JPS6112282A - Microorganism producing alkaline cellulase - Google Patents

Abstract

PURPOSE:To novel microbial strain which is an actinomycete separated from the soil collected in Haga district, Tochigi Prefecture, Japan, and capable of producing alkaline cellulase. CONSTITUTION:Examples of the above microbial strain capable of producing alkaline cellulase are Streptomyces sp-KSM-9 (FERM-P No.7620) and Streptomyces sp-KSM-2 (FERM-P No.7621). The separation of the above strain from the soil of the above district is carried out by conventional procedure using an agar medium. Alkaline cellulase can be prepared by inoculating and culturing the novel microbial strain belonging to Streptomyces in a proper medium. The mutant of the above strain may be used for the purpose, and the obtained culture product can be used as it is as an enzyme source. The living microbial cell separated from the culture medium or its treated product (e.g. freeze-dried product) can be used also as an enzyme source.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a novel alkaline cellulase producing bacterium.

[Conventional technology]

Alkaline cellulase has recently attracted attention as a new ingredient in laundry detergents. However, in nature, most of the cellulases produced by microorganisms are so-called neutral or acidic cellulases, which have stable enzymatic activity in neutral to acidic conditions, and alkaline cellulases are extremely rare.

Therefore, there are only two conventional methods for producing alkaline cellulase using alkaline cellulase-producing bacteria:
A method in which cellulase-producing bacteria belonging to the genus Bacillus and having an optimum pH on the alkaline side is cultured and harvested from the medium (Japanese Patent Publication No. 1983-28515), and a method in which alkaline-loving bacteria belonging to the genus Cellulomonas are cultured to produce alkaline cellulase 30
Only a method for producing 1-A (Japanese Unexamined Patent Publication No. 58-224686) was known.

[Problem that the invention seeks to solve]

Therefore, an object of the present invention is to provide a novel microorganism that produces alkaline cellulase.

[Failure to solve the problem]

The present inventor has searched nature for microorganisms that produce alkaline cellulases, and has continued to search for them. Recently, actinomycetes collected from soil in Haga District, Tochigi Prefecture have been found to be alkaline cell 2-
The present invention was completed based on the discovery of the ability to produce zeolite.

Conventionally, neutral or acidic cellulases produced by actinomycetes were rarely known (for example,
No. 032), it has not been known until now that actinomycetes produce alkaline cellulase as in the present invention.

The microorganism of the present invention that belongs to the genus Streptomyces and has alkaline cellulase productivity includes, for example, Streptomyces sp.
m mp+) KSM m 9 (Feikoken Bakuyori No. 7620
8M-2 (No.) and Streptomyces sp.
7621). These two strains were isolated by the present inventor from the soil of Haga-miyako, Tochigi Prefecture, and have the mycological properties shown in Tables 1 and 2 below. The strain has morphological characteristics of actinobacteria. Also, it is classified into the genus Streptomyces because its cell wall contains L-L-cyaminopimelic acid.

The KSM-2 strain belongs to the yellow series of strains based on the color of aerial mycelia on various agar media, the surface of the spores is smooth, the spore chains are slightly bent or generally straight, and there is no melanin. Based on the fact that similar pigments are not produced and the results of carbon source assimilation tests, we searched for similar strains of this strain among known strains according to Virginie's Manual, 8th edition, and found that Streptomyces canescens
tomyc@i Canaeens) is mentioned as a similar strain. However, whereas the KSM-2 strain produces alkali-resistant cellulase, Streptomyces
Both strains differ in that K. canescens does not produce alkaline-resistant cellulase, and the KSM-2 strain is a new bacterial species.

In addition, KSM-9 is also available in Virginie's Manual No. 8.
When searching according to edition, Streptomyces Punlceus is listed as a similar strain.

While Shikashi and Streptomyces zoniceus are characterized by the production of purple to red pigments, KBM-9 does not have this property, and only KSM-9 produces alkaline-resistant cellulase. Therefore, the X5M-9 strain is also a new bacterial species.

Isolation of this bacterial strain from soil as an isolation source was carried out by a conventional method using an agar medium, for example, as shown in Example 1 below.

〔Effect of the invention〕

The novel microorganism belonging to the genus Streptomyces of the present invention produces alkaline cellulase when it is inoculated into an appropriate medium and cultured.

As the microorganism, not only the above-mentioned strains but also mutant strains thereof can be used.

The culture medium may be any medium as long as it is utilized by the strain used and contains carbon sources, nitrogen sources, organic nutrients, inorganic salts, etc. necessary for the successful production of alkaline cellulase. .

As a carbon source, for example, carboxymethyl cellulose (
Soluble cellulose derivatives such as CMC), cellulose such as solid cellulose such as No9 Luso powder, mouth paper powder, and Avicel; carbohydrates such as glucose, functose, sucrose it, and sorbitol; citric acid, Kono\ Organic acids such as phosphoric acid; hydrocarbons such as n-dodecane and n-hexadecane, and any other organic acids that can be assimilated can be used. Among these carbon sources, a medium using cellulose or the like, especially a soluble cellulose derivative, is preferable because it produces a large amount of alkaline cellulase.

Examples of nitrogen sources or organic nutrient sources include nitrates such as sodium nitrate, potassium nitrate, and ammonium nitrate.
Salts: yeast extract, meat extract, pezotone, etc.

Examples of inorganic salts include carbonates such as sodium carbonate, potassium carbonate, sodium hydrogen carbonate, and potassium hydrogen carbonate; sodium diphosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium phosphate, potassium pyrophosphate, and sodium birophosphate; ,bird? sodium lyphosphate,
Phosphates such as sodium hexametaphosphate and inorganic salts such as magnesium sulfate can be used. Among them, carbonate is 0.
A medium containing 1 to 1.5% by weight is suitable because it produces a large amount of alkaline cellulase. In addition, trace amounts of heavy metal salts are used, but do not require additions as extended in media containing natural products.

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

For culture, after sterilizing the medium by heating etc., inoculate the bacteria and inoculate it for 25 minutes.
What is necessary is just to shake or aerate and stir at ~35°C for 2 to 4 days. p
Good results can be obtained by adjusting H to about 8 to 11. When using a carbon source that is poorly soluble in water, it is also possible to add various surfactants such as polyoxyethylene lupitan to the medium.

The culture obtained as described above can be used as it is as a prime image. Furthermore, live bacterial cells isolated from a culture or processed products thereof (lyophilized product, etc.) can also be used as an enzyme source. To separate K from the culture, ordinary solid-liquid separation means are used. Furthermore, the culture solution can be used as it is as an enzyme source, and the target substance, alkaline cellulase, can be collected and purified for use.

Collection and purification of alkaline cellulase from the culture solution can be performed according to general enzyme collection and purification methods.

〔Example〕

Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1 One cup of Soil Suno Chill from Haga District, Tochigi Prefecture (approximately 0.5 f
) was suspended in 1Od sterile water, thoroughly stirred, and left to stand. The soil suspension supernatant thus obtained is 0. 1- was applied to a separation agar medium having the following composition.

Composition: CMC2Of Peptone 1 (1 Meat Extract 1oy KH, PO41f N&Ctlof NalC0310f Agar 7.52 Water 1000-pH1
0,5 Next, this was cultured at 30°C for 3 days, and C was added around the colony.
Confirm that bacteria with a pellucid zone appear due to the dissolution of MC, collect colonies that form a clear pellucid zone, inoculate onto a slanted agar medium with the same composition as the above isolation agar medium, and incubate at 30°C.
The strains on the 10 slanted media were confirmed macroscopically and microscopically to be the same strain.

It was also confirmed that these 10 strains had the same properties on each medium and physiological properties.

The properties and physiological properties of the above-mentioned strains on each culture medium are as follows.
This is as shown in Table 2 and Table 2, and the present inventor has discovered that Streptomyces sp.
em ap, ) KSM-2.

Streptomyces sp. K8M-9
was similarly isolated from soil in Haga District, Tochigi Prefecture.

As a result of the above test, it was found that each of the 10 cultured bacteria was a single strain isolated from nature.

Next, a loopful of the pure cultured bacterial strain on the slant medium was suspended in a cryopreservation vial containing a sterilized 10% aqueous glycerin solution (2 m/liter) and stored frozen at -80°C.

After 3 months of frozen storage, a loopful of the suspension obtained by rapid thawing was resuscitated on an ordinary agar medium, and the properties and physiological properties were examined on each medium under the same conditions as above. No change was observed.

In addition, when the properties and physiological properties of the strains were similarly examined on each culture medium after the above-mentioned freezing and thawing was repeated five times every month, no changes were observed.

[Reference example]

Next, the results of producing alkaline cellulase using the strain obtained in Example 1 will be cited as a reference example. In addition, in the reference example, cellulase activity was measured by the following method.

[Cellulase activity measurement method]

CMC 2.5% solution, glycine NaCt-NaOH buffer (500mM, pH 9.0), ion exchange water
: Mix at a ratio of 1:1 and use it as a substrate.

Substrate 0.4 m/! Add enzyme solution o and 1- to the mixture and heat at 40°C.
Let it react for 20 minutes. After the reaction is completed, the amount of reducing sugar is determined using 3,5-dinitro-salicylic acid (DNS method). That is, add 1- of the DNS reagent to 0.5- of the reaction solution,
Color is developed by heating at 100° C. for 5 minutes, and after cooling, 4.5 m of ion-exchanged water is added to dilute. This is measured colorimetrically at a wavelength of 535 mμ.

For control, add 0.1 ml of enzyme solution to 0.4 ml of substrate DNS
Add 0.5 - of the reagent at the same time, immediately heat to develop color at IOOO°C for 5 minutes, and perform colorimetric determination in the same manner.

The unit of the enzyme titer was 100 units when reducing sugar comparable to the amount of glucose in the enzyme was produced in 1 minute under the above conditions. Furthermore, the cellulase activity of the purified concentrate was evaluated using the amount of reducing sugar produced by the enzyme 1Tq per minute under the above conditions as the amount equivalent to glucose.

Reference Example 1 A liquid medium containing 1% CMC', 1.5% meat extract, 0.5% yeast extract, 0.1% potassium dihydrogen phosphate, and 0.5% sodium carbonate in a 500-capacity plate flask. (
pH 9.5), Streptomyces sp. KSM
-2 shares? The cells were inoculated using sucinto, and after culturing at 30°C for 2 days, the bacterial cells were removed by centrifugation and the upper groove was collected.

When the cellulase activity was measured, it was found to be 32,000 units/l (<pH 9,0).

Furthermore, this supernatant was fractionated with ammonium sulfate, and the resulting solid fraction was freeze-dried to obtain enzyme powder. The cellulase activity of the obtained enzyme was % 0.90 N and 0 at pH 7.0 and 9.0, respectively.
．． 44 mg glucose equivalent/qmin. Furthermore, the results of investigating the pH dependence of the cellulase activity of this enzyme are shown in FIGS. 1 and 2. However, Fig. 1 shows that in the cellulase activity measurement method described above, the substrate and enzyme solution were heated at 40°C for 6
Figure 2 shows the result when the reaction was carried out for 0 minutes, and Figure 2 shows the result when the reaction was carried out at 20°C for 24 hours.

Reference example 2 8M-2 to Streptomyces sp. of reference example 1
Streptomyces sp. KSM-9
The supernatant of the culture solution was collected using the button operation in the same manner as in Reference Example 1, except that strain T was inoculated.

The cellulase activity was measured and found to be 45,000 units/l (pH 9.0).

Furthermore, an enzyme powder was obtained from this supernatant in the same manner as in Reference Example 1. The cellulase activity of the obtained enzyme was determined at pH 7.0 and 9.
．． 0 and 2 each. O1 and 1.49 glucose phases/η°min.

Furthermore, the results of investigating the pH dependence of the cellulase activity of this enzyme are shown in FIGS. 3 and 4. Note that the measurement conditions are the same as in FIGS. 1 and 2, respectively.

[Brief explanation of drawings]

Figures 1 and 2 are diagrams showing the pa dependence of the activity of alkaline cellulase obtained in Reference Example 1, Figures 3 and 4
The figure is a drawing showing the pH dependence of the activity of alkaline cellulase obtained in Reference Example 2. that's all

Claims (1)

[Claims] 1. An alkaline cellulase-producing bacterium belonging to the genus Streptomyces. 2. The alkaline cellulase-producing bacterium according to claim 1, which is Streptomyces sp. KSM-9 (Feikoken Bibori No. 7620). 3. The alkaline cellulase-producing bacterium according to claim 1, which is Streptomyces sp. KSM-2 (Feikoken Bibori No. 7621).