JP3070290B2 - Microorganism producing thermostable alkaline xylanase and its use - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel microorganism producing thermostable alkaline xylanase and a method for utilizing the microorganism. More specifically, the present invention relates to Bacill, a microorganism that produces thermostable alkaline xylanase.
us) belonging to the genus Bacillus sp. BX-1 (No. 13052), Bacillus sp. BX-2 (No. 13053), Bacillus sp. BX-
No. 3 (No. 13054), Bacillus SP BX-4 (No. 13055), a method for producing a thermostable alkaline xylanase using the thermostable alkali xylanase producing bacterium, and a thermostable alkali xylanase The present invention relates to a method for bleaching pulp with a producing bacterium and / or a thermostable alkaline xylanase produced by the microorganism.

[0002] Xylanase is an enzyme that decomposes xylan in hemicellulose, but its effective use has not been developed so far. However, when kraft pulp is treated with xylanase, the bleachability is improved, and it is possible to reduce chemicals such as chlorine used in the pulp bleaching process and AOX in effluent, and since the usefulness of functional oligosaccharides has been clarified, Xylase is an enzyme that has gained attention.

[0003]

2. Description of the Related Art In the pulp manufacturing process, there are many treatments under alkaline or high temperature conditions such as kraft cooking. Therefore, in order to carry out the enzyme treatment efficiently in the paper pulp manufacturing process, heat resistance and alkali resistance are required. Xylanase is required. If such a thermostable alkaline xylanase can be used, chemicals for pH adjustment can be reduced, and the reaction can be carried out under high temperature and alkali conditions, thereby preventing propagation of various bacteria.

As a microorganism producing xylase, a review of xylanase (Microbiological Reviews Sept., 3
05,1988) and Aspergillus (Aspergillus)
Genus, genus Trichoderma, genus Aureobasidium, genus Streptomyces, Schizophyllum co
A large number of microorganisms such as filamentous fungi such as B. mmune) and bacteria such as Bacillus and Clostridium are known. Microorganisms that produce xylanase include Bacillus (Bacill).
us) genus (System.Appl.Microbiol., 8,152,1986, Appl.Mic
robiol.and Biotechnol., 19,335,1984), Aeromonas
(Agric. Biol. Chem., 40, 3037, 1985),
Streptomyces (Biotechnol.L
et., 12, 225, 1990) and the like.

[0005] In addition, there have been reported several attempts to reduce bleaching chemicals and AOX by causing xylanase to act on pulp. For example, Viikali et. Al Bleachi
ng with enzymes, 3rd international conference on
biotechnology in the pulpand paper industry p.67
1988, JP-A-2-21086, JP-A-2-2-286
JP-A-21482, JP-A-2-264087, JP-A-2-293486, JP-A-3-40887, and JP-A-3-505785, all of which relate to acid xylanase.

On the other hand, as an example using alkaline xylanase, there is Pulpzyme HB from Novo, and LSPe
derson et.al "Bleach boosting of kraft pulp using
alkaline hemicellulase ", Production of bleached ch
emical pulp in the futureinternational pulp bleach
ping conference 1991, Vol.2 p.107 and Pulp and Paper Technology Times, 5, 20-25, 1992.
This enzyme has a neutral to weakly alkaline optimum pH and has almost no activity at pH 9.

Another example using alkaline xylanase is "Stefan Hogman et. Al" Bleachability improvem
ent of softwood kraft pulp through treatment with
an alkali and thermostable xylanase ", 5th internat
ional conference on biotechnology in the pulp and
There is a paper industry 1992 p.5, and when the oxygen-bleached kraft pulp is treated with alkaline xylanase having an optimum pH of 9 and an optimum temperature of 65 ° C., the kappa number becomes 17.
1 to 15.6.

[0008] Also, Viikali et.al "Important propert"
ies of xylanases for use in thepulp and paper indu
stry ", 5th international conference on biotechnolo
gyin the pulp and paper industry 1992 p.4
Treating unbleached hardwood kraft pulp with alkaline xylanase from Bacillus stearothermophilus at a temperature of 5 to 8 and a temperature of 50 to 80 ° C. to reduce chlorine gas in chemical bleaching to 2
5% can be reduced.

Xylanase is generally the most productive when xylan is used as a carbon source, and although the productivity is low, agricultural waste such as wheat bran or the like containing xylan or plant fiber such as pulp or bagasse is used. It can also be produced using a carbon source. However, xylan-free glucose produces very little.

[0010]

In the paper pulp manufacturing process where there are many conditions of alkali and high temperature, when acidic xylanase is used, the temperature and pH are reduced to the extent that the enzyme reaction is possible, and the temperature and the temperature during the enzyme reaction are reduced. It is necessary to control the pH. In addition, depending on the process, it is necessary to return the pH to the conventional level by further adding an alkali after the end of the enzymatic reaction, or to raise the temperature.
And equipment are required.

If a thermostable alkaline xylanase can be used, it is not necessary to adjust and control the temperature and pH, so that the xylanase treatment can be efficiently incorporated into a conventional production line. However, currently readily available xylanase is an acidic to neutral medium temperature xylanase. Since these xylanases are extremely unstable at alkaline or high temperatures, they cannot be used for reactions at alkaline or high temperatures.

A thermostable alkaline xylanase having an optimum reaction temperature of 70 ° C. or higher is difficult to obtain because it is not commercially available, and Bacill is also used as a producing bacterium.
us) Only some strains belonging to the genus etc. are known. Xylanase can be produced using xylan and a carbon source containing xylan, but xylan is expensive, and in order to produce xylanase efficiently and economically, alkali xylanase that can be produced with an inexpensive carbon source other than xylan such as glucose. Producing bacteria are desired.

Accordingly, an object of the present invention is to provide a novel microorganism that produces a thermostable alkaline xylanase.

[0014]

Means for Solving the Problems The present inventors have searched for a xylanase-producing bacterium having heat resistance and alkali resistance, and conducted extensive screening. As a result, the soil in a pulp mill was excellent in heat resistance and alkali resistance. A strain belonging to the alkalophilic Bacillus genus which produces thermostable alkaline xylanase having the above properties was found.

The present invention relates to Bacillus sp.BX-1 and Bacillus sp.BX-2 belonging to the genus Bacillus, which are microorganisms producing thermostable alkaline xylanase. ), Bacillus sp. BX-3, Bacillus sp. BX-4, a method for producing a thermostable alkaline xylanase using the thermostable alkaline xylanase-producing bacterium, and the microorganism And a method for bleaching pulp with a thermostable alkaline xylanase.

The strain of the present invention is an alkaline medium containing 1% sodium carbonate (glucose or xylan).
1.0, peptone 0.5, yeast extract _{0.5, K 2 HPO 4 0.1,} M
_{gSO 4 · 7H 2 O 0.02,} Na 2 CO 3 1.0, numerical values of the component composition of pH10.3 are by weight%. The same applies to the following. ) Grows well. The mycological properties of this strain are as follows.

First, regarding (a) morphological properties, (1) Bacillus sp. BX-1 and Bacillus sp. BX-
2. Both Bacillus sp. BX-3 and Bacillus sp. BX-4 are bacilli in form, and the size of Bacillus sp. BX-1 is 2.5-5 × 0.3-0.7 μm.
m, Bacillus SP BX-2 is 2.5-5 × 0.4
0.7 μm, Bacillus SP BX-3 is 2.5 to
5.5 × 0.4 to 0.7 μm, Bacillus SP BX-
4 is 2.5-5 × 0.4-0.7 μm. (2) Bacillus sp. BX-1, Bacillus sp. BX-
2. Bacillus sp. BX-3 and Bacillus sp. BX-4 are all egg-shaped endogenous endospores (1.0 to
2.5 × 0.4 to 1.0 μm), and (3) it has mobility. (4) Bacillus sp. BX-1, Bacillus sp. BX-2, and Bacillus sp. BX-3 are single cells, but may be several chains. Bacillus sp. BX-3 is a single cell, but may be tens of linked.

(5) Bacillus sp. BX-1, Bacillus sp. BX-2, Bacillus sp. BX-3,
Gram stain of Bacillus sp. BX-4 is positive or indefinite.

(B) The growth state and the like in the various media are as follows (hereinafter, the numerical values indicating the component compositions in the media are% by weight). (1) Gravy agar plate medium (Difco nutrient broth 0.8,
Bacillus sp. BX-1, Bacillus sp. BX-2, Bacillus with agar 1.5, Na ₂ CO ₃ 1.0, pH 10.3)
It grows well with SP BX-3 and Bacillus SP BX-4. The colony shape of all four strains is circular or irregular, but the surface is smooth but becomes rough as the growth grows, and the periphery becomes smooth but becomes irregular as the growth grows. The color tone is Bacillus SP BX-1, Bacillus SP BX-
2. Bacillus sp. BX-4 is light-ocher opaque and slightly dull. Bacillus SP BX-3 is slightly white, transparent and glossy.

(2) A broth agar plate medium containing 7.5% NaCl (Difco nutrient broth 0.8, NaCl 7.5, agar 1.5,
Na ₂ CO ₃ 1.0, pH 10.3) Bacillus sp. BX-
1, Bacillus sp. BX-2, Bacillus sp. BX-3, and Bacillus sp. BX-4 grow well.

(3) A broth agar plate medium containing 12% NaCl (Difco nutrient broth 0.8, NaCl 12, agar 1.5, Na ₂ C
O ₃ 1.0, pH 10.3) Bacillus sp. BX-1,
Bacillus SP BX-2, Bacillus SP BX
-3 and Bacillus sp. BX-4 have poor growth.

(4) Gravy agar plate medium (Difco nutrient
(Broth 0.8, agar 1.5, pH 7.0), Bacillus sp. BX-2 and Bacillus sp. BX-3 grow, but Bacillus sp. BX-1 and Bacillus sp. BX-4 grow poorly.

(5) Meat gelatin medium (Difco beef ext)
ract 1.0, peptone 1.0, NaCl 0.5, gelatin 12.0
, Na ₂ CO ₃ 1.0, pH 10.3)
X-1, Bacillus sp. BX-2, Bacillus sp. BX-3, and Bacillus sp. BX-4 also grow and liquefy. The following table shows (c) physiological properties

[0024]

[Table 1] Table 1 SP SP SP SP BX-1 BX-2 BX-3 BX-4 (1) Nitrate reduction +-+-(2) VP test + + + + (3) Formation of indole---- (4) Production of hydrogen sulfide----(5) Hydrolysis of starch ++ ++ (6) Utilization of citric acid Koser---Christensen ++ ++ (7) Oxidase ++ ++ (8) Catalase +++++ (9) Utilization of saccharides Arabinose ++++++ galactose ++++++ glucose ++++ maltose ++++-mannitol ++++++ mannose ++++-sucrose +++++ xylose + − + − + −

The generation of gas is Bacillus SP BX-
1, Bacillus sp. BX-2, Bacillus sp. BX-3, and Bacillus sp. BX-4 were not recognized.

(10) The temperature range for growth of Bacillus sp. BX-1, Bacillus sp. BX-2, Bacillus sp. BX-3, and Bacillus sp.
The optimal growth temperature is 55 to 50 ° C.

(11) The pH range for growth of Bacillus sp. BX-1, Bacillus sp. BX-2, Bacillus sp. BX-3 and Bacillus sp. BX-4 is 6-1.
2. The optimum pH for growth is 9-10.5.

(12) The GC content is Bacillus sp. BX-
1 is 49.4% and Bacillus SP BX-2 is 43.
1%, Bacillus SP BX-3 was 43.8%, and Bacillus SP BX-4 was 51.1%.

About the above mycological properties, Bergy's Manual
As a result of an examination with reference to the Systematic Bacteriology, these four strains are aerobic Gram-positive rods that form spores, and since they are catalase-positive, it is clear that they belong to the genus Bacillus. There was no corresponding item, and these strains were judged to be new strains.

These strains were transformed into Bacillus sp. BX-
1, named Bacillus sp. BX-2, Bacillus sp. BX-3, and Bacillus sp. BX-4, and deposited with the Research Institute of Microbial Technology. Accession number is Bacillus
SP-BX-1 (Bacillussp.BX-1) is the first
No. 3052, Bacillus sp. BX-2 (Bacillussp.
BX-2) is No. 13053, Bacillus sp. BX-3 is Bacillus sp. 1305.
No. 4, Bacillus sp. BX-4
No. 13055 is a microtechnical laboratory.

A known alkaline xylanase producing bacterium C-125 of the genus Bacillus (System. Appl.
robiol. 8, 152, 1986) and the present invention.
Indicates that the VP test is negative, whereas Bacillus sp. BX-1, Bacillus sp. BX-2, Bacillus sp. BX-3, and Bacillus sp. BX-4 are all positive. Since it is about 1/100 of the present invention, Bacillus sp. BX-1, Bacillus sp. BX-2,
Bacillus SP BX-3, Bacillus SP BX
-4 and C-125 are distinctly different strains.

A known heat-resistant alkali xylanase-producing bacterium belonging to the genus Bacillus (Appl. Microbio
l. and Biotechnol., 19, 335, 1984) W1, W2, W
3, W2 and W4 are oxidase-negative, whereas Bacillus sp. BX-1, Bacillus sp. BX-2, Bacillus sp. BX-3,
All Bacillus sp. BX-4 are oxidase positive.

W1, W2, W3 and W4 are all 12% NaC
whereas Bacillus sp. BX-1, Bacillus sp. BX-2, Bacillus sp. BX-3, and Bacillus sp. BX-4 do not grow.

While W1, W2, W3 and W4 are neutral spores, Bacillus sp. BX-1, Bacillus sp.
SP BX-2, Bacillus sp. BX-3, and Bacillus sp. BX-4 are all standing spores.

From the above, the Bacillus sp. BX-1, Bacillus sp. BX-2, and Bacillus sp.
SP BX-3, Bacillus SP BX-4 is a known alkaline xylanase-producing bacterium, Bacillus (Baci
llus), which are distinctly different from W1, W2, W3, W4.

By culturing these new strains, a thermostable alkaline xylanase can be produced. Carbon source,
Any nitrogen source can be used as long as it can assimilate and produce thermostable alkaline xylanase. For example, as the carbon source, use is made of xylan or agricultural waste such as wheat bran containing xylan, pulp, vegetable fiber such as bagasse, etc., or monosaccharides such as molasses, starch, glucose, etc. which do not contain xylan. be able to.
For example, nitrogen compounds such as yeast extract, peptone, various amino acids, soybean, corn steep liquor, and various inorganic nitrogen can be used as the nitrogen source. In addition, various salts, vitamins, minerals, and the like can be used.

The culture temperature and pH may be any range as long as the bacteria grow and produce alkaline cellulase. The culture temperature is 20 to 55 ° C, preferably 40 to 50 ° C, and the pH is 6 to 12, preferably 9 to 10 ° C. .5. After culturing the microorganism of the present invention, the cells are separated, and the culture filtrate can be used as it is as a heat-resistant alkaline xylanase crude enzyme solution. The thus obtained thermostable alkaline xylanase has an optimum reaction temperature of 50 to 70 ° C. and an optimum pH of 7-1.
1.5.

The crude enzyme can be concentrated or solidified by means such as dialysis, salting out, ultrafiltration, and lyophilization. Further, the crude enzyme can be purified by molecular weight fractionation by gel filtration, various ion exchange resins, hydroxyapatite, isoelectric point fractionation and the like.

In the process of producing chemical pulp and mechanical pulp, bleaching can be performed by subjecting the pulp to the heat-resistant alkali xylanase treatment of the present invention. The pulp can be bleached by incorporating chemical bleaching or alkali extraction before and after the bleaching treatment with the xylase. The alkaline xylanase treatment can be performed twice or more.

Next, examples are shown, but the present invention is not limited by these examples.

[Example 1] Xylan (derived from birch wood, manufactured by Sigma) 1.0
%, Peptone 0.5%, yeast extract 0.5%, K ₂ HPO ₄ 0.1
_{%, MgSO 4 · 7H 2 O} 0.02%, Na 2 CO 3 1.0%, pH 10.
Bacillus sp. BX-1, Bacillus sp. BX-2, Bacillus sp. BX-3, and Bacillus sp. BX-4 were inoculated in the liquid medium of No. 3 and cultured at 45 ° C for 24 hours. After culture, centrifuge (10,000rpm)
× 10 min.) To separate the culture supernatant to obtain a thermostable alkaline xylanase.

The thermostable alkaline xylanase in the culture supernatant was measured by the following method. 1% xylan solution (beechwood xylan, Sigma pH 8.0, 1/10 McIlvai
ne Buffer) Add 50 μl of enzyme solution to 200 μl,
Incubate at 70 ° C. for 10 minutes. 500 μl of DNS reagent
After adding 1 l and boiling for 5 minutes, immediately cool on ice, add 4 ml of distilled water and measure the absorbance at 500 nm. The calibration curve is prepared using xylose of known concentration. The xylanase activity is defined as the amount of an enzyme that produces 1 μmol of reducing sugar per minute under the above conditions, as 1 Unit. As a result, the thermostable alkaline xylanase activity in the culture supernatant was 51 U / ml for Bacillus sp. BX-1, and 37 U / ml for Bacillus sp. BX-2.
3 is 34 U / ml, Bacillus SP BX-4 is 31
U / ml.

[0042]

EXAMPLE 2 1.0% glucose, 0.5% peptone, yeast extract _{0.5%, K 2 HPO 4 0.1} %, MgSO 4 · 7H 2 O 0.02
%, Na ₂ CO ₃ 1.0%, pH 10.3, Bacillus sp. BX-1, Bacillus sp. BX-2, Bacillus sp. BX-3, Bacillus sp. Cultured for 24 hours. After completion of the culture, the culture supernatant was separated by centrifugation (10,000 rpm × 10 min.) To obtain a heat-resistant alkaline xylanase. As a result of measuring the thermostable alkaline xylanase in the culture supernatant in the same manner as in Example 1, Bacillus sp.
ml, Bacillus SP BX-2 is 20 U / ml, Bacillus SP BX-3 is 18 U / ml, Bacillus
SP BX-4 was 14 U / ml.

[0043]

Example 3 Xylan (from birch wood, manufactured by Sigma) 1.0
%, Peptone 0.5%, yeast extract 0.5%, K ₂ HPO ₄ 0.1
%, MgSO ₄ .7H ₂ O 0.02%, pH 7.0 in a liquid medium, Bacillus SP BX-1, Bacillus SP BX-2,
Bacillus SP BX-3, Bacillus SP BX
-4 was inoculated and cultured at 45 ° C. for 24 hours. After completion of the culture, the culture supernatant was separated by centrifugation (10,000 rpm × 10 min.) To obtain a heat-resistant alkaline xylanase. As a result of measuring the thermostable alkaline xylanase in the culture supernatant in the same manner as in Example 1, Bacillus sp.
/ Ml, Bacillus SP BX-2 is 23U / ml,
Bacillus sp. BX-3 was 17 U / ml, and Bacillus sp. BX-4 was 18 U / ml.

[0044]

Example 4 1.0% glucose, 0.5% peptone, yeast extract _{0.5%, K 2 HPO 4 0.1} %, MgSO 4 · 7H 2 O 0.02
%, PH 7.0 in a liquid medium, Bacillus SP BX-1,
Bacillus SP BX-2, Bacillus SP BX
-3, Bacillus sp. BX-4 inoculated,
The cells were cultured at 45 ° C for 24 hours. After culture, centrifuge (1
(000 rpm × 10 min.) To separate the culture supernatant to obtain a thermostable alkaline xylanase. As a result of measuring the thermostable alkaline xylanase in the culture supernatant in the same manner as in Example 1, Bacillus sp. BX-1 was 30 U / ml, Bacillus sp. BX-2 was 20 U / ml, and Bacillus sp.
X-3 was 17 U / ml, and Bacillus sp. BX-4 was 15 U / ml.

[0045]

Example 5 <Improvement of pulp bleachability by heat-resistant alkali xylanase treatment> The heat-resistant alkali xylanase obtained in Example 1 was added to hardwood unbleached kraft pulp at 10 U per pulp absolute dry weight, and the pulp concentration was 3%. pH 9, 60 ° C
For 4 hours. After the completion of the reaction, bleaching was carried out by a conventional method in the order of chlorine-alkali extraction-hypochlorite-chlorine dioxide.

As a result, the amount of chlorine was 26% for the thermostable alkaline xylanase of Bacillus sp. BX-1 and 24% for the thermostable alkali xylanase of Bacillus sp. , Bacillus sp. BX-3 with 26% thermostable alkaline xylanase, Bacillus sp.
With the thermostable alkaline xylanase of X-4, the reduction was 25%.

[0047]

The present invention provides a novel microorganism which produces a thermostable alkaline xylanase. According to the present invention, it is possible to produce a thermostable alkaline xylanase, which contributes to industrial production of a thermostable alkaline xylanase, and also contributes to saving of chemicals such as chlorine in paper pulp production and AOX in wastewater. It was done.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int. Cl. ⁷ Identification code FI C12R 1:07) (56) Reference Agric. Biol. Chem. , vol. 49, no. 11 (1985) p. 3165-3169 Ferment. Techno l. , Vol. 64, no. 5 (1985) p. 373-377 (58) Fields surveyed (Int. Cl. ⁷ , DB name) WPI (DIALOG) BIOSIS (DIALOG)

Claims (2)

(57) [Claims] 1. Bacillus sp. BX-1 and Bacillus sp. BX- strains belonging to the genus Bacillus, which are microorganisms producing thermostable alkaline xylanase.
2, Bacillus sp. BX-3, Bacillus sp. BX-4. 2. A method for producing a thermostable alkaline xylanase, comprising culturing the heat-stable alkaline xylanase-producing bacterium according to claim 1, and collecting the thermostable alkaline xylanase from the culture.