces and Thermoactinomyces. However, at present, there is not much use of cellulases for biomass on an industrial scale.

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. Also,
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 alkali cellulase and an alkali resistant 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.
No. 28515), a method of culturing an alkalophilic bacterium belonging to the genus Cellulomonas to produce alkaline cellulase 301-A (JP-A-58-224686), an alkalophilic Bacillus No. A method for culturing 1139 to produce carboxymethylcellulase (Fukumori,
F. , Kudo, T .; and Horikoshi,
K. , J. et al. Gen. Microbiol. , 131, 3
339, (1985)) and a method of producing an alkaline cellulase using a Streptomyces genus (Japanese Patent Application Laid-Open No. Sho 61-19483).
None of them were suitable for industrial fermentative 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 at an alkaline level 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. In parallel with the means of gene recombination, we have continued to search for the bacterium in nature.

As a result, it was found that the strain isolated from the soil in Haga-gun, Tochigi Prefecture produces a so-called alkali-tolerant cellulase which has a sufficient action even on the alkaline side and retains stability. Was completed.

The strain of 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) (% by weight; the same applies below) 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; 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 results The size of the cells is 0.6 to 0.8 μm × 1.5 to 6.5.
It is a rod-shaped bacterium having a diameter of μm and produces columnar or elliptical endospores (0.6 to 1.2 μm × 2.0 to 4.0 μm) in the center of the bacterium. No flagella and no motility. Gram staining is positive. There is no acid resistance.

(B) Growth condition in various media (1) Meat agar plate culture (medium 1) Growth condition is normal. The shape of the settlement is circular or irregular, the surface is smooth or rough, 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. (2) Meat broth agar slant culture (medium 1) Growth is normal, and its state is spreading, glossy, and pale yellow and translucent. (3) Broth liquid culture (medium 2) Grow. No surface growth, but upper growth is observed. (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 reaction (medium 5, medium 6) Nitrate reduction is positive and denitrification reaction is 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 Kristensen 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) Temperature range of growth is 15 to 50 ° C, optimum temperature range of growth is 25 to 40 ° C pH range of growth is pH 5 to 9, optimum pH range of growth is pH 6 to 9
Met.

(14) Attitude toward oxygen Aeration anaerobic. (15) OF test (medium 19) Both aerobic and anaerobic grow, and gas is generated. (16) Utilization of sugar (medium 20) (+: used, −: not used)

[0020]

[Table 1]

(17) pH in VP medium (medium 7) pH 5.2 (18) Growth in medium containing salt (modified medium 1) Grow at 5%. Grows at 7%. Grows at 10%. (19) Growth at pH 5.7 (Medium 21) Grow.

(20) Growth 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.

Regarding the above-mentioned mycological properties,
Manual of Date Native Bacteriology (Bergey's Manual of De)
terminating Bacteriology)
Eighth Edition and The Genus Bacillus ("The Gen
us Bacillus ", Ruth, E. Gordo
n by Agricultural Handbook N
o. 427, Agricultural Research
ch Service, U. S. Department
of Agricultural, Washingto
n D. C. (1973)), the strain of the present invention was found to be Bacillus (B.
It is recognized as a kind of genus acillus). And this strain has recently been found to be in the dig and Akiba (“Alkalophil
ic Microorganism ”, Japan S
scientific Society Press (T
(Okyo), published in 1982), a so-called alkalophilic microorganism grows in an alkaline medium having a pH of 8 or higher, and cannot grow in a neutral pH range lower than this, but from a weakly acidic range. Since it can grow in the alkaline region (pH 5-9), it is clearly different from this alkalophilic microorganism,
It can be judged as a general neutral and viable Bacillus microorganism.

Further, when this strain is compared with other known strains of the genus Bacillus, Bacillus licheniformis is the most closely related species.
s). However, when the above-mentioned known strain is compared with this strain, the known strain does not produce at least alkali-resistant cellulase. Therefore, the present inventors determined that this strain was a new strain, and Bacillus sp. KSM-344.
And was deposited with the Institute of Microbial Technology, National Institute of Industrial Science and Technology (FERM P-9008).

In order to obtain the alkali-tolerant cellulase, cellulase K-344, using the strain of the present invention, a medium may be inoculated with the strain and cultured according to a conventional method. In the medium,
It is preferable that an appropriate amount of assimilable carbon source and nitrogen source be contained. 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 meal,
Examples include cultivators, amiflex and adipron, zest, azix and the like. Further, as carbon sources, rice husks, malts, filter papers, general papers, vegetable fibers such as sawdust, molasses, invert sugar, carboxymethyl cellulose (C
MC), Avicel, cellulose cotton, xylan, pectin, and carbon sources that can be assimilated, for example, arabinose, xylose, glucose, mannose, fructose, galactose, sucrose, maltose, sorbitol, mannitol, inositol, glycerin, soluble starch and Examples thereof include organic acids that can be assimilated, such as acetic acid and citric acid. In addition, phosphoric acid, Mg ²⁺ , Ca ²⁺ , Mn ²⁺ ,
Inorganic salts such as Zn ²⁺ , Co ²⁺ , Na ⁺ , 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-344, from the thus obtained culture can be carried out according to a general enzyme collection and purification means. That is, the cells can be removed from the culture solution by ordinary solid-liquid separation means such as centrifugation or filtration to obtain a crude enzyme solution. 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 or an ultrafiltration method, and further purified and crystallized by a known method, It is also possible to use it as a purified enzyme.

The thus obtained alkaline cellulase K-
344 has the following enzymatic properties. The enzyme activity was measured according to the following method, and the buffer solutions used were as follows.

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.), 1
00 μmol 0.1 ml of the enzyme solution was added to 0.9 ml of a substrate solution containing various buffers (McClubein, phosphoric acid, glycine-NaOH, etc.) and reacted at 30 ° C. for 20 minutes. After the reaction,
3,5-dinitro-salicylic acid (3,5-dinitr
The reducing sugar was quantified by the o-salicyclic acid (DNS) method. That is, 1.0 ml of reaction solution
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 of p-nitrophenyl cellobioside (manufactured by Sigma) and 100 μmol of phosphate buffer (pH 7.0) in 1.0 ml of reaction solution. After allowing an appropriate amount of enzyme solution to act at 30 ° C., 0.3 ml of 1M Na ₂ CO ₃ and 1.
Add 7 ml sequentially to release 400 parts of p-nitrophenol.
Colorimetric determination in 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, and filter paper degrading activity 20 mg Avicel (manufactured by Merck & Co., Inc.), 200 μmol phosphate buffer (pH 7.0) to 2.0 ml of a reaction solution, an appropriate amount of enzyme solution was added, It was made to act while shaking at 30 ° C. and 250 rpm. After the reaction, cooling centrifugation (5 ° C., 3000 rpm,
20 minutes) and 1.0 ml of the supernatant was added to 3,5-dinitro-salicylic acid (3,5-dinitro-salicy).
The reducing sugar was quantified by the lic acid (DNS) method. Cellulose powder decomposition activity was measured by using cellulose powder (manufactured by Toyo Roshi Kaisha, Ltd.), and filter paper decomposition activity was measured by 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 An appropriate amount of enzyme solution was allowed to act at 30 ° C. in 1.0 ml of a reaction solution containing 10 mg cellobiose (manufactured by Kanto Chemical Co., Inc.) and 100 μmol phosphate buffer (pH 7.0). After deactivating the enzyme by treating at 100 ° C. for 2 minutes, the amount of glucose produced was measured by the mulotase / GOD method (Glucose C-Test, manufactured by 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 in addition to 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 6, which has a relative activity of 50% or more of the activity at the optimum pH even in the range of 4.5 to 11, and is the most alkaline side among the cellulases studied in the past. Therefore, it can be said that the enzyme has a wide range of pH (Fig. 1).

(4) pH stability The pH stability was examined by measuring the residual activity after holding at 30 ° C for 1 hour at each pH. As a result, it is extremely stable at pH 5-10 and does not deactivate, and even at pH 4.5-12 it is about 50%.
The above activity was maintained. The enzyme is sufficiently stable even in the highly alkaline region (Fig. 2).

(5) Optimum temperature The working temperature was in a wide range of 15 to 80 ° C, and the optimum temperature was 70 ° C. Further, it had 50% or more of the activity at the optimum temperature even in the range of 45 to 75 ° C (Fig. 3).

(6) Temperature stability After being treated at each temperature for 30 minutes at the optimum pH, the residual activity was measured, and as a result, it was stable at 30 ° C and was about 50% at 55 ° C. It had residual activity (Figure 4).

(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, and a main peak at about 16,000 was observed.

(8) Effect of metal ion Regarding 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, Hg ²⁺ and P
Inhibition was observed with d ²⁺ and activation with Co 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. Almost no inhibition of activity was observed even with the strong detergent Sodium dodecyl sulfate.

(10) Protease resistance Detergent proteases such as API-21 (manufactured by Showa Denko KK), Maxatase (manufactured by Gist) and Alcalase (manufactured by Novo Co.) are allowed to coexist (0.1 mg / ml) during activity measurement. As a result of examining its effect, 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 were allowed to coexist during the activity measurement, and the effect was examined, but almost no inhibition was observed.

[0044]

The cellulase K-344 obtained from the microorganism of the present invention has a relative activity of 60% or more of the optimum pH even at a high alkaline pH of 10, despite having an optimum pH of 6. It is extremely stable at pH 5-10. Further, it is hardly inhibited by detergent 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 the strain of the present invention, Bacillus sp. KSM-344, is a strain that grows neutrally, it is possible to industrially produce an alkali-resistant cellulase more easily than an alkaliphilic bacterium.

[0046]

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-treated solution was appropriately diluted and applied to an agar medium for separation (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. By the method described above, the Bacillus sp. KSM-344 strain (FERM P-90 of the present invention is used.
08) could be obtained.

[0048]

[Table 2] 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

[0049]

[Table 3] 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-344 obtained in Example 1 was inoculated into the liquid medium 2 of Example 1 and shake-cultured at 30 ° C. for 3 days. After the culture, the 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 obtain 10.5 g of cellulase K-344 as a dry powder (specific activity 6 units / g). The same applies for the measured values at pH 9).

Reference Example 2 Using liquid medium 2 in which CMC was changed to 1% sucrose and polypeptone was changed to 7% CSL (corn steep liquor), shaken at 30 ° C. for 2 days according to Reference Example 1. Cultured. Regarding the supernatant obtained by centrifugation of the obtained culture broth, C
When the MCase activity was measured, it was 2900 units / l.

[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.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location C12R 1:07) C12R 1:07) (72) Inventor Kei Mori 396-Minemachi, Utsunomiya City, Tochigi Prefecture 12

Claims (1)

(57) [Claims] 1. Bacillus sp. Which has the ability to produce an alkali-resistant cellulase K-344 and has been deposited as Microindustrial Research Institute No. 9008.
p. ) KSM-344.