JPH0375152B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a microorganism that produces a novel alkaline cellulase, belongs to the genus Bacillus, and grows in a neutral medium.

[Conventional technology]

The development of cellulase, a fibrinolytic enzyme, has traditionally been carried out with the primary goal of effectively utilizing biomass resources, particularly cellulose resources. There are many types of bacterial strains that have been isolated as cellulase-producing bacteria.
Mainly filamentous fungi such as Aspergillus, Penicillium, Trichoderma, Fusarium, Humicola, Acremonium, Pseudomonas,
It has also been reported in bacteria such as Cellulomonas, Ruminococcus, and Bacillus, as well as actinobacteria such as Streptomyces and Thermoactinomyces. However, at present, there are not many uses of cellulase for biomass on an industrial scale.

On the other hand, as a new industrial use of cellulase,
Its use as a compounding ingredient in laundry detergents has been studied and is attracting attention (Japanese Patent Publication No. 59-49279, Japanese Patent Publication No. 60-23158, 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 the existence of so-called alkaline cellulases and alkali-resistant cellulases, which have the conditions for inclusion in detergent compositions, that is, exhibit maximum activity in the alkaline region or have alkali resistance, is extremely rare. . Here, alkaline cellulase refers to one whose optimal pH is in the alkaline region, and alkaline-tolerant cellulase refers to one whose optimal pH is in the neutral to acidic region, but which has an optimal pH in the alkaline region. It refers to something that has sufficient activity compared to the activity and maintains stability. Further, neutral refers to a pH range of 6 to 8, and alkaline refers to a higher pH range.

That is, conventional methods for producing alkaline cellulase and alkali-resistant cellulase that can be used in laundry detergent compositions include a method in which cellulase A is collected by culturing alkaliphilic Bacillus bacteria (Japanese Patent Publication No. 50-28515); Method for producing alkaline cellulase 301-A by culturing alkalophilic bacteria belonging to the genus Cellulomonas (Japanese Patent Application Laid-open No. 1983-
224686), a method for producing carboxymethyl cellulase by culturing alkalophilic Bacillus No. 1139 (Fukumori, F., Kudo, T. and
Horikoshi, K., J.Gen.Microbiol., 131, 3339,
(1985)) and a method for producing alkaline cellulase using a species of the genus Streptomyces (Japanese Unexamined Patent Publication No. 61
-19483) have been reported, and none of them are suitable for industrial fermentation production.

However, the present inventors recently discovered that Bacillus sp. KSM-635, a type of alkalophilic bacterium,
(Bacillus sp.KSM-635) (FERM P-8872)
discovered that by producing alkaline cellulase K, which is suitable as a component in laundry detergents, in good yield and by selecting culture conditions, productivity can be further increased and industrial fermentation production of alkaline cellulase becomes possible. Ta.

[Problem that the invention seeks to solve]

However, the above Bacillus sp.
The culture conditions for KSM-635 cannot necessarily be said to be industrially advantageous. In other words, it is necessary for alkaline-loving bacterial strains to maintain an alkaline pH during cultivation, but to date, the so-called alkaline fermentation method using alkaline-loving strains has only a short history, and compared to normal neutral microorganisms, these alkaline-loving Knowledge about the physiology and biochemistry of microorganisms has not been sufficiently accumulated, and medium preparation and culture methods have been operational difficulties when carrying out industrial fermentation production.

Furthermore, among the reported examples mentioned above, the original alkaline cellulases whose optimal pH is in the alkaline region are those produced by Bacillus strains N1, N2, and N3 (Japanese Patent Publication No. 1983-28515). 8-9, 9, 8-9 enzymes, Bacillus no.
1139 produces the optimal PH9 and Bacillus
There is an alkaline cellulase K (Japanese Patent Application No. 61-257776) with an optimum pH of 10 produced by SP KSM-635, but the optimum pH is on the alkaline side and can be used in detergent compositions. Its action
There has been a demand for an alkaline cellulase with a wide pH range.

[Means for solving problems]

Under these circumstances, the present inventors conducted various studies in order to obtain a strain that can grow in a neutral medium and produce alkaline cellulase with excellent activity.

To solve this problem, it is possible to use a so-called genetic recombination method in which the relevant cellulase gene is cloned using a bacterial strain that grows in a neutral region as a host, but it is best suited for an alkaline region.
It is more effective to search for neutral microorganisms in nature that produce alkaline cellulases with pH values and to isolate them. As a result of searching for the above-mentioned microorganisms in nature, the present inventors discovered that a group of microorganisms belonging to the genus Bacillus produce a certain amount of alkaline cellulase even though they grow in a neutral medium. completed.

That is, the present invention provides a microorganism that belongs to the genus Bacillus, grows in a neutral medium, and produces alkaline cellulase having the following enzymatic properties.

(1) It has a wide optimum PH range of PH7 to 10, and its optimum
PH is around PH10.

(2) The presence of Hg ²⁺ inhibits its activity;
Activated by the presence of Ca ²⁺ .

(3) Its activity is hardly inhibited by proteases, surfactants, and chelating agents.

(4) CMCase activity (Cx activity) is the main activity,
Filter paper disintegration activity and avicelase activity ( _C1 activity)
(5) Molecular weight: approximately 31,000 (as determined by gel filtration using Cephadex G-100).

The microorganism of the present invention that produces such alkaline cellulase is Bacillus sp.
(FERM R-9009).

This strain exhibits the mycological properties shown below. In addition, the following culture media 1 to 25 were used for classifying the strains. (Displayed as weight%) Medium 1 Meat extract, 1.0; Bactopeptone, 1.0;
NaCl, 0.5; Bacto agar, 1.5 (PH7.2) Medium 2 Meat extract, 1.0; Bacto peptone, 1.0;
NaCl, 0.5 (PH7.2) Medium 3 Meat extract, 1.0; Bactopeptone, 1.0;
NaCl, 0.5; Gelatin, 1.0 (PH7.2) Medium 4 Bactolithomus milk, 10.0 Medium 5 Bacto peptone, 1.0; KNO ₃ , 0.1 Medium 6 Bacto peptone, 1.0; NaNO ₃ , 1.0 Medium 7 Bacto peptone, 0.7; NaCl, 0.5; Glucose, 0.5 (PH7.0) Medium 8 Bactopeptone, 1.0 Medium 9 TSI agar (manufactured by Eiken Chemical): indicated amount medium 10 Meat extract, 1.0; Bactopeptone, 1.0;
NaCl, 0.5; Soluble starch, 0.2; Agar,
1.5 Medium _{11 NaNH4HPO4}・_4H2O , 0.15; _{KH2PO4 ,}
0.1; MgSO ₄ 7H ₂ O, 0.02; Sodium citrate, 0.25 (PH6.8) Medium 12 Christensen medium (manufactured by Eiken Chemical): indicated amount medium 13 Glucose, 1.0; KH ₂ PO ₄ , 0.1;
_MgSO4.7H2O , 0.05; HCl, 0.02; Nitrogen _source , 0.1 (PH7.2) Sodium nitrate and ammonium sulfate were used as nitrogen sources.

Medium 14 King A medium “Eiken” (manufactured by Eiken Chemical): Indicated amount medium 15 King B medium “Eiken” (manufactured by Eiken Chemical): Indicated amount medium 16 Urea medium “Eiken” (manufactured by Eiken Chemical) : Indicated amount medium 17 Filter paper for cytochrome oxidase test (manufactured by Nissui Pharmaceutical) Medium 18 3% hydrogen peroxide aqueous medium 19 OF basal medium (manufactured by Difco): Indicated amount 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 (manufactured by Difco): indicated amount medium 22 Meat extract, 0.3; Bacto peptone, 0.5;
Yeast extract, 1.0; Glycerin, 2.0 Medium 23 Phenylalanine malonate medium (Nissui Pharmaceutical Co., Ltd.): Indicated amount medium 24 Skim milk, 5.0; Bacto agar, 1.5 Medium 25 Meat extract, 0.3; Bacto peptone, 0.5;
L-tyrosine, 0.5; Bacto agar, 1.5 (Mycological properties) (a) Microscopic observation results The size of the bacterial body is 0.6-0.8 μm x 1.0-2.0 μm
It is a bacillus and produces cylindrical or oval endospores (0.4-0.8 μm x 1.0-2.0 μm) in the center of the bacterial body. It has periflagella and is motile. Gram staining was positive. It has no anti-acid properties.

(b) Growth status in various media Meat juice agar plate culture (medium 1) Grows well. The shape of the village is circular,
The surface is smooth and the periphery is smooth or lobed.
The color tone of the village is pale yellow, translucent and shiny.

Broth agar slant culture (medium 1) Grows. Its condition is spread-like, glossy, and pale yellow and translucent.

Meat juice liquid culture (medium 2) It grows and becomes cloudy.

Meat juice gelatin puncture culture (medium 3) Grows on the surface layer, and liquefaction of gelatin is observed.

Litmus milk medium (medium 4) Liquefaction of milk is observed, and no discoloration of litmus is observed.

(c) Physiological properties Nitrate reduction and denitrification reaction (medium 5, 6) Both tested negative.

MR test (medium 7) Positive.

VP test (medium 7) Positive.

Production of indole (medium 8) negative.

Generation of hydrogen sulfide (medium 9) negative.

Hydrolysis of starch (medium 10) negative.

Utilization of citric acid (medium 11, 12) Positive in Christensen medium, negative or positive in Cosa medium cannot be determined.

Utilization of inorganic nitrogen sources (medium 13) Both nitrate and ammonium salts were negative.

Pigment production (medium 14, 15) Positive.

Urease (medium 16) negative.

Oxidase (medium 17) There were no negative or positive results.

catalase Positive.

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

The PH range for growth is PH5-10, and the optimum PH range for growth is PH6-10.

attitude towards oxygen Aerobic.

O-F test (medium 19) oxidation.

Production of acids and gases from sugars (Medium 20)
(+: produced, -: not produced) Acid production Gas production 1 L-arabinose + - 2 D-xylose + - 3 D-glucose + - 4 D-mannose + - 5 Fructose + - 6 D-galactose + - 7 Maltose - - 8 Sucrose + - 9 Lactose - - 10 Trehalose + - 11 D-sorbitol - - 12 D-mannitol + - 13 Inosyte - - 14 Glycerin + - 15 Starch - - PH in VP medium ( Medium 7) Growth in PH5.0 salt-containing medium (modified from medium 1) All grow in the presence of 5%, 7%, and 10% NaCl.

Growth at PH5.7 (Medium 21) Grow.

Production of dihydroxyacetone (medium 22) negative.

Deamination of phenylalanine (medium 23) negative.

Degradation of casein (medium 24) Positive.

〓〓 Degradation of tyrosine (medium 25) negative.

Judging from the above taxonomic considerations, KSM-521
The strain is easily recognized as a species of the genus Bacillus, which is a spore-bearing bacillus.

Furthermore, regarding mycological properties, Bergey's Manual of Determinative Bacteriology (Bergey's Manual of Determinative Bacteriology)
Determinative Bacteriology) 8th edition and the
“The Genus Bacillus”
Ruth, E. Gordon Agriculture Hand−book No.
427, Agricultural Research Service, US
Department of Agriculture Washington DC;
(1973)), this strain was recently discovered by Horikoshi and Akiha (“Alkalophilic
Microorganism”, Japan Scientific Society
Press (Tokyo, 1982) claims that it belongs to the so-called Alkalophilic microorganisms, that is, microorganisms that grow in alkaline media with a pH of 8 or higher, but cannot grow in the neutral pH range below this. from the weakly acidic region to the alkaline region (PH
5 to 10), it can be judged that it is a common Bacillus microorganism that grows in neutral conditions.

Furthermore, when this strain is compared with other known strains of the genus Bacillus, Bacillus pumilus is listed as the most related species. However, when this strain is compared with known strains belonging to Bacillus pumilus, the above-mentioned known strains do not produce at least alkaline cellulase, so the present strain is judged to be a new strain.

In order to obtain alkaline cellulase using this strain as described above, the strain may be inoculated into a medium and cultured according to a conventional method. It is preferable that the medium contains appropriate amounts of assimilable carbon sources and nitrogen sources. There are no particular restrictions on the carbon and nitrogen sources, but examples include corn gluten meal, soybean flour, corn steep liquor, casamino acids, yeast extract, firma media, sardine meal, meat extract, and peptone. , Hypro, Azipower, corn soybean meal, coffee grounds, cottonseed oil meal, Cultivator, Amiflex, Aziprone, Zest, Ajitsux, and the like. In addition, carbon sources include rice husk, starch, filter paper, general paper, vegetable fibers such as sawdust, molasses,
In addition to invert sugar, CMC, Avicel, cellulose cotton, xylan, and pectin, assimilated carbon sources such as arabinose, glucose, mannose, fructose, maltose, sucrose, mannitol, sorbitol, inosyte, glycerin, soluble starch, and assimilated The organic acids obtained include, for example, citric acid and acetic acid. In addition, phosphoric acid,
Inorganic salts such as Mg ²⁺ , Ca ²⁺ , Mn ²⁺ , Zn ²⁺ , Co ²⁺ , Na ⁺ , K ⁺ and, if necessary, inorganic and organic trace nutrients should be appropriately converted into the medium. You can also do it.

Collection and purification of the target substance alkaline cellulase from the culture thus obtained can be carried out in accordance with common enzyme collection and purification methods. That is, a crude enzyme solution can be obtained by removing the bacterial cells from the culture solution 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 further purified and crystallized by a known method.
It is also possible to use it as a purified enzyme.

A typical alkaline cellulase thus obtained is one named alkaline cellulase K-521, and the present invention will be further explained below using this cellulase as an example.

The enzyme activity was measured according to the following method, and the following buffer was used.

PH3-8 McLuvain buffer PH8-11 Glycine-sodium hydroxide buffer PH12-13 Potassium chloride-sodium hydroxide buffer Enzyme activity measurement method: (1) CMCase activity 10mg CMC (A-01L, Sanyo Kokusaku Pulp Co., Ltd.) ,
100 μmol various buffer solutions (macluvain, phosphoric acid,
0.9 ml of substrate solution containing glycine-NaOH, etc.)
0.1 ml of enzyme solution was added and reacted at 30°C for 20 minutes.
After the reaction, 3,5-dinitro-salicylic acid (3,
Reducing sugars were quantified using the 5-dinitro-salicylic acid (DNS) method. That is, after the reaction, 1.0
1.0 ml of DNS reagent was also added to the solution, and color was developed by heating at 100° C. for 5 minutes. After cooling, 4.0 ml of deionized water was added to dilute. This was measured colorimetrically at a wavelength of 535 nm. The enzyme titer was determined in 1 minute under the above conditions.
The amount of enzyme that produces reducing sugar equivalent to 1 μmol of glucose was defined as 1 unit.

(2) p-nitrophenyl cellobioside degrading activity 0.1 μmol p-nitrophenyl cellobioside (Sigma), 100 μmol phosphate buffer (PH7.0)
Add an appropriate amount of enzyme solution to 1.0 ml of reaction solution containing
After the reaction, 0.3 ml of 1M Na ₂ CO ₃ and 1.7 ml of deionized water were sequentially added, and the liberated p-nitrophenol was determined colorimetrically at 400 nm. For the enzyme titer, one unit was defined as the amount of enzyme that liberated 1 μmol of p-nitrophenol per minute under the above conditions.

(3) Avicel, cellulose powder, and filter paper degrading activity Add an appropriate amount of enzyme solution to 2.0 ml of a reaction solution containing 20 mg Avicel (Merck) and 200 μmol phosphate buffer (PH7.0), and incubate at 30°C and 250 rpm. It was allowed to work while being shaken. After the reaction, cooled centrifugation (5
℃, 3000 rpm, 20 minutes) and 1.0 ml of the supernatant.
3,5-dinitro-salicylic acid (3,5-
Reducing sugars were determined using the dinitro-salicylic acid (DNS) method. For cellulose powder decomposition activity, use cellulose powder (Toyo Roshi Co., Ltd.), and for filter paper decomposition activity, use filter paper (filter paper for cellulase activity assay, Toyo No.
51-Toku) in the same manner as for Avicelase activity. For the enzyme titer, one unit was defined as the amount of enzyme that produced reducing sugar equivalent to 1 μmol of glucose per minute under the above conditions.

(4) Cellobiase activity 10mg cellobiose (Kanto Kagaku Co., Ltd.), 100μmol
After reacting an appropriate amount of enzyme solution in 1.0 ml of reaction solution containing phosphate buffer (PH7.0) at 30°C,
After treating for 2 minutes to inactivate the enzyme, the amount of glucose produced was measured using the mulotase/GOD method (Glucose C
-Test, Wako Pure Chemical Industries, Ltd.). For the enzyme titer, one unit was the amount of enzyme that produced 2 μmol of glucose per minute under the above conditions.

(Enzymatic properties) (1) Action It acts well on cellulose such as CMC, cellulose powder, filter paper, and Avicel, and dissolves them to produce reducing sugars such as glucose.

(2) Substrate specificity In addition to CMC, this enzyme had activity against cellulose powder, Avicel, filter paper, p-nitrophenyl cellobioside, and cellobiose.

(3) Effective PH and optimal PH The effective PH range was extremely wide, from 3 to 12.5. The optimum pH ranges from 7 to 10, with a pH of 4.5.
Even in the range of ~10.5, it has a relative activity that is more than 50% of the activity at the optimal pH, and therefore, among the alkaline cellulases studied in the past, it is the enzyme that exhibits the most activity at the alkaline side. (Figure 1).

(4) PH stability PH stability was investigated by measuring the residual activity after holding at each PH for 1 hour at 30°C. As a result, it is extremely stable at pH 5 to 12 and does not lose its activity.
Even in the range of 4.5 to 12.5, approximately 50% or more activity was maintained. This enzyme is sufficiently stable even in a highly alkaline region (the second
figure).

(5) Optimum temperature The operating temperature ranges from 15 to 80℃,
The optimum temperature was 60°C. Also, 45-65℃
50% of the activity at the optimum temperature even in the range of
It had the above (Figure 3).

(6) Temperature stability As a result of measuring the residual activity after treatment at each temperature for 30 minutes at the optimum pH, it was stable at 40℃, and even at 55℃, the residual activity was about 50%. (Figure 4).

(7) Molecular weight Sephadex G-100
When the molecular weight was measured based on the gel filtration method using (Sephadex G-100), it was approximately 31,000.

(8) Effects 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 allowed to coexist during activity measurement to examine their effects (K ⁺ ,
For Na ⁺ the concentration was 50mM and for other ions it was 1mM). the result,
Inhibition was observed with Hg ²⁺ and activation with Ca ²⁺ .

(9) Effect of surfactants Various surfactants (e.g. LAS, AS,
ES, AOS, α-SFE, SAS, soap, polyoxyethylene secondary alkyl ether)
We investigated the effect of the enzyme on the enzyme activity. The enzyme was treated with a 0.05% surfactant solution at 30°C for 15 minutes, and then the activity was measured. As a result, there was almost no inhibition by any of the surfactants. No inhibition of activity was observed even with sodium dodecyl sulfate, a strong detergent.

(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 their effects were investigated. It was found to have strong resistance to all proteases.

(11) Influence of chelating agents We examined the effects of chelating agents EDTA, EGTA, sodium tripolyphosphate, zeolite, and citric acid when measuring activity, but almost no inhibition was observed.

〔Effect of the invention〕

The alkaline cellulase obtained by the microorganism of the present invention has an optimum pH on the high alkaline side (PH10) compared to conventional alkaline cellulases. Moreover, it has an optimum pH within a wide range of PH7.0 to 10, and is extremely stable over an even wider range.

Furthermore, it is hardly inhibited by detergent ingredients such as surfactants, proteases, and chelating agents. Therefore, this enzyme can be advantageously used as a component of cleaning compositions.

Furthermore, since the microorganism of the present invention grows in neutral conditions,
Alkaline cellulase can be produced industrially more easily than with alkaliphilic strains.

〔Example〕

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

Example 1 A spoonful of soil (approximately 0.5
g), suspended in sterile physiological saline and heat-treated at 80°C for 10 minutes. The supernatant of this heat-treated solution was diluted appropriately and applied to a separation agar medium (medium 1). Next, this was cultured at 30°C for 3 days to form colonies. We selected bacteria that formed a pellucid zone around the colony due to CMC dissolution, and obtained CMCase-producing bacteria. Furthermore, the obtained bacteria were inoculated into a liquid medium (medium 2) and cultured with shaking at 30°C for 3 days. After culturing,
The CMCase activity of the centrifuged supernatant was determined by
The pH was measured at 3 to 13, and alkaline cellulase producing bacteria were screened.

By the method described above, the KSM-521 strain (FERM P-9009) of the present invention could be obtained.

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% PH6.8 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% PH6.8 Example 2 Bacillus sp. KSM− obtained in Example 1
The 521 strain was inoculated into liquid medium 2 of the same example, and cultured 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 part of this crude enzyme solution, 3 parts of ethanol was added in dry ice-ethanol, and the resulting precipitate was centrifuged and freeze-dried to form a dry powder of alkaline cellulase K-521 (specific activity * 20 units). /g) 9g was obtained.

* Enzyme activity is a value measured at PH9 (the same applies below) Example 3 CMC was replaced with 1% sucrose, and polypeptone was replaced with 7% sucrose.
KSM-521 strain was inoculated into a medium with the same composition as liquid medium 2 of Example 1 except that CSL was replaced with
Cultured with shaking for 1 day. This culture was centrifuged and
The CMCase activity of the obtained supernatant was measured and found to be 100 units/unit.

[Brief explanation of drawings]

FIG. 1 is a drawing showing the relationship between the enzymatic reaction PH and relative activity of alkaline cellulase K-521. Second
The figure 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 diagram showing the relationship between treatment temperature and relative activity of the same enzyme.

Claims (1)

[Scope of Claims] 1. Bacillus that grows in a neutral medium and produces alkaline cellulase having the following enzymatic properties.
SP KSM−521. (1) It has a wide optimum PH range of PH7 to 10, and its optimum
PH is around PH10. (2) The presence of Hg ²⁺ inhibits its activity;
Activated by the presence of Ca ²⁺ . (3) Its activity is hardly inhibited by proteases, surfactants, and chelating agents. (4) Main activity is CMC decomposition activity (Cx activity), and also has filter paper disintegration activity and abselase activity (C ₁ activity). (5) Molecular weight: Approximately 31,000 (by gel filtration method using Cephadex G-100).