JPS6024711B2 - New cellulase and its production method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a novel cellulase A and a method for producing the same, more specifically, a method for culturing a novel cellulase A belonging to the genus Ceratocystis, and separating and collecting the novel cellulase A from the culture. The present invention relates to a method for producing a novel cellulase A characterized by the following.

In the present invention, "cellulase A" refers to an enzyme having the physicochemical properties described below.

Cellulase A obtained by the present invention specifically acts on natural cellulose and cellobiose, and its 8-1,4
-It is an enzyme that has substrate specificity to cleave glucoside bonds from the end (exo type) and cleave carboxymethyl cellulose (CMC) and Avicel internally (endo type) in an extremely short time of about 60 minutes.

Cellulases with such characteristic substrate specificity are
It has not been found in conventional cellulases originating from fungi, bacteria, molluscs, higher animals, etc. Therefore, the cellulase A of the present invention is a novel enzyme in its substrate specificity. The present invention will be explained in detail below.

First, the microorganism used in the present invention is Ceratocystis (Ce-toc$t), which has the ability to produce cellulase A.
is), and one example is Ceratocystis densiflora knob sp.
toc$tis densiflora skin v sp)
(Hereinafter, referred to as "Ceratocystis sp.").

). The Ceratocystis bacterium was isolated from the inside of the material that caused pine wilt, and was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on February 26, 1980, and its microbial accession number is This is FERMP-6365.

The mycological properties of the Ceratocystis bacterium are as follows.

[a-Growth status in each tower site ■ Abek agar medium medium composition Growth status KOZO. 59, MgS04 After culturing at 20-2.5°C, 10.59, K2HP04 forms a round, white, 1.09 cm diameter 5.5 cm in 10.59 weeks.

NaN032.09 in the middle of the fungus, gray in the center, generally woolly. The back side of the bar is 0.019, sucrose 309, the outer edge is dark yellow,
The central part exhibits gray agar 15-209 and water IZ purple-yellow. Growth is good at pH 6.8, after which the color of the bacteria changes from the center to a deep gray blue (almost black).

After 10 days of culture, the The diameter is 10 skin and wool-like. It grows and is dark near the center and outer edge. Creates a black discolored trench to promote good growth. The most part of the white bacteria is dense.

On the back side, the outer edge is white, The round part of the hair is almost dark black, There are parts with a slight yellowish-blue tinge. Ru.

After that, the hair gradually turns black.

■ Culture medium used with the mycological properties listed in the ``Fermentation Products of Microorganisms'' in the Industrial Examination Standards for ``Fermented Products of Microorganisms'' (excluding ``Tsabek Agar Toji'').

), the following culture medium was used. Medium composition: Growth condition: KO20.59. When cultured at around 200C, IMgS040.59, white woolly droplets K2HP041.09, are suspended on the liquid surface within a week, and after 10 days, K becomes liquid NaN032.0g, and yeast surface becomes white. The bacterial flora is amazing.

After 20 days of Extract 2.59 and Bebuton, the bacterial flora was grayish in color, and in Liquid 59 and Glycerin 5mZ, it was yellowish brown.
Approximately half of the furnace paper that was put in furnace paper 109 and water IZ disappears. After that, the ash pH was 6.5 and the black color became stronger, and after one month, the entire K became a gray-black liquid. Most of the fibers disappear.

oH went from 6.5 to 8.2-8.1 month later. It will be about 8.5.

■ Matsuba broth tower medium composition and growth. Condition KOZO. 59, MgS04 Good growth, wet woolly 0.59, K2HP04 Mold-like, 1.09, NaN0320 dark to black color, 509, Beptone 59, Yeast.

On the medium, Chararaekis 2.59, agar (ohal
ara) type conidiophore of 15 to 20 g, and separate pine needle oval conidia 1.
5~2.5〃Spout 2~3〃 of water IZ in 2002. In addition, after boiling for 2 hours, a small number of zygotic mycelium, which appeared to be tied together with a solution suitable for the cloth furnace, were seen, although in small numbers, in the bacterial flora. When old, cocoon bed medium pH7
．． A black cocoon nucleus-like substance is formed in the dry area of 0. The ascus is about 250 mm wide, has an eggplant flask shape with a length of about 400 mm, has a short neck of 50 to 60 mm, and has a 5 x 8 ascospore inside, and 6 to 8 ascus spores. Yes, it's a cool color.

The rhombospores are pillow-shaped, somewhat narrow in the center, and large. The size is 1.0~1.5mm x 4~ It is 6.

'2} Physiological properties ■ Optimum growth conditions pH: PH6.8 Temperature: 20°C ■ Growth range pH: PH6.5-8.5 Temperature: 20-25qo Based on the above properties, the Ceratocystis sp.
T. R. NagRaiand Boys Kendr
ick;“ A Monograph of Ch.
alara and AmedCEnera''Wil
frid Lamier UniV Press, Ca
As a result of a comparative search according to the classification method described in Hada Da (1975), no matching species was found among the known bacterial species, and it was decided that the Ceratocystis genus bacteria could be designated as a new bacterial species belonging to the genus Ceratocystis. It was concluded that it was reasonable.

The Ceratocystis genus bacteria produce and accumulate high units of cellulase A in their culture solution, and their culture includes:
It is preferable to use various carbohydrate raw materials such as sucrose, glycose, starch, cellulose powder, and bushes as carbon sources in the culture medium, and organic substances such as beptone, meat extract, corn steep liquor, and defatted soybeans as nitrogen sources. Inorganic substances such as salts, phosphates, nitrates, etc. can also be used.

In addition, it is recommended to add appropriate amounts of inorganic metal salts, vitamins, yeast extract, etc. In addition, when culturing, 20~
Although it is preferable to perform static culture near 25:00 o'clock, shaking face or aerated rope culture may also be used, and in the case of static culture, approximately 4
In the case of shaking face or aerated fly Azusa culture, the accumulation of cellulase A reaches its maximum in approximately 1 to 2 weeks. A crude enzyme solution can be obtained by adding a furnace auxiliary agent to the obtained culture solution and filtering it, or by separating it halfway.

This crude enzyme solution may be used as it is, but it is better to use a crude enzyme solution obtained by applying a known method such as ammonium sulfate salting out method, solvent precipitation method, or dialysis method, or use a further known method. It can also be used as a purified enzyme after being purified and crystallized. Furthermore, the activity of the cellulase A preparation obtained according to the present invention is measured as follows.

[Measurement method and unit of activity]

Add this enzyme solution 1' to 0.5' of 1% CMC/(or 1% Avicel or 10M cellobiose) and acetate buffer (pH 5.7), and flood at 37°C for 1 hour.

After the reaction is completed, the Nelson-Somogyi method (Nelson-S
Quantitative determination of reducing sugars is carried out using the omoliu method).

That is, 0.5 of Somogyi's reagent was added to 0.5 of the reaction solution, heated at 100 qC for 10 minutes to develop color, and after cooling, 0.5 of Nelson's reagent was added and diluted with distilled water in step 5. This is measured colorimetrically at a wavelength of 50 nm. The unit of enzyme titer was 100 units when reducing sugar equivalent to 1 part of glucose was produced in 1 minute under the above conditions. The specific activity of cellulase A of the present invention against cellobiose is 10
,000 to 20,000 units/night.

Cellulase A is a novel enzyme with physicochemical properties, particularly action and substrate specificity, as described below. [Physicochemical properties of the enzyme] [11-acting cellulase A specifically acts on natural cellulose and cellobiose and hydrolyzes their 8-1,4-glucoside bonds.

■ Substrate-specific cellulase A cleaves the 6-1,4-glucoside bonds of natural cellulose and cellobiose from the terminal (exo type), while cleaving carboxymethyl cellulose (CMC) and Avicel internally in an extremely short time (endo type). ) has substrate specificity.

That is, when the Ceratocystis genus bacteria were cultured using furnace paper scraps as the only carbon source, and the activity of the culture furnace liquid after one week was measured by the Somogyi method using CMC as a substrate, it was found that the activity was measured by the Somogyi method using CMC as a substrate. The amount of glucose equivalent is determined. In addition, 125mutano' activity was shown for cellobiose, and both exhibited characteristic substrate specificity that exerted an endo-type effect at an extremely slow rate. Glue Optimal pH and stable pH range PH3-8 is Mcllivaine buffer, pH 8-11 is Glycyl (GIycjne)
Each was adjusted using a buffer solution.

The lowest pH of cellulase A was pH 5.75 to 6.0 (see attached drawing).

In addition, when we investigated the residual activity when cellulase A was incubated at 30q○ for 60 minutes, the pH was 4.0 to 9.
It was found to have a stable pH range of 0.

'4' Titer determination method 1% CMC/', acetate buffer (p
H5.7) Add the top of this enzyme solution 1 to 0.5 chickens, salt and react at 370°C for 1 hour, then add 0.5 parts of Somogyi's reagent to 0.5 parts of the reaction solution, and incubate for 1 blow. , heated at 10000,
After color development and cooling, add 0.5'Nelson's reagent,
Dilute with 5' of distilled water.

This is determined colorimetrically using a wavelength of 500 Yanagi (Nelson-Somogyi method). '5} Range of suitable temperature for action The suitable temperature for action of cellulase A is in the range of 30 to 37q0.

'6} Conditions for disqualification due to pH and temperature Inactivation due to PH: As a result of examining the conditions for disqualification due to pH for cellulase A under the same conditions as the measurement of the stable pH range described above, it was found that pH9.
0 or more and pH 3.0 or less is disqualified.

Temperature stability: Regarding cellulase A, the conditions for disqualification due to concentration were investigated by varying the temperature under the condition of pH 6.0, and as a result, the temperature at which thermal disqualification begins was 50 qo.

[7] Purification method The culture solution of Ceratocystis bacteria was concentrated by ultrafiltration method, and hydroxylapatite (Hydroxylapatite) was equilibrated with phosphate buffer (adjusted to pH 7.0).
The mixture is adsorbed onto a calcium phosphate (calcium phosphate) column and eluted with the above buffer.

DEAE-Sephadex (“Sephadex”) equilibrated with phosphate buffer (adjusted to pH 7.0) was then added.
ep-hade (manufactured by Swedish Pharmacia), and after thorough washing with the buffer solution, 0.8
Elute with the above buffer containing M NaCl. After elution, biogel (Bi
A purified enzyme preparation of cellulase A is obtained by gel filtration using a P-60Blogel (manufactured by Bio-Rad) column.The molecular weight of cellulase A is obtained by gel filtration using Sepharose fat. The molecular weight was estimated to be about 90,000, and based on SDS-acrylamide electrophoresis, it was estimated to be about 95,000-100,000.

{9} Is the crystal structure of cellulase A crab? Its crystal structure is unknown because it is difficult to produce crystals.

'IQ Elemental Analysis Cellulase A has a molecular weight of approximately 90, based on its behavior through gel filtration.
,000, and the molecular weight was estimated to be approximately 95,000 to LOOO,000 from the behavior by SDS-acrylamide electrophoresis, but it is difficult to discover the characteristics of such polymeric enzymes even by calculating elemental analysis values. Since this is not possible, this measurement was not performed.

As detailed above, according to the present invention, cellulase A-producing bacteria belonging to the genus Ceratocystis can be cultured, and cellulase A, a novel enzyme, can be advantageously produced from the culture solution.

The method of the present invention will be explained below using examples.

Example KCI O. 5 evening, MgS04 0.5 evening, K2
HP041.0 evening, NaN032.0 evening, Glysil 5
2.5 nights of yeast extract, 5 nights of bepton, 10 parts of cellulose (Fragment of Furnace), 1 part of water, and inoculated the Ceratocystis sp. , 25q0, flat static culture was performed for 5 weeks.

After culturing, the culture solution was concentrated 5 times by ultrafiltration, adsorbed onto a Hydro-based lapa-tite column equilibrated with a phosphate buffer (adjusted to 7.0), and eluted with the buffer ( Concentration gradient: 0.0-0.2 mol). DEAE-S equilibrated with phosphate buffer (adjusted to a plaque of 7.0)
It is adsorbed onto an ephadex column, thoroughly washed with the above buffer, and then eluted with the above buffer containing 0.9 M NaCl (concentration gradient: 0.0 to 1.0 mol).

After elution, gel filtration was performed using a Biosaddle IP-60 column equilibrated with an acetate buffer (adjusted to pH 5.7), and ethanol was further added to precipitate it into a dry powder. Ten female purified enzyme preparations of cellulase A (specific activity: 20,00 hits/night) were obtained per night of the culture solution.

[Brief explanation of drawings]

The accompanying drawing is a graph showing the optimum pH of cellulase A of the present invention.

Claims (1)

[Claims] 1. A novel cellulase A having the following physical and chemical properties. (1) Action: Acts specifically on natural cellulose and cellobiose, hydrolyzing their β-1,4-glucoside bonds. (2) Substrate specificity While the β-1,4-glucoside bond of natural cellulose and cellobiose is cleaved from the terminal (exo type), carboxymethylcellulose (CMC) and Avicel are
Cuts inside in an extremely short time of about 0 minutes (end type)
It has substrate isomerism. (3) Optimal pH and stable pH range The optimal pH range is between pH 5.75 and 6.0, and the stable pH range is between pH 4.0 and 9.0. (4) Range of suitable temperature for action The suitable temperature for action is in the range of 30 to 37°C under the condition of pH 6.0. (5) Living conditions It is inactivated at 50°C and at pH 99.0 or higher and 3.0 or lower. (6) Molecular weight: Approximately 90,000 by gel filtration and approximately 95,000 to 100,000 by SDS-acrylamide electrophoresis. 2. A method for producing a novel cellulase A, which comprises culturing a novel cellulase A-producing bacterium belonging to the genus Ceratocystis, and separating and collecting the novel cellulase A from the culture. 3. A novel cellulase A-producing bacterium belonging to the Ceratocystis genus is Ceratocystis densiflora knob sp.
sdensiflora nov sp. (Feiko Kenboku No. 6365).