JPH08173148A - Bacterium of genus bacillus producing xylanase having activity in alkali range, production of the same xylanase and method for using the same - Google Patents

Abstract

PURPOSE: To obtain Bacillus sp.NUF (3) 4-4 suitable for recycle of waste paper, capable of readily deinking waste paper pulp, capable of producing xylanase having activity in an alkali range. CONSTITUTION: This Bacillus sp.NUF (3) 4-4 produces xylanase having activity in an alkali range and is deposited as FERM-BP-4674. The Bacillus sp.NUF (3) 4-4 is cultured, xylanase having activity in an alkali range is obtained from the culture product and waste paper is deinked by using the xylanase.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel Bacillus bacterium and a method of using the same. More specifically, the present invention provides a bacterium belonging to the genus Bacillus that produces xylanase having an activity in the alkaline region (hereinafter referred to as alkaline xylanase), a method for producing xylanase using the bacterium belonging to the genus Bacillus, and removal of waste paper using the xylanase. It relates to the ink method.

[0002]

2. Description of the Related Art When manufacturing recycled paper from waste paper, a deinking step is essential in order to improve the whiteness of the recycled paper. This deinking process mainly involves swelling the fibers of the waste paper pulp and the ink binder under strong alkalinity, peeling the ink from the fibers by mechanical force such as a rotor or a rotor, and removing the peeled ink by flotation or It is separated by washing. Attempts have been made to accelerate the deinking by making the deinking process efficient, saving processing time and energy, and further acting an enzyme to improve the whiteness of recycled paper. For example, Japanese Examined Patent Publication No. 3-57235 and Japanese Patent Laid-Open No. 59-929.
No. 9, JP-A No. 2-80683, and JP-A No. 3-8.
No. 82 discloses a pulp treatment method using cellulase, JP-A No. 2-80684 discloses a pulp treatment method using lipase, and JP-A No. 4-2895 discloses a pulp treatment method using protease. However, most of the enzymes used in these treatment methods are active in the acidic and neutral regions, and some of them are inactivated in the strongly alkaline region.

Therefore, in the deinking process carried out under alkaline conditions, conventional enzymes cannot be used directly, and once an acidic or neutral active enzyme, especially xylanase, is used, the To an appropriate range for the enzyme, and at the end of the enzymatic reaction, it is necessary to add an alkali again to return to an appropriate pH for the next step, which causes a great loss of chemicals. Therefore, in order to perform the enzyme treatment efficiently by incorporating the xylanase treatment step into the conventional deinking step as it is, an enzyme that is stable and active in the alkaline region has been demanded, but conventionally, medium-weak alkaline Only the thing was marketed. In order to solve such a problem, Japanese Patent Laid-Open No. 6-6
Japanese Patent No. 2839 discloses a method for efficiently performing an enzyme treatment in a pulp manufacturing process performed under high temperature and alkaline conditions by using a microorganism that produces a thermostable alkaline xylanase and its enzyme. However, a xylanase having an activity in the alkaline region and a bacterium producing the xylanase, which are particularly useful in the deinking step of used paper purple for producing recycled paper, have not been known.

[0004]

In particular, a Bacillus bacterium that produces a xylanase having an activity in an alkaline region, which can be used in the deinking step of waste paper pulp, a method for producing xylanase using the Bacillus bacterium, and It is an object to provide a method for deinking waste paper using the xylanase.

[0005]

DISCLOSURE OF THE INVENTION The present inventors have searched for a xylanase-producing bacterium having an activity in the alkaline region, and as a result of extensive screening, have grown under alkaline conditions from the soil in a pulp mill, and in the alkaline region. We have found a new bacterium of the genus Bacillus that produces xylanase with excellent enzymatic activity. The present invention was made based on the finding that xylanase produced by this new strain of bacteria exerts an excellent enzyme action in the pulp treatment step, particularly in the deinking step of waste paper pulp. Therefore, the present invention provides Bacillus sp. NUF (3) 4-4 that produces xylanase having activity in the alkaline region. Further, the present invention is a method for producing an alkaline xylanase, which comprises culturing the Bacillus sp. NUF (3) 4-4, and obtaining a xylanase having an activity in an alkaline region from the culture, and a method for producing the same. The present invention provides a pulp treatment method characterized by adding xylanase, particularly a method for deinking waste paper. The mycological properties of the NUF (3) 4-4 strain isolated as a bacterium producing xylanase having an activity in the alkaline region were as follows.

[Bacteriological properties] (a) Morphological properties Cell size 0.9 to 1.1 × 2.7 to 4.6 μ Cell shape Straight rod morphology Presence or absence of cell polymorphism Presence or absence of motility (movement by periflagellates) With or without spores (0.7 to 1.0 × 1.2 to 1.4 μ elliptic and edible spores are formed. Sporangium is swollen and the size is 1.3 to 1.5 × 1.6 to 1.9 μ. .) Gram stain positive (incubation time) with alkalinity

(B) Growth condition in various media (1) Growth condition of broth agar plate culture medium Good surface condition Smooth color, glossy shape, ridge, rim, and other All well-developed at pH 9.0, characteristic colony pigment and diffusible pigment generation is not a recognized.

(2) Degree of turbidity of broth liquid medium No growth of liquid surface (no formation of surface film is observed) Medium precipitation Other At pH 9.0, growth and precipitation of the entire medium are observed.
It (3) Litmus milk growth is not observed.

(4) Separation Medium The strain of the present invention has a pH of 10 containing 50 mM sodium carbonate.
Alkaline medium (glucose 0.5% or wheat bran 5%, peptone 0.5%, yeast extract 0.05%, K ₂ HPO ₄ , 0.
_{1%, MgSO 4 · 7H 2} O, grow well in 0.05%). Growth precipitation of degree liquid surface of the turbid

(C) Physiological properties Denitrification reaction-MR test-Indole production NG (not grown in test medium) Gelatin liquefaction-Hydrogen sulfide production-TSI agar-Lead acetate agar-Use of citrate -Koser medium-Christensen medium-Use of inorganic nitrogen source Nitrate-Ammonium salt-Dye formation-Urease-Oxidase-Catalase Weak growth range pH (optimal) 8.5-10.0 (9.0-9.5) ) Temperature (optimal) 17 to 45 ° C (36 to 41 ° C) Attitude toward oxygen Facultative anaerobic Growth under anaerobic + OF test F

Acid formation Glucose + arabinose + xylose + L-arabinose + D-xylose + D-mannose-D-fructose + D-galactose-maltose-sucrose + lactose-trehalose + D-sorbitol-D-mannitol-inositol. -Glycerin-Starch-Gas formation None of the above sugars is found.

Regarding the above-mentioned mycological properties, Bergy's Manu
As a result of searching with reference to al of Systematic Bacteriology, this bacterium is an aerobic gram-positive bacterium that forms spores, and is considered to belong to the genus Bacillus because its catalase is weak. However, there is no corresponding type, so we judge that it is a new bacterial species, and
p. NUF (3) 4-4 (hereinafter referred to as Bacillus bacterium of the present invention). This bacterium belonging to the genus Bacillus has been deposited as FERM BP-4674 at the Institute of Biotechnology, Institute of Industrial Technology, Ministry of International Trade and Industry. For example, when comparing the known alkaline-growing Bacillus alcalophilus JCM5265 of the genus Bacilus sp. NUF (3) 4-4 of the present invention, JCM5265 shows negative anaerobic growth while Bacilus SP. NUF (3) 4-
4 is +, JCM5265 is + for gelatin liquefaction, whereas Bacilus sp. NUF (3) 4-4 is −. Bacilus SP. NUF (3) 4-4 is Bacilus alcalophilus J
It is a distinct bacterial species from CM5265.

By culturing the Bacillus bacterium of the present invention, alkaline xylanase can be produced. Carbon source,
Any nitrogen source can be used as long as it can be assimilated to produce alkaline xylanase. For example, as the carbon source, agricultural waste such as xylan or wheat bran containing xylan, plant fiber such as pulp bagasse, and molasses containing no xylan can be used.
As the nitrogen source, yeast extract, peptone, various amino acids, soybean, various inorganic nitrogen compounds and the like can be used.

The culture temperature and pH may be in the range where the Bacillus bacterium of the present invention grows and produces alkaline xylanase. The culture temperature is 25 to 45 ° C., preferably 30 to
40 ° C. is suitable, and the pH is 8.5-11, preferably 9-10.5. After culturing the microorganism of the present invention, the bacterial cells can be separated, and the culture filtrate can be used as it is or concentrated by boiling under reduced pressure and used as a crude alkaline xylanase enzyme solution. The alkaline xylanase thus obtained has an optimum reaction temperature of 30 to 50.
C., preferably 35 to 45.degree. C., and pH is 6.5 to 11, preferably 8 to 10.5. The alkaline xylanase produced by the Bacillus bacterium of the present invention does not inactivate at 60 ° C. or lower under the conditions of pH 5 to 12 for at least 60 minutes, and at 30 to 50 ° C., pH 6 to 10. The activity does not drop below 50% for 10 to 60 minutes under the conditions.
Therefore, the alkaline xylanase of the present invention can be used also in the pulp processing step employing such conditions. For example, pulp disintegration process, beating process,
It is a pulp production process such as a preparation process and a bleaching process with hydrogen peroxide.

In addition, dialysis, salting out, ultrafiltration, freeze-drying,
The crude enzyme can be concentrated or solidified by such means. As an example, in the case of ultrafiltration, the culture solution is
A method of obtaining a concentrate by centrifuging at 500 rpm for 20 minutes, concentrating and filtering with an ultra filter, and further repeating twice hydration and concentration (ultra filter) twice, can be used.

In deinking used paper pulp, the enzyme of the present invention can be used in steps such as disaggregation with pulper, before alkali immersion, and alkali immersion step. The deinking process for waste paper is all performed under alkaline conditions. In addition, since the currently known highly effective xylanase burys its potency under acidic conditions, in order to use it, it requires an extra step of lowering the pH to be acidic and returning it to alkaline again after xylanase treatment. And As in the present invention, if a xylanase having high potency under alkaline conditions is used, the enzyme treatment can be easily used in any current deinking process.
Although it is known to use cellulases that can act under alkaline conditions in the deinking process, the xylanase of the present invention is superior in this respect because cellulases also cause decomposition of pulp fibers. Next, the present invention will be described in detail based on examples.

[0017]

【Example】

[Example 1] Glucose 0.5%, polypeptone 0.5
%, Yeast extract 0.05%, K ₂ HPO ₄ 0.1%, MgSO ₄
・ 7H ₂ O 0.05%, Na ₂ CO ₃ 1.0%, pH 10.4
In the liquid medium of Bacillus sp. NUF of the present invention.
(3) 4-4 was inoculated and cultured at 30 ° C. for 3 days. After the completion of the culture, the culture supernatant was separated by centrifugation (10000 rpm × 10 minutes) to obtain a crude enzyme solution of alkaline xylanase. The activity of alkaline xylanase in the culture supernatant was measured by the following method. 0.5% xylan solution (beechwood xylan, manufactured by Sigma, pH 10.0, 50 mM sodium carbonate buffer) 20
50 μl of enzyme solution is added to 0 μl and reacted at 40 ° C. for 10 minutes. After the reaction, it is boiled for 10 minutes to inactivate the enzyme, 250 μl of Somogyi reagent is added, and further boiled for 20 minutes and immediately cooled with ice. Nelson reagent 500 in the liquid
Add μl and 3 ml of pure water and measure the absorbance at 500 nm. Xylanase activity is 1 μmol / min under the above conditions
The amount of the enzyme that produces the reducing sugar of is 1 unit (U).
As a result, the alkaline xylanase activity in the culture supernatant was 0.7 U / ml.

Example 2 5.0% wheat bran, polype
Puton 0.5%, yeast extract 0.05%, K₂ HPO _Four0.
1%, MgSO_Four・ 7H₂ O 0.05%, Na₂CO₃1.0%,
Inoculation of Bacillus sp. NUF (3) 4-4 into liquid medium with pH 10.4
And cultured at 30 ° C. for 3 days. After culturing, centrifuge (1
(0000 rpm x 10 minutes) to separate the culture supernatant,
Lanase was obtained. Alkaline xylanase in the culture supernatant
The result of measurement in the same manner as in Example 1 was 0.9 U / ml.
Was.

[Example 3] <Improvement of deinking property of waste paper by alkaline xylanase treatment> Water was added to waste printing paper consisting of offset printing newspaper waste paper and flyer waste paper, and disintegrated with a test pulper under the following conditions to give a density of 20. Dehydrated to%. Pulp concentration 4.2% by weight Surfactant addition rate 0.25% by weight (against pulp, the same applies below) The surfactant is polyoxyethylene nonylphenyl ether (mol number of ethylene oxide n = 9) Furthermore, alkali dipping step (soaking) (Synonymous with process) was carried out under the following processing conditions. pH Approx. 11 Pulp concentration 15% Treatment time 4 hours Treatment temperature 80 ° C Caustic soda addition rate 2.8% by weight (to pulp, the same below) Sodium silicate addition rate 10% by weight (JIS3 weight% in terms of silicic acid) Surface activity Agent addition rate 0.07% by weight Hydrogen peroxide addition rate 2.5% by weight This pulp liquid was diluted to 4% and the xylanase crude enzyme liquid obtained in Example 1 was pulped without adjusting the pH. After adding 45 U per kg, carrying out an enzyme reaction at 40 ° C. for 2 hours, diluting to 1%, and treating with a test floater for 5 minutes to separate and remove the floating ink,
The whiteness of the pulp obtained by dehydration to 0% was measured. The whiteness was 63.3%.

Comparative Example 1 The whiteness of the obtained pulp was measured by the same procedure as in Example 3 except that no enzyme was used. The whiteness was 60.3%. [Comparative Example 2] A pH of 5.
After preparing to 5, commercially available cellulase Vesselex (purified joint liquor) was added 45 U per kg of pulp,
After performing the enzyme reaction for 2 hours, the pH was adjusted to 10.
Readjusted to 3. It was diluted to 1% and the same operation as in Example 3 was performed below to measure the whiteness of the obtained pulp. The whiteness was 61.8.

[0021]

INDUSTRIAL APPLICABILITY The present invention provides a novel microorganism that produces alkaline xinase. INDUSTRIAL APPLICABILITY According to the present invention, it becomes possible to produce alkaline xylanase, particularly alkaline xylanase useful in the deinking step of waste paper pulp. Therefore, the present invention enables efficient deinking in the production of paper pulp, particularly in the recycling of used paper.

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Claims (3)

[Claims] 1. Bacillus sp. NUF (3) 4-4 producing xylanase having activity in the alkaline region. 2. A method for producing alkaline xylanase, which comprises culturing Bacillus sp. NUF (3) 4-4 according to claim 1 and obtaining xylanase having an activity in an alkaline region from the culture. 3. A method for deinking waste paper, which comprises adding xylanase produced by the method of claim 2.