JPH06292576A - Novel cellulase - Google Patents

Abstract

PURPOSE:To provide a novel cellulase which is isolated from the culture mixture of Streptomyces sp. KSM-26 and useful as an engyme to be added to a cloth detergent containing protease, because it has resistance to protease and broad bubstrate specificity. CONSTITUTION:Streptomyces sp. KSM-26 (FERM P-13548) is isolated from the soil in Ibraking Prefecture, Japan is cultured and the product is isolated from the culture mixture to give the novel cellulase S-1 which acts on carboxymethylcellulose to form redudcing sugar, further acts on xylan and lichenan well, and on filter paper, phosphoric acid-swollen cellulose, and laminarin a little, and on CMC sustrate in a pH range from 4 of 9, optimally at 6.5 at a temperature from 20 to 70 deg.C, optimally 40 deg.C and has more than 80% remaining activity in 3 days when treated with protease and a molecular weight of 22,000+ or -2,000 (gel filtration and SDS electrophoretic method).

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel cellulase, and more particularly to a novel cellulase S-2 which exhibits protease resistance and broad substrate specificity.

[0002]

2. Description of the Related Art The development of fibrinolytic enzymes (cellulases) has hitherto been promoted with the major goal of effectively utilizing biomass resources. Mainly filamentous fungi such as Trichoderma spp. And Humicola spp., Pseudomonas spp., Bacillus spp. Cellulases derived from bacteria such as genus and actinomycetes such as Streptococcus and Actinomyces have been reported. However, the action specificity and various physicochemical properties,
It is hard to say that the actual situation has been clarified.

Of the above cellulases, those having a particularly high action on carboxymethylcellulose (CMC) are C
Although referred to as MCase, in recent years, the use of this CMCase as a detergent for clothes has been increasing year by year as an industrial use (Japanese Patent Publication No. 59-49279, Japanese Patent Publication No. 60-).
23158, Japanese Patent Publication No. 60-36240).

As a method for producing cellulase which can be used as a detergent composition for clothes, a method of culturing an alkalophilic bacterium belonging to the genus Bacillus and collecting cellulase A from a medium (Japanese Patent Publication No. 50-28515), Cellulomonas Method for producing alkaline cellulase 301-A by culturing alkalophilic bacterium belonging to the genus (JP-A-58-224)
No. 686), alkalophilic Bacillus No. 1139
To produce carboxymethylcellulase (Horikoshi et al., J. Gen. Microbi
ol. , 131, 3339, (1985)), a method for producing alkaline cellulase using a Streptomyces genus (Japanese Patent Application Laid-Open No. 61-19483), a alkalophilic bacterium Bacillus sp. KSM-63.
5 (Bacillus sp. KSM-635) is cultured to produce an alkaline cellulase K or the like (Japanese Patent Laid-Open No. 6-58242).
3-1099776-109978), a method of culturing a Bacillus bacterium to produce alkali-tolerant cellulase K-522 or K-588 (JP-A-64-37285, JP-A-64-37286), etc. Has been reported to produce the so-called alkali-tolerant cellulase.

[0005]

However, these cellulases do not have resistance to protease, which is a proteolytic enzyme, and therefore, for example, they may be simultaneously added to a liquid laundry detergent containing protease. could not. Further, even if they have protease resistance, they were not satisfactory in terms of enzyme activity and the like. Therefore, it has been desired to newly provide a novel cellulase which has protease resistance and can be added to a detergent for clothes together with protease to enhance the cleaning effect.

[0006]

[Means for Solving the Problems] The inventors of the present invention have sought, in nature, a microorganism capable of producing a cellulase having a surfactant resistance and a protease resistance, and as a result of continuing an earnest search, the soil around Kashima-gun, Ibaraki Prefecture is now being found. It was found that the microorganisms belonging to the genus Streptomyces that were collected from the above produced a novel protease-resistant cellulase and that the cellulase was further purified to obtain a novel cellulase as a main component thereof, and thus the present invention was completed.

That is, the present invention provides a novel cellulase S-
2 is provided.

The cellulase S-2 of the present invention is isolated from the culture of Streptomyces sp. KSM-26 which is a microorganism belonging to the genus Streptomyces. Below, this Streptomyces SP KS
The mycological property of M-26 is shown. The methods for testing and classifying the mycological properties described below are as follows: "The Japan Patent Office edited industry-specific examination criteria applied microbial industry", "Experimental method for identifying actinomycetes" (ed. Classification experiment method ”(edited by Kazuo Komagata).

(A) Morphology The branching method of spore-forming hyphae is a pseudo-rotational bifurcation in which both simple bifurcation and axle bifurcation are observed, and the hyphae are loop-shaped and spiral-shaped. The number of spores is 10 spores or more, the spore surface is smooth, the size is about 0.5 μm in minor axis and 1.2 to 1.
It is a 4 μm elliptical shape. No flagella spores or sporangia. The spore stalk is located on the aerial mycelium.

(B) Growth state in medium Table 1 shows.

[0011]

[Table 1]

(C) Physiological properties (1) Growth temperature range: 20-40 ° C. (2) Liquefaction of gelatin (glucose, peptone gelatin medium): Liquefaction was not observed in any medium at 20 ° C and 28 ° C. (3) Starch hydrolysis (starch agar medium): Decomposes. (4) Coagulation of skim milk and peptone conversion (skim milk medium):
After culturing at 30 ° C., peptone formation was observed after coagulation. (5) Generation of melanin-like pigment (tyrosine agar medium and peptone yeast iron agar medium): Generate.

(D) Assimilation of carbon source (Pridham-Godlieve agar medium) (++: good +: normal)

[Table 2] L-arabinose ++ D-xylose ++ D-glucose ++ D-fructose + sucrose ++ inositol ++ L-rhamnose ++ raffinose ++ D-mannitol ++

The KSM-23 strain is morphologically characteristic of actinomycetes. It also contains LL-diaminopimelic acid in the cell wall, develops well without dividing the basal hyphae normally, does not form sclerotia, conidia shell, sporangia, and creates long segmented spores in aerial mycelia. Since the spore stalk is a pseudo-rotational branch, it can be identified as Streptomyces genus according to the search formula mainly on the cell wall of Kiyono and Hasegawa.

Furthermore, the color of the aerial mycelium on oatmeal agar medium is red, the spore chains are loop-shaped and spiral-shaped, the spore surface is smooth, and a soluble pigment is not formed,
When melanin-like pigment is produced and the result of the carbon source assimilation test is searched according to Nonomura's or Quester's search formula, this strain is identified as a new strain of Streptomyces. It was named SP KSM-26. This strain is FER
Deposited as MP-13548 to the Institute of Biotechnology, Industrial Technology Institute, AIST.

To produce the cellulase S-2 of the present invention, Streptomyces sp. KSM-26 may be cultured and cellulase S-2 may be collected from the culture.

The cellulase S of the present invention using the above strains
-2 can be obtained by inoculating the strain in an appropriate medium and culturing according to a conventional method. As the culture medium, one prepared by appropriately combining assimilable nitrogen source and carbon source is used. The carbon source and the nitrogen source are not particularly limited. For example, as a nitrogen source, ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium phosphate, sodium nitrate, corn gluten meal, soybean powder, corn steep liquor, casamino acid, yeast extract, pharmamedia, sardines, meat extract,
Peptone, Hypro, Aji Power, Corn soybean meal, Coffee meal, Cottonseed meal, Calcibeta, Azix, Zest, Sodium glutamate, etc. Quality, molasses, invert sugar, carboxymethyl cellulose (CMC), Avicel, cellulose cotton, xylan,
Pectin, ribose, arabinose, xylose, glucose, mannose, fructose, galactose, lactose, maltose, sucrose, trehalose, mannitol, glycerin, starch, acetic acid, citric acid and the like can be mentioned. In addition, phosphoric acid, Mg ²⁺ , Ca ²⁺ , Mn ²⁺ , Zn ²⁺ , Co ²⁺ , Na ⁺ ,
Inorganic salts such as K ⁺ and, if necessary, inorganic and organic micronutrient sources can be added.

Cellulase S- which is the desired product from the culture
Collection and purification of 2 can be performed according to a general enzyme collection and purification means. That is, the crude enzyme solution can be obtained by separating the cells by centrifugation or filtration of the culture, for example. This crude enzyme solution can be used as it is, but if necessary, for example, a protein is precipitated by a salting out method, a solvent precipitation method (methanol, ethanol, isopropanol, etc.), or ultrafiltration (for example, Asahi Kasei Kogyo). The crude enzyme powder can be obtained by freeze-drying after concentration with a molecular weight cut-off of 13,000) produced by the company. Specifically, in the case of the salting-out method, for example, it can be made into a freeze-dried powder by precipitating with ammonium sulfate (30 to 50% saturated fraction), followed by filtration or centrifugation and desalting. As a desalting method, a general method such as dialysis or a gel filtration method using Sephadex G-25 (manufactured by Pharmacia) is used. Further, the purification of the target product, cellulase S-2, is carried out, for example, by ion exchange using DEAE-Toyopearl under a weakly acidic condition to adsorb the dye. The adsorbed fraction is again subjected to ion exchange chromatography on DEAE-Toyopearl under mildly alkaline conditions and then gel filtration on Sephacryl S-200. It is also effective to utilize affinity chromatography, hydroxyapatite chromatography, hydrophobic chromatography and the like, if necessary, in order to increase the degree of purification.

The cellulase S-2 thus obtained has the following enzymatic properties. The enzyme activity was measured according to the following method.

Enzyme activity measurement method (1) CMC decomposition activity CMC in reaction system (A01MC, Sanyo Kokusaku Pulp Co., Ltd.)
Final concentration of 1%, phosphate buffer (pH 7) final concentration is 40%
0.1 ml of the enzyme solution appropriately diluted is added to 0.9 ml of the substrate solution having a mM concentration, and the mixture is reacted at 40 ° C. for 20 minutes. After the reaction, add 1 ml of DNS solution and boil at 100 ° C. for 5 minutes.
Immediately after boiling, cool with ice, add 4 ml of deionized water,
The absorbance at 535 nm is measured. At the same time, a glucose calibration curve was measured and the enzyme titer was 1 μm / min under the above conditions.
The amount of enzyme that produces a reducing sugar corresponding to mol of glucose was defined as 1 unit.

(2) p-Nitrophenyl cellobioside degrading activity A substrate solution in which the final concentration of the substrate (manufactured by Sigma) in the reaction system is 10 mM and the final concentration of the phosphate buffer solution (pH 7) is 40 mM. Add 0.1 ml of the enzyme solution appropriately diluted to 9 ml to 40
React at 10 ° C for 10 minutes. Then, ₃ ml of 1M Na ₂ CO 3
In addition, after stopping the reaction, the absorbance at 405 nm is quickly measured. 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) p-Nitrophenyl glucopyranoside decomposition activity In 0.9 ml of a substrate solution such that the final concentration of the substrate (manufactured by Sigma) in the reaction system is 10 mM and the final concentration of phosphate buffer solution (pH 7) is 40 mM. Add 0.1 ml of the enzyme solution diluted appropriately to 40
React at 10 ° C for 10 minutes. Then, ₃ ml of 1M Na ₂ CO 3
In addition, after stopping the reaction, the absorbance at 405 nm is quickly measured. 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.

(4) p-Nitrophenyl xylopyranoside decomposing activity A substrate solution 0 such that the final concentration of the substrate (manufactured by Sigma) in the reaction system is 10 mM and the final concentration of phosphate buffer solution (pH 7) is 40 mM. Add 0.9 ml of appropriately diluted enzyme solution to 9 ml of 40
React at 10 ° C for 10 minutes. Then, ₃ ml of 1M Na ₂ CO 3
In addition, after stopping the reaction, the absorbance at 405 nm is quickly measured. 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.

(5) Avicel Degrading Activity The same procedure as for CMC degrading activity was performed except that 5% Avicel (manufactured by Merck) was used as a substrate.

(6) Phosphoric Acid Swelling Cellulose Degrading Activity The same procedure as for CMC degrading activity was performed except that 0.4% phosphoric acid swollen cellulose was used as a substrate.

(7) Degradation activity of filter paper The same activity as the decomposition activity of CMC was carried out except that 0.1% filter paper was used as the substrate.

Enzymatic properties (1) Action It acts well on CMC and dissolves them to form reducing sugar.

(2) Substrate specificity It has been found that, in addition to CMC, xylan and lichenan have a clearly strong resolution. It also has some activity against filter paper, phosphoric acid swollen cellulose, curdlan and laminarin. However, it does not have the activity of degrading crystalline fibers such as Avicel.

(3) Working pH and optimum pH When CMC was used as a substrate, the working pH was 4-9 and the optimum pH was 6.5. At pH 4.5 to 8.0, it has 50% or more of the activity at the optimum pH (Fig. 1).

(4) pH stability When treated at 40 ° C. for 2 hours, it does not deactivate at pH 3.0 to 10.0 (FIG. 2).

(5) Working temperature and optimum temperature As a result of measuring the working temperature in a phosphate buffer of 40 mM and pH 7, the working temperature when CMC is used as a substrate is 20 to 70.
The optimum temperature was 40 ° C. Even at 30 to 50 ° C, it has a relative activity of 50% or more of the activity at the optimum temperature (Fig. 3).

(6) Temperature stability When treated in a 40 mM, pH 7 phosphate buffer for 30 minutes at each temperature, it is stable between 0 and 50 ° C (Fig. 4).

(7) Effect of surfactant When stored at 40 ° C. with a 10% aqueous solution of alkylsulfonic acid sodium salt (SAS), alkyl sulfate sodium salt (AS) or polyoxyethylene alkyl sulfate sodium salt (ES). It has a residual activity of 50% or more even after 2 weeks.

(8) Protease resistance It has a residual activity of 80% or more in 3 days in the presence of 0.1% of various proteases.

(9) Effect of metal ion As a result of studying the effect by adding 2 mM various metal ions,
Sn²⁺, Fe²⁺, Cu²⁺, Zn ²⁺And Hg²⁺Inhibited by Mn²⁺
Activated by.

(10) Molecular weight It has a molecular weight of 38,000 ± 2,000 by the gel filtration method using Sephacryl S-200 and the SDS electrophoresis method.

(11) Content of Neutral Sugar No color is observed in the phenol-sulfuric acid method.

[0038]

INDUSTRIAL APPLICABILITY The novel cellulase S-2 of the present invention exhibits extremely strong protease resistance despite containing almost no sugar. Furthermore, regarding the substrate specificity, it has a much wider range of action than conventional cellulases, and it is a more natural cellulose (filter paper, phosphate swollen cellulose).
Also works.

Although it has an optimum pH around pH 7,
The working pH is 4 to 9, and it is recognized that it works even on the alkaline side, and it is extremely stable in a wide range.

Therefore, the cellulase of the present invention can exert a cleaning effect in the presence of protease, and thus can be effectively used as an additive enzyme for detergents for clothes and the like.

[0041]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

Example 1 About 1 g of collected soil around Kashima-gun, Ibaraki prefecture was suspended in 5 ml of sterilized water containing 1% NaCl, and the mixture was allowed to stand with stirring, and the supernatant was applied to an agar medium containing CMC for screening (medium 1). After culturing at 30 ° C. for 2 to 7 days, halo-forming colonies are selected,
A cellulase-producing bacterium was obtained. Acquired bacteria are macroscopic and
By repeating single colony separation until it became microscopically single, pure isolates could be obtained. Further, the obtained bacterium was inoculated into a liquid medium (medium 2) and shake-cultured at 30 ° C. for 3 days. After culturing, 1% ES and savinase (manufactured by Novo Co.) were added to the supernatant liquid after centrifugation at 1 AU / ml, and 4
It was stored at 0 ° C., the residual activity of CMCase activity at pH 9 was measured, and a protease-resistant cellulase-producing bacterium was selected. According to the method described above, Streptomyces sp. KSM-26 (FERM P-1354) of the present invention is used.
8) was able to be acquired.

TABLE 3 Medium 1 CMC-containing agar medium (HA-Agar) CMC 3% 0.1% humic acid _{KH 2 PO 4 0.2% MgSO 4} · 7H 2 O 0.05% NaCl 0.1% Agar 1% 0.05% medium 2 CMC 1% polypeptone 0.1% yeast extract _{KH 2 PO 4 0.1% MgSO 4} · 7H 2 O 0.02%

Example 2 Streptomyces sp. KSM obtained in Example 1
-26 was replaced by 3 in place of CMC in the medium 2 of Example 1.
% Bran was replaced with polypeptone in a liquid medium containing 0.5% nitric acid, and the aeration rate was 0.5 vvm at 30 ° C. for 3 days.
The cells were cultivated in a 30-liter fermenter with stirring at 200 rpm.
After culturing, the bacterial cells and insoluble matter were removed by a filter press to obtain a crude enzyme solution. 15 l of this crude enzyme was concentrated and desalted by ultrafiltration (produced by Asahi Chemical Industry Co., Ltd., molecular weight cutoff of 6,000) to obtain 1 l of crude enzyme solution. Further, freeze-drying was performed to obtain a dry powder, which was used as a starting material for purifying cellulase S-2.

Example 3 Dry powder obtained in Example 2 (specific activity 10.8 KU / mg
Protein) was purified by the following procedure. Dissolve 5 g of the dry powder in 500 ml of 0.02 mM phosphate buffer (pH 6), centrifuge at 9,000 xg for 10 minutes, and then exchange the supernatant with anion exchanged with DEAE Toyopearl 650M. Subjected to chromatography. The adsorbed fraction is eluted by concentration gradient elution with sodium chloride to obtain the cellulase S-2 fraction. This fraction was lyophilized and then gel filtered on Sephacryl S-200. As a result, cellulase S-2 (5.15 KU / mg)
Could be separated.

Example 4 Cellulase S-2 obtained in Example 3 and JP-A-63-
Regarding CMCase I described in Japanese Patent No. 109777, enzyme activity against various substrates was measured using a pH 7 phosphate buffer. As a result, as shown in Table 4, cellulase S-2 has a clearly high resolution for xylan and lichenan in addition to CMC, and also shows activity for filter paper, phosphate swollen cellulose and laminarin. It was In addition, it has no resolution for Avicel and p-nitrophenyl cellobioside. As is clear from this result, the cellulase S-2 of the present invention has a broad substrate specificity as compared with CMCase I.

[0046]

[Table 4]

Example 5 With respect to the cellulase S-2 obtained in Example 3, the protease resistance was measured. Trypsin (manufactured by Sigma) and savinase (manufactured by Novo) were added to a final concentration of 0.1%, and the mixture was added at 40 ° C. in 0.1M phosphate buffer (pH 7.0).
Saved in. The results are shown in Table 5.

[0048]

[Table 5]

[Brief description of drawings]

FIG. 1 is a diagram showing the relationship between the enzyme reaction pH and relative activity of cellulase S-2.

FIG. 2 is a diagram showing the relationship between the treatment pH and relative activity of cellulase S-2.

FIG. 3 is a diagram showing the relationship between the reaction temperature and relative activity of cellulase S-2.

FIG. 4 is a graph showing the relationship between cellulase S-2, treatment temperature and relative activity.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Naoto Yamada 1-8762-23 Doihonhonmachi, Hasaki-cho, Kashima-gun, Ibaraki 1-207 Kao Dogosha House 1-201

Claims (2)

[Claims] 1. Cellulase S-2 having the following physicochemical properties. (1) Action It works well on carboxymethylcellulose (CMC),
These are dissolved to produce reducing sugar. (2) Substrate specificity In addition to CMC, it acts very well on xylan and lichenan, and also slightly on filter paper, phosphate swollen cellulose and laminarin. (3) Working pH and optimum pH When CMC is used as a substrate, the working pH is 4 to 9, and the optimum pH is 6.5. At pH 4.5 to 8.0, it has 50% or more of the activity at the optimum pH. (4) pH stability When treated at 40 ° C for 2 hours, it does not deactivate at pH 3.0 to 10.0. (5) Working temperature and optimum temperature When CMC is used as a substrate, the working temperature is 20 to 70 ° C and the optimum temperature is 40 ° C. It has a relative activity of 50% or more of the activity at the optimum temperature even at 30 to 50 ° C. (6) Temperature stability When treated for 30 minutes at pH 7.0, it is stable between 0 and 50 ° C. (7) Effect of surfactant When stored at 40 ° C. with a 10% aqueous solution of alkyl sulfonic acid sodium salt (SAS), alkyl sulfate sodium salt (AS) or polyoxyethylene alkyl sulfate sodium salt (ES) Two weeks later However, it has a residual activity of 50% or more. (8) Protease resistance It has 80% or more residual activity in 3 days in the presence of protease. (9) Effect of metal ions ^{Sn 2+, Fe 2+, Cu 2+} , is inhibited by Zn ²⁺ and Hg ^2+, Mn ²⁺
Is activated by. (10) Molecular weight 38,000 ± 2,000 (by gel filtration method with Sefacryl S-200 and SDS electrophoresis method). (11) Sugar content No color was observed by the phenol-sulfuric acid method. 2. Streptomyces sp. KSM
The cellulase S-2 according to claim 1, which is isolated and obtained from a culture of -26 (FERMP-13548).