JPH034788A - Alkali cellulase and bacterium producing the same cellulase - Google Patents

Abstract

NEW MATERIAL:Alkali cellulase K-520. Action: well dissolving cellulose such as CMC to form reducing sugar. Substrate specificity: having activity against CMC or Avicel(R). Action pH and optimum pH: 3-5 and 8.5-9.5, respectively. pH Stability: pH6-11 when CMC is used as a substrate. Optimum temperature: 10-70 deg.C action temperature on CMC and 50 deg.C optimum temperature. Molecular weight: 170,000+ or -10,000 and 80,000+ or -200 (very small peak) when CMC is used as a substrate. Influence of metallic ion: being inhibited by Hg<2+> and activated by Co<2+> when CMC is used as a substrate. Influence of surfactant, protease and chelating agent: hardly being inhibited. USE:A component for cleaning agent composition for cloth. PREPARATION:Bacillus sp. KSM-520 (FERM P-10483) is cultured and the objective substance is collected from the culture mixture.

Description

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

[Conventional technology]

Conventionally, the development of cellulase, a fibrinolytic enzyme, has been carried out with the major goal of effectively utilizing biomass resources (particularly cellulose resources), and in many cases molds have been sought as a supply source. There are many types of bacterial strains that have been isolated as cellulase-producing bacteria, including Aspergillus,
Mainly filamentous fungi (molds) such as Penicillium, Trichoderma, Fusarium, Humicola, and Acremonium, as well as bacteria such as Shutomonas, Cellulomonas, Ruminococcus, and Bacillus, as well as Streptomyces and Thermos. It has also been reported in actinomycetes such as the genus Actinomyces.

On the other hand, as a new industrial use of cellulase, its use as a compounding ingredient in laundry detergents is being studied and attracting attention (Japanese Patent Publication No. 59-49279, Japanese Patent Publication No. 60-23
158, Japanese Patent Publication No. 60-36240). However, in the natural world, most cellulases produced by microorganisms are classified as so-called neutral or acidic cellulases, which exhibit maximum and stable enzymatic activity in neutral to acidic regions, and are suitable for use in clothing. The reality is that there are very few cellulases that meet the requirements for inclusion in detergent compositions, that is, so-called alkaline cellulases and alkali-resistant cellulases that exhibit maximum activity in the alkaline region or have alkali resistance. Here, alkaline cellulase refers to a cellulase whose optimum pH is in an alkaline region.

That is, conventional methods for producing alkaline cellulases and alkali-resistant cellulases that can be used in laundry detergent compositions include a method of collecting cellulase A by culturing alkaliphilic Bacillus bacteria (Japanese Patent Publication No. 50-285
15), a method for producing alkaline cellulase 301A by culturing alkalophilic bacteria belonging to the genus Cellulomonas (Japanese Unexamined Patent Publication No. 58-224686), producing carboxymethyl cellulase by culturing alkalophilic Bacillus No. 1139 Method (Pukumori, F.,
Kuclo, T. and 1lorikoshi
.

K,,J,Gen,Microbiol,,13
1.3339. (1985)) and a method for producing alkaline cellulase using a species of Streptomyces (1985)).
JP-A-6119483) has only been reported, and none of them are suitable for industrial fermentation production.

However, recently, the present inventors have applied 5M-635 (Bacillus sp.) to Bacillus sp., a type of alkalophilic bacterium.
lus sp, KSM-635, FBRM [IP
-1485, Japanese Unexamined Patent Publication No. 63-109771) is an alkaline cellulase K (
It has been found that by producing JP-A No. 63-109776) in good yield and by selecting culture conditions, productivity can be further increased and industrial fermentation production of alkaline cellulase becomes possible. Furthermore, alkaline cellulase (JP 63-137677.240785.24
Publication No. 0?77.240786, JP-A-64-3728
Publication No. 5, 3. l(awai et a+
, 8gric, Dial, Chem,.

52, 1425 (1988)) and alkali-resistant cellulase (JP-A-63-141586.146786.2)
73474.273475.279790, Japanese Unexamined Patent Publication No. 64-37286).

[Problem to be solved by the invention]

However, the above-mentioned examples are the only examples of alkaline cellulases suitable for industrial fermentation production to date, and there has been a desire to develop alkaline cellulases with various characteristics and their producing bacteria.

[Means for resolving categories]

The present inventor has continued to search for microorganisms that produce alkaline cellulase in the natural world, and has recently discovered microorganisms that succumb to the genus Bacillus collected from soil in Nasu District, Tochigi Prefecture.
The present invention was completed by discovering the production of a new alkaline cellulase that is effective as an additive component for laundry detergent compositions.

Therefore, the present invention provides a novel alkaline cellulase and a microorganism that produces it.

The microorganism producing alkaline cellulase of the present invention exhibits the following mycological properties. In addition, the following 21 types of media were used for classifying the bacterial strains, medium 1 to 21 below, all of which were separately sterilized sodium carbonate (Naa [1
, 03) in an amount of 1.0% by weight (hereinafter simply expressed as %).

Composition of the medium used (displayed as %): Medium 1. Nutriendo broth, 0.8 bacto agar, 1.5 medium 2. Two neat triendo broth, 0.8 medium 3.
2 neat triendo broth, 0.8 bacto gelatin, 2
0.0; Bacto agar, 1.5 Medium 4. Bata Toli Tomas Milk, 10.5 Medium 5.
Nutriendobroth, 0.8; KNO, 0.1 Medium 6. Batatobeptone, 0.7; NaC
j! .

0.5; Glucose, 0.5 Medium 7. 3IM agar medium, indicated amount medium 8. TSI agar (manufactured by Eiken Chemical), indicated amount medium 9
, Batatopebutone, 1.5; Yeast extract, 0.5
Soluble starch, 2.0; LtlPO-, 0,1: Mg5O-.7+1.0°0.02; Bacto agar, 1.5 Medium 10. Koser medium, indicated amount medium 11. Christensen medium (manufactured by Eiken Chemical).

Instruction m Medium 12. ■Yeast extract, 0.05: Na2SO4
°0.1; H2PO, 0.1; glucose.

1.0 ■Yeast extract, 0.05:Na2SO4゜0.1;K
H□PO,, 0,1; glucose.

1.0; CaC12-21120,0,05;Mn
5O=・4～6N20. 0.001;Fe5O=
-7H20,0,02 As nitrogen sources, sodium nitrate, sodium nitrite, ammonium chloride, and ammonium phosphate were used in addition to the medium of (1) and (2) above so that the total nitrogen content was 0.0412N%.

Medium 13. King A medium “Eiken” (manufactured by Eiken Chemical), indicated amount medium 14. King B medium “Eiken” (manufactured by Eiken Chemical), indicated amount medium 15. Urea medium “Eiken” (manufactured by Eiken Chemical).

Indicated amount medium 16. Filter paper for cytochrome oxidase test (manufactured by Hinaga Pharmaceutical Co., Ltd.) Medium 17.3% hydrogen peroxide aqueous medium 1B, Bactobebutone, 0.5° yeast extract, 0.5; glucose.

1, Q, LIIPO,, o, t; Mg5O<・71120.　0.02 Medium 19. Batatobebuton, 2.7; NaCl.

5.5; Glucose, 0.5; KlhPO,.

0.3; Bromthymol Blue 0.06; Bacto Agar, 1.5 Medium 20. (N114)211PO,,0,1;
KCl, 0.02; Mg5O<・71120.0.0
2; Yeast extract.

0.05; sugar, 1.0 Medium 21. Casein, G, 5; Nokuto agar.

1.5: yeast extract, 0.5; glucose, 1.0; K2+1PO, 0,1; Mg5O<
・71hO,0.02 (Mycological properties) (a) Microscopic observation results: The size of the bacterial cells is 0.5-1.2 μm x 1.2-5.0 μm
It is a bacillus with an oval shape at the quasi-end of the bacterial body (0,
7-1.2 μm x 1.2-1.6 μm). It has periflagella and has vitality.

Durham staining was positive.

0]) Growth status in various media ■ Broth agar plate culture (medium 1) Growth status is good. The shape of the settlement is irregular, with a smooth surface and wavy edges.

The color of the village is milky white and shiny.

■ Broth liquid culture (medium 2) Growth is good.

■Meat juice gelatin hole culture medium (medium 3) Growth is good. Liquefaction of gelatin was observed.

■ Lidomus milk medium (medium 4) Coagulation and peptonization of milk were observed.

IJ) Discoloration of trout cannot be determined because the medium is alkaline.

(C) Physiological properties ■ Nitrate reduction and denitrification reaction (medium 5) Nitrate reduction is positive. Denitrification reaction was negative.

■MR test (medium 6) It is easy to determine whether it is negative or positive.

■VP test (medium 6) Positive.

■Generation of indole (medium 7) negative.

■Generation of hydrogen sulfide (medium 8) negative.

■Starch hydrolysis medium 9) Positive.

■Use of citric acid (medium 10.11) Positive in Cosa medium. Christensen's medium cannot determine whether it is positive or negative.

■Use of inorganic nitrogen sources (medium 12) It is used as nitrate, salt, and salt.

■Pigment production (medium 13.14) A yellow pigment is produced in King B medium.

[Stock] Urease (medium 15) negative.

■Oxidase (medium 16) Positive.

@ Catalase (medium 17) Positive.

■Growth range (medium 18) The temperature range for growth is 15-40℃, the optimal temperature range for growth is 3
0-37℃. The pH range for growth was ρ117-11, and the optimum pH for growth was pH 10.5.

■Attitude towards oxygen Aerobic.

@O-F test (medium 19) Due to its alkaline nature, discoloration cannot be determined.

Grows only under aerobic conditions.

■Sugar utilization (medium 20.0: Grows: No growth) 1, L-arabinose +2, D-xylose +3, D-glucose
+4, D-mannose +5,
D-fructose +6, D-galactose +7, maltose 18, sucrose 19, lactose
+10. Trehalose +11, D-
Sorvit +12, D-Mannit +13, Inosit 14, Glycerin +15, Starch 10 Growth in a medium containing salt (modified medium 1) Grows at a salt concentration of 7% and does not grow at a salt concentration of 10%.

■Decomposition of casein (medium 21) Positive.

[Phase] G + C content (Tm method) The G content of the DNA of the bacterium according to the present invention is approximately 37.6
The mo is 1%.

Based on the above two studies on mycological properties, I have published the following book: []ergey's Manual of Systemic Bacteriology.
Systematic Bacteriology
) Volume 2 and The Genus Bacillus (“The
Genus Bacillus” Ruth, B.
Gordon.

8gr+culture 1landbook N
α427. EightgriculturalResearch
5service, II, S, Departme
nt of^griculture Washing
ton D, C1 (1973)),
As a result of a comparative search, this strain was recognized to be a type of Bacillus a, which is a spore-bearing bacillus.

However, since this strain cannot grow in a neutral region and only grows well in an alkaline region, recently 1lori
kosi and ^kiba (”Alkalophili
c Microorganism Japan 5cie
ntific 5ociety Press (Tok
yo).

It belongs to the so-called alkalinophilic bacteria, as claimed by the author (published in 1982), and is distinguished from conventional Bacillus bacteria that grow in neutral conditions.

Since the above-mentioned mycological properties of this strain do not match those of any known alkaliphilic bacilli, we judged this strain to be a new strain and named it Bacillus sp. 5M-520. It was deposited at the Research Institute as Microtechnical Research Institute No. 10483 (F-port RM P-10483).

The alkaline cellulase of the present invention is, for example, the alkalophilic Bacillus sp. KSM-520 (Ell!
1M P-10483) and various mutant strains derived therefrom that have high titer productivity of the enzyme.

In order to obtain the alkaline cellulase of the present invention using the above-mentioned bacterial strain, the strain may be inoculated into an appropriate medium and cultured according to a conventional method. As the culture medium, one containing an appropriate combination of assimilable nitrogen sources and carbon sources is used. There are no particular restrictions on the carbon source and nitrogen source.

For example, nitrogen sources include ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium phosphate, sodium nitrate, corn gluten meal, soy flour, corn steep liquor, casamino acids, yeast extract,
Pharmamedia, sardine meal, meat extract, Hefton, Vibro, Ajipower, corn soybean meal, coffee grounds, cottonseed oil cake, cultivator, Ajiburon, zest, etc. Carbon sources include rice husk, wheat gluten, pond paper, and general paper. , vegetable fibers such as sawdust, blackstrap molasses, invert sugar, CMC
, Avicel, cellulose cotton, xylan, pectin, ribose, arabinose, xylose, glucose, mannose, fructose, galactose, maltose, sucrose, lactose, trehalose, mannitol, sorbitol, glycerin, starch, acetic acid, citric acid. . In addition, phosphoric acid, N g x + , Ca 2 “Mn 2+ ,
Inorganic salts such as ln2°, Co'', Na'', K+, and, if necessary, inorganic or organic micronutrient sources for fleas can also be added.

Collection and purification of the target substance alkaline cellulase from the culture thus obtained can be carried out using conventional enzyme collection and purification methods. That is, the crude enzyme solution can be obtained by removing the bacterial cells from the culture using conventional solid-liquid separation means such as centrifugation or filtration. This crude enzyme solution can be used as it is, but if necessary, the crude enzyme can be obtained by separation means such as salting-out method, precipitation method, ultrafiltration method, etc., and then purified and crystallized by a known method. It is also possible to use it as a purified enzyme.

Next, the enzymatic properties of the novel enzyme alkaline cellulase-520 provided by the present invention thus obtained will be explained.

Note that the enzyme activity was measured according to the following method.

(1) CMCase activity 2.5% CMC (Sunrose AOIL, manufactured by Nanyo Kokusaku Pulp Co., Ltd.) '0.4mf, 5001 glycine buffer (p
Add 0.1 me of the enzyme solution to a substrate solution consisting of 0.2 ml of f19) and 0.3 d of deionized water, and incubate at 30°C for 2 ml.
The reaction was allowed to proceed for 0 minutes. After the reaction, 3,5 dinitro-salicylic acid (D
Reducing sugars were determined by the NS) method. That is, 1.0 mg of DNS reagent was added to the reaction solution 1.0-1, and the mixture was incubated for 5 minutes at 1.0 mg.
Heated to develop color at 00℃, 4.0 mj after cooling! of deionized water was added to dilute it, and colorimetric determination was performed at a wavelength of 535 nm.

For the enzyme titer, one unit was the amount of enzyme that produced reducing sugars equivalent to 1 μmol of glucose per minute.

The buffer solution used is as follows.

pH 3-8 Macilpine buffer p] 18-11
Glycine-sodium hydroxide buffer pH 12-13 Potassium chloride-sodium hydroxide buffer (2) p-nitrophenyl glucoside and p-nitrophenyl cellobioside degrading activity 0.8 μmol p-nitrophenyl cellobioside (manufactured by Sigma) Or p-nitrophenyl glucoside (manufactured by Sigma) and 50 μmol phosphoric acid warm solution (pH 7.0)
Alternatively, reaction solution 1 containing 100 μmol glycine buffer (pH 9). After allowing an appropriate amount of the enzyme solution to act in Od at 30°C, 0.4-lM 1lazcO* was added, and p-nitrophenol liberated was determined colorimetrically at 410 nm. For the enzyme titer, one unit was defined as the amount of enzyme that liberated 1 μmol of p-nitrophenol per minute.

(3) Avicel, cellulose powder, phosphoric acid W-wetted cellulose, alkali-swollen cellulose and filter paper degrading activity 10+I1g Avicel (manufactured by Merck & Co., Ltd.) and 100 μm
Reaction solution containing ol glycine buffer (pf19) 1.
Add an appropriate amount of enzyme to 0- and incubate at 28 Orpm at 30°C.
The reaction was performed while shaking. After the reaction, reducing sugars were quantified using the DNS method. For the enzyme titer, one unit was the amount of enzyme that produced reducing sugar equivalent to 1 μmol of glucose per minute.

Other activities were also carried out according to the above method. As substrates, cellulose powder manufactured by Toyo Roshi Co., Ltd., filter paper for cellulase activity assay (Toyo N [LSI-Toku), and the method of Tomikoku et al. (Tomita, Y.) were used.

at al.: J.Fermant, Techn.
Alka IJ Og wet cellulose, phosphoric acid swollen cellulose treated according to J.D. ol., 52.235° 1974) was used.

(4) β-1,3-; 1,4-glucanase, β-1,
3-glucanase activity β-1,3-;1,4-glucanase, β-1,3-glucanase activity was determined using lichenan (from L
lsnera barbata, manufactured by Sigma) and laminarin (from L filter paper inariadigi)
(3) D4'
Activity was measured by t.

(Enzymatic properties) (1) Action: Acts on fibrous substances such as CMC, cellulose powder, and Avicel, dissolves them, and produces reducing sugars.

(2) Substrate specificity An example of the substrate specificity of this enzyme is shown in Table 1. In addition to CMC, this enzyme also contains laminarin, Rikenan, 11 papers, p.
It had some activity against -nitrophenyl glucosi)' and hI)-nitrophenyl cenobioside.

Table 1: p-nitrophenyl cellobioside (3) Action pH and optimum pH The action pH range for CMC is wide, from 3 to 13.0, and the optimum action pH is 8 .5 to 9.5. Moreover, even in the range of 7.0 to 11.0, it has a relative activity that is 50% or more of the activity at the optimum pH (Fig. 1).

(4) pH stability The residual activity after holding each pH at 30°C for 1 hour was measured to examine the pH stability. As a result, ρ116.
Extremely stable in the range of 0 to 11.0, pH 5.0 to
11.5, the activity is maintained at about 50% or more (Figure 2).

(5) Action temperature and optimum temperature This enzyme acts over a wide range of 10 to 75°C, and its optimum temperature is 50°C. Moreover, even in the range of 35 to 60°C, it has 50% or more of the activity at the optimum temperature (Fig. 3).

(6) 1 at each temperature in temperature-stable glycine-Na leaf buffer (pH 9.0)
As a result of measuring the residual activity after heat treatment for 0 minutes, it was found that this enzyme was hardly inactivated even at 40°C, and remained active at about 50°C even at 50°C.
It had 0% residual activity (Figure 4).

(7) Molecular weight This enzyme is Sephacryl S-300 (manufactured by Pharmacia)
According to the gel filtration method using
It peaks at ±0.2 million.

(8) Effect of metal ions Various metal ions (Na+, K”, Ca”, Cu
“Co”, Cd”, MT+”, Mg”, Da”
, Ni”, l1g”pe2+, Pb”, ln2
+. As a result of examining the effects of coexisting AI'' and Pe3(-, ) during activity measurement, we found that l g2 +, Cd''
, Pb” (1 mM each) gave an inhibitory effect,
Addition of Co2+, Mn2+ (1 mM) gave an activating effect.

(9) Effect of surfactants This enzyme contains 0.005% of various surfactants (such as LAS).
, ^S, ES, 80S, S to S, soap, polyoxyethylene secondary alkyl ether) was not found to inhibit the enzyme activity.

Color detergent proteases such as HachiPI-21 (Showa Denko), Maxatase (IBIs), Savinase, Alcalase, and Esperase (Novo) were coexisted during activity measurement (0
．． 2AU#) and examined its influence, it was found that it has strong resistance to all proteases.

Effects of chelating agents [iDTA, IEGT8, sodium tripolyphosphate, zeolite, citric acid, etc.] were present in the presence of chelating agents during activity measurement, and their effects were investigated, but no inhibition was observed.

〔Effect of the invention〕

The cellulase of the present invention has an optimum pH on the alkaline side,
A novel alkaline cellulase that is stable over a wide pH range. Furthermore, this enzyme contains surfactants, proteases,
It is hardly inhibited by cleaning agent ingredients such as chelating agents, and can be advantageously used as a cleaning agent ingredient.

〔Example〕

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

Example 1 A spoonful (approximately 0.5 g) of soil from Nasu District, Tochigi Prefecture was suspended in sterile physiological saline and heat-treated at 80° C. for 10 minutes. The supernatant of this heat-treated liquid was diluted appropriately and applied to a separation agar medium (medium A). Next, this was cultured at 30°C for 3 days to form colonies. Bacteria that formed a pellucid zone around the colony due to CMC' dissolution were selected to obtain CMCase-producing bacteria. Furthermore, the obtained bacteria were inoculated into a liquid medium of j8 soil B, and cultured with shaking at 30°C for 3 days. After culturing, the centrifuged supernatant was tested for CMCase activity at pH 3 to 1.
3, and alkaline cellulase producing bacteria were selected.

By the method described above, the 5M-520 bacterium (F[!
RM P-10483).

Medium A, CMC 2% Polypeptone 0.5% Yeast extract 0.5% KIl, PO, O, l % Mg5O= ・711zυ D, 02% Na
2CO-0.8% Agar o, 75% p+-+ approx. 10 Medium B, CMC 1% Meat extract 1.5% Yeast extract 0.5% KIl, PO40.1% Na2CO, 0.5% pH approx. 10 Examples 2 Bacillus sp. KSM520 obtained in Example 1 was inoculated into liquid medium B of Example 1, and cultured with shaking at 30°C for 3 days. After culturing, the bacterial cells were removed by centrifugation to obtain a crude fermentation liquid. Furthermore, the cellulase enzyme preparation (Table 2) was obtained by converting it into ethanol dry smoke powder according to a conventional method.

The following is a margin: Table 2 Figure 1 (Enzyme activity is the measured value at pH 9.) Example 3 In the liquid medium of Example 1, 1% cellobiose was used instead of CMC, and C6L was used instead of polypeptone. KSM520 bacteria was inoculated into a medium supplemented with 5% of the following, and cultured with shaking at 30°C for 2 to 3 days. As a result of measuring the CMCase activity of the centrifugation supernatant, a CMCase activity of 3542 U/1 was obtained.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the relationship between the reaction ρ■ of the alkaline cellulase of the present invention with respect to CMC and relative activity, and FIG.
A drawing showing the relationship between treatment pH and residual activity for C anus,
FIG. 3 is a diagram showing the relationship between reaction temperature and relative activity of the alkaline cellulase of the present invention, and FIG. 4 is a diagram showing the relationship between treatment temperature and residual activity. 810 Reaction pH 2 4 Figure 2 11 pH Procedure correction 1 (spontaneous) Figure 3 C%) 0 10 20 30 40 50 60 70 80
Reaction 1 degree (@C) Figure 4 (%)

Claims (1)

[Claims] -520 to an alkaline cellulase having the following physicochemical properties. 1) Action carboxymethyl cellulose (CMC), cellulose,
It acts well on cellulose such as filter paper and Avicel, dissolving them and producing reducing sugars. (2) Substrate specificity In addition to CMC, it has activity against cellulose powder, phosphoric acid-swollen cellulose, alkali-swollen cellulose, Avicel, filter paper, p-nitrophenyl glucoside, and p-nitrophenyl cellobioside. (3) Working pH and optimum pH The working pH range for CMC is 3 to 13.0. The optimum pH is 8.5 to 9.5, and even in the range of 7.0 to 11.0, the relative activity is 50% or more of the activity at the optimum pH. (4) pH stability When CMC is used as a substrate, it is extremely stable at pH 6.0 to 11.0 and does not deactivate, and even at pH 5 to 11.5, it is
Maintain activity above 0%. (5) Optimum temperature The operating temperature for CMC is over a wide range of 10 to 70°C, and the optimum temperature is 50°C. Also, 35-60℃
Even within this range, it has 50% or more of its activity at the optimum temperature. (6) When molecular weight CMC is used as a substrate, approximately 17 ± 10,000 and 8.0 ± 0
．． 20,000 (by gel filtration method using Biogel A 0.5m). (7) Effect of metal ions When CMC is used as a substrate, Hg^2^+, Cd^2^+
, Pb^2^+, inhibited by Co^2^+, Mn
Activated by ^2^+. (8) Effect of surfactants When CMC is used as a substrate, linear alkylbentenesulfonates, alkyl sulfates, polyoxyethylene alkylsulfonates, α-olefin sulfonates, secondary alkanesulfonates, soaps, etc. , polyoxyethylene secondary alkyl ether hardly inhibits the activity. (9) Effect of protease When CMC is used as a substrate, it has resistance to protease. (10) Effect of chelating agents When CMC is used as a substrate, EDTA, EGTA, citric acid, sodium tripolyphosphate, and zeolite do not inhibit the activity. 2 -520-producing alkaline cellulase bacteria that belong to the genus Bacillus and grow in an alkaline medium. 3. The alkaline cellulase K-520-producing bacterium according to claim 2, which is named Bacillus sp. KSM-520 and deposited as FAIMS Deposit No. 10483.