JPH02265479A - Production of cellulase - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for producing cellulase, and more specifically, it involves culturing neutral and alkalophilic producing bacteria belonging to the genus Bacillus under specific conditions, and extracting neutral from the culture medium. The present invention relates to a method for producing cellulase, which comprises collecting cellulase, alkaline cellulase, or alkaline-resistant cellulase.

[Conventional technology]

Traditionally, cellulase converts cellulose to glucose.

It is also understood as a complex group of enzymes that catalyze the enzymatic reaction that breaks down to lobiose or cellooligosaccharides.
Its mechanism of action is Eri, C7 enzyme 1cx enzyme and β-gluco/
Dase, or exo-β-glucanase, endo-β
-It is said to contain enzymes called glucanase, cellobiase, etc. Research on cellulases over the past several decades has been focused exclusively on the effective use of biomass resources.

For example, molds such as Trichoderma, Aspergillus, Acremonium, and 7 Mycolas are sought after as sources! ! Nine.

Most of the cellulases produced by these fungi have optimal and stable enzymatic activity at neutral to acidic pH. It is a so-called neutral or acidic cellulase.

On the other hand, most of the cellulases derived from bacteria are neutral or acidic cellulases, and there are only a few examples of production of alkaline cellulases, such as culturing bacteria belonging to the alkalophilic Bacillus group and producing cellulase A from the medium. Method (Japanese Patent Publication No. 50-28515), alkaline cellulase 301-A was obtained by culturing bacteria belonging to Cellulomonas.
ft production method (Japanese Unexamined Patent Publication No. 58-224686)
, A method for producing carboxymethyl cellulase by culturing alkaliphilic Bacillus 41139 strain (Fukumor
i F.

Kudo T. and Horikoshi K.
J. Gen. Mlcrobiol.

131.3339. (1985)) and a method for producing alkaline cellulase using Stretmyces sp. However, the productivity of Bacillus sp., which is a type of alkaliphilic bacterium, has been developed very recently.
cillus sp, ) KSM-635 (FEBM
P-8872) was found to produce a significant amount of alkaline cellulase K suitable as a laundry detergent composition, and a 1% patent application was filed (Patent Application No. 257776/1983).
.

[The problem that the invention attempts to solve]

However, there is no example of culturing cellulase-producing bacteria on an industrial scale to produce cellulase with high productivity, and there is an urgent need to develop a technology for culturing with high productivity.

[Elaborate means to solve the problem, S″]

The present inventors have conducted intensive research to produce cellulase in an industrially advantageous manner, and have discovered that the productivity of recellulase is significantly improved by including iron-containing substances in the medium. A friend who completed the invention.

That is, one aspect of the present invention provides a method for producing cellulase, which is characterized by culturing cellulase-producing bacteria in a medium containing an iron-containing substance.

Used in the method of the present invention. Examples of iron-containing substances added to the medium include iron powder, Fed, F'eSO, and the like.

FeC/, , Fe(OH)y, Fe5(PO<
)2 and other divalent iron compounds: Fezes, Fez
(Son)a, FeC15, Fe(OH)s
.

F e P Os, F eNHL (804')2
Examples include trivalent iron compounds such as iron threonine; iron-containing proteins such as hemoglobin, myoglobin, cytochromes, and catalase. These iron-containing substances are contained in the medium in an amount of 0.1 ppm or more as Fe, preferably lpp.
It can be formulated at m to 1000 ppm.

The cellulase-producing bacteria used in the method of the present invention may be any cellulase-producing bacteria, but preferably cellulase-producing bacteria belonging to the genus Bacillus, such as Bacillus sp.
sp, ) KSM-635 (FERMP-8872
), KSM-635n (FERM P-9083),
The same KSM-635v (FERM P-9084).

Same KSM-635r (FERM P-9085),
KSM-635nv (FERM P-9086), KSM-425 (FERM P-9007), KS
M-534 (FERMP-'JOIO), KSM-5
21 (F'ERM P-9009).

KSM-580 (FEBM P-9013), I (
SM-344 (FEBM P-9008), KSM-
539 (FERM P-9011), KSM-577
(FERM P-9012), KSM-522 (FERM
RM P-9370).

KSM-588 (FERM P-9371), KS
M-597 (FEBM P-9573) and the like.

The mycological properties of Uchibacillus sp. KSM-635 are as follows.

Mycological properties: +1) Microscopically observed bacteria size is 0.5-1.2 /jfiX 1.5
~4.0 μm rod, endospores (0.7 μm) at one end of the bacterial body.
Forms ~1.2 μmXl, Q ~2.0/jfi)'(z). Has periflagellates, is motile, and Durham staining is positive.

r21 Growth status in various media The pH range for growth is 8 to IL&Ci1. Usually, the best growth is achieved in Ishireto medium supplemented with 0.5 to 1.0% of sodium carbonate (Na1COs). The properties shown below are
All are mycological properties in the presence of 1.0 weight 5 Na1COs.

■ Meat juice agar medium The shape of the colony is circular and the surface is flat. The color of the village is white to yellow, translucent, and glossy.

■ Broth grows in liquid medium and becomes cloudy. It will not grow if the pH of the medium is neutral.

It grows in a 7% salt broth liquid medium and becomes cloudy.

Meat juice gelatin puncture culture It doesn't grow.

■ Ridmus milk medium Milk coagulation and begtonization were negative. (3) Physiological properties Although it has nitrate reducing ability, it does not perform denitrification reaction.

MR reaction is unknown, but vP reaction is positive.

Does not produce indole.

No hydrogen sulfide formation was observed.

Hydrolysis and assimilation of starch are not possible.

Use citric acid.

Assimilates nitric acid and nitrite. Although the usability of ammonium chloride is weak, am72ram phosphate can be used.

■ Shows pale yellow color in King B medium but has no fluorescence 0 ■Urease test is negative.

0 Oxidase is coagulation positive.

■ Catalase is positive ◎ Range of growth The temperature range for growth is 20-45℃, and the optimum temperature for growth is 29-37℃. The pH range for growth is 8-11, and the optimum pH for growth is 9.5-10.2°O.The attitude towards oxygen is aerobic.

■ Sugar utilization D-ribose, L-arabinose, D-xylose, D
-4 lucose, D-mannose.

Assimilates D-fructose, maltose, sucrose, trehalose, D-mannitol, ynontol, and glycerol. D-galactose, lactose,
I)-Sorbitol. Starch, dextrin, and rough-enose are not available.

■Hydrolyzability of casein and gelatin is negative.

0 Requires pyotin (or desthiobiotin) for growth.

Bacillus sp. KSM-4'! 5 p'
Examples of monological properties are as follows.

Mycological properties: (1) Microscopically observed bacterial cell size is 0.4-0.8 μm x 1, 5-
4.0/Jffi bacilli, oval endospores (0.8-1.2 pm x 1.2-1.5 μm) at one end of the bacterial body.
make. It has periflagella and is motile. Durham staining is indeterminate. It has no anti-acid properties.

(2) Growth status in various media ■ Broth agar plate medium Growth condition is weak. The shape of the village is circular.

The surface is smooth and the edges are also smooth. The color of the village is white, translucent and glossy.

■ Growth on gravy agar slant medium is weak, and the condition is spread-like, glossy, white and translucent.

■ Broth liquid medium It grows and becomes cloudy.

■ Meat juice gelatin puncture culture grows, but the condition is weak and liquefaction of gelatin is not observed.

■ Lido 1 Milk Medium No coagulation or liquefaction of milk was observed. Father, IJ) Discoloration of the trout was also observed.90 (3) Physiological properties■ Nitrate reduction and denitrification reactions were both negative.

■ The 5i4R test can be negative, positive, or equivocal (pH
5,2).

■ VP test was negative (pH 5,2).

■ Indole production was negative.

■ The production of hydrogen sulfide was negative.

■ Starch hydrolysis is positive.

■ The use of citric acid was negative in both Kosa medium and Christensen medium.

■ Regarding the use of inorganic nitrogen sources, both nitrates and ammonium salts were negative.

Pigment production was negative.

Urease test was negative.

Oxidase is positive.

Catalase is positive.

Growth range The temperature range for growth is 15-37℃, the optimal temperature range for growth ￥i
25-30℃.

The pH range for growth was pI (5 to 10). Optimum growth was achieved at pH 6 to 9.

■ The attitude toward oxygen is facultatively anaerobic.

0 0-F test It grows, but grows poorly in both aerobic and anaerobic conditions.

0 Sugar utilization (+: dual benefit; -2 not utilized) IL-arabinose + 2, D-xylose + 3, D-glucose + 4, 0-mannose + 5, fructose + 6, D-galactose + 7 , maltose (-8° sucrose 19, lactose 110, trehalose + 11, D-sorbitol + 12, D-mannitol 13, inosit - 14, glycerin ± 15, starch + pl in OvP medium ( pIf 5 .2 [Phase] No growth at 5% growth in salt-containing medium.

No growth at 7%.

No growth at 10%.

@Growth at pH 5 and 7 Not growing.

[Phase] Negative and positive for zohydroxyacetone production,
Not clear.

Deamination of O-phenylalanine was negative.

[Phase] Casein decomposition is negative.

[Phase] Tyrosine degradation is negative.

In addition to the above-mentioned saccharides, other appropriate assimilated carbon sources, nitrogen sources or organic nutrient sources, inorganic salts, etc. can be added to the medium used in the method of the present invention, if necessary.

Examples of carbon sources include plant fibers such as rice husk, wheat gluten, filter paper, patterned papers, sawdust, blackstrap molasses, (soluble) denzone,
Invert sugar, carboxymethyl cellulose (CMC'), Avicel, cellulose cotton, xylan, pectin, as well as assimilable carbon sources such as ribose, arabinose, xia-sose, glucose, mannose, fructose, galactose, maltose, sucrose. , lactose, trehalose, mannitol, ino/nodo, glycerin, and assimilated organic acids such as malic acid, acetic acid, and citric acid. Ninth, organic nutrient sources include nitrates such as sodium nitrate, potassium nitrate, and ammonium nitrate; yeast extracts from various sources, extracts from various animal tissues, extracts from various plant tissues, and animal or vegetable proteins. and its peptone. Examples of inorganic salts include carbonates such as sodium carbonate, potassium carbonate, carunium carbonate, sodium hydrogen carbonate, and potassium hydrogen carbonate; Potassium acid, sodium pyrophosphate, tri? Phosphates such as sodium lyphosphate and sodium hexametaphosphate, and inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium sulfate, and magnesium sulfate can be used. In addition, heavy metal salts are used, but do not necessarily require addition in media, including natural media.

Furthermore, when using a mutant strain that requires nutritional sources other than those mentioned above, a substance that satisfies the nutritional requirements is added to the medium.

Cultivation according to the method of the present invention can be carried out according to conventional methods, except for aeration and stirring under the above-mentioned conditions. For example, after completely heating the medium and sterilizing it, cellulase-producing bacteria are inoculated and kept at 15-50°C for half a day to 7 days, preferably for 1-7 days.
This can be carried out by culturing for 3 days. In addition, the p of the medium
Although H differs depending on the microorganism used, good results can be obtained by adjusting it to 5 to 12.58 degrees. In addition, when using a carbon source that is poorly soluble in water, various surfactants such as 1? Lioxyethylene sorbitan fatty acid ester.

Sorbitan fatty acid ester? It is also possible to add a surfactant such as lyoxyethylene alkyl ether, and the amount added is 0.01 to 2%, preferably 0.1 to 1%.
It's %.

〔Effect of the invention〕

According to the method of the present invention, cellulase can be produced on an industrial scale.

〔Example〕

Next, the present invention will be explained with reference to examples. In addition.

In the Examples, CMCase activity was measured as follows.

CMCase activity measurement method: 10xgCMC (Deyu Kokusaku) Q Rusosha'), 100p1
Substrate solution containing 00p glycine-NaOH buffer (pH 9,0) 0.91! 0.1 g Lt of enzyme solution was added to Lt and reacted at 30°C for 20 minutes. After the reaction, 3,5-dinitro-salicylic acid (3,5-dintro-5al
Reducing sugars were quantified using the icylic acid (DNS) method. In other words, 1 reaction solution 1.01 part DNS reagent
．． Od gold was added, and the mixture was heated at 100° C. for 5 minutes to develop color. After cooling, 4.0 II lj of deionized water was added to dilute.

This can be used for colorimetric determination at a total wavelength of 535 nm. The enzyme titer is
Under the above conditions, the amount of enzyme that produces all the reducing sugars equivalent to 1 μm 01 of glucose per minute is defined as 1 unit.

Example 1 (1) Bacillus sp. KSM-63 was added to the medium shown below.
5nv (FEBM P-9086)'.

A seed medium was obtained by culturing with shaking (or aerobically) at 30°C for an appropriate period of time.

Flavor liquid 8% (manufactured by Ajinomoto ■) Glucose 0.5% (separately sterilized) Fish meat extract
0.5% yeast extract 0.05% sodium carbonate 0.5% (separately sterilized) (11) Next, inoculate 1% of the nine types of culture medium obtained in (1) into the main medium shown below,
Aerated culture was performed at 30° C. for 2 days using a 2001 culture tank. This culture yielded 50,000 units of alkaline cellulase per ml of culture.

Flavor liquid 8% Fructose 2.2% Glucose 0.3% F e S 04 ・7 L 0 5 ppm Yeast extract 0.1% Sodium carbonate 0.5% Example 2 (1) Add Chillus to the medium shown below.・Varnish f-KSM-4
25 (FERM P-9007) was inoculated and cultured with shaking (or aerobically) at 30°C for an appropriate time to obtain a seed medium.

Sucrose 1% Cornstarch 7% Kerr yeast extract 0.5% KH, PO, 0.1% Na! HPO,・12H100,25%MgSO4・7
H, 00,02% pH 6,8 (Ill) Next, the main medium (control) with the same composition as in (1) was added to the nine types of culture medium obtained in (1), and to this control medium was added F e S
04.04.7H20 was inoculated at 1% into a main medium supplemented with 5 ppm, and cultured for 2 days at 30°C using a 2001 culture tank. Through this culture, cellulase' shown in Table 1 below was obtained.

Table 1 Examples 3 to 11 Fe50. *The alkaline cellulases shown in Table 2 below were obtained by culturing in the same manner as in Example 1, except that the components listed below were used instead of 105 ppm of 7H.

Table 2 that's all

Claims (1)

[Scope of Claims] 1. A method for producing cellulase, which comprises culturing cellulase-producing bacteria using a medium containing iron-containing substances at an amount of 0.1 ppm or more as iron. 2. The method for producing cellulase according to claim 1, wherein the cellulase-producing bacterium belongs to the genus Bacillus. 3. The method for producing cellulase according to claim 1, wherein the iron-containing substance is selected from iron powder, divalent iron ions, trivalent iron ions, iron-containing proteins, and iron threonine. 4. The method for producing cellulase according to claim 3, wherein the cellulase is selected from iron-containing proteins such as hemoglobin, myoglobin, cytochromes, and catalase. 5.Claim 1 in which the culture is carried out at 15 to 50°C
Method for producing cellulase as described in Section 1. 6. The method for producing cellulase according to claim 1, wherein culturing is carried out for half a day to seven days. 7. The method for producing cellulase according to claim 1, wherein the pH of the medium is 5 to 12.5. 8. The method for producing cellulase according to claim 1, wherein 0.01 to 2% of a surfactant is added to the medium. 9. Cellulase producing bacteria is Bacillus sp. KSM-6
35 (FERM P-8872), KSM-635n
(FERM P-9083), KSM-635v (FERM
ERM P-9084), KSM-635r (FER
MP-9085), KSM-635nv (FERM
P-9086), KSM-425 (FERM P-9
007), KSM-534 (FERM P-9010
), KSM-521 (FERM P-9009), KSM-580 (FERM P-9013), KSM
-344 (FERM P-9008), KSM-53
9 (FERMP-9011), KSM-577 (FE
RM P-9012), KSM-522 (FERM
P-9370), KSM-588 (FERM P-9
371) and KSM-597 (FERM P-957)
3) The method for producing cellulase according to claim 1, which is any one of 3). 10. In the culture of cellulase-producing bacteria, rice husk, wheat gluten, filter paper, general paper, sawdust, blackstrap molasses, (soluble) starch, invert sugar, carboxymethyl cellulose (C
MC), Avicel, cellulose cotton, xylan, pectin, ribose, arabinose, xylose, glucose,
Mannose, fructose, galactose, maltose, sucrose, lactose, trehalose, mannitol, inositol,
The method for producing cellulase according to claim 1, characterized in that one or more compounds selected from glycerin, malic acid, acetic acid, and citric acid are used. 11. In the culture of cellulase-producing bacteria, compounds selected from sodium nitrate, potassium nitrate, ammonium nitrate, yeast extract, animal tissue extract, plant tissue extract, and animal or vegetable protein and its peptone as a nitrogen source or organic nutrient source. The method for producing cellulase according to claim 1, characterized in that one or more of these are used. 12. In the culture of cellulase-producing bacteria, inorganic salts include sodium carbonate, potassium carbonate, calcium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, sodium phosphate, sodium monohydrogen phosphate, sodium dihydrogen phosphate,
One or more inorganic salts selected from potassium phosphate, potassium pyrophosphate, sodium pyrophosphate, sodium tripolyphosphate, sodium hexametaphosphate, sodium chloride, potassium chloride, calcium chloride, sodium sulfate, and magnesium sulfate are used. A method for producing cellulase according to claim 1.