JPH0662839A - A microorganism producing heat-resistant alkali xylanase - Google Patents

Abstract

PURPOSE:To provide a microorganism producing heat-resistant alkali xylanase and its use. CONSTITUTION:A strain in Bacillus such as Bacillus sp-BX-1, Bacillus sp-BX-2, Bacillus sp-BX-3 or Bacillus sp-BX-4 is cultured and heat-resistant alkali xylanase is collected from the culture mixture. Further, the heat-resistant alkali xylanase can be used to bleach wood pulp.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel microorganism which produces thermostable alkaline xylanase and a method for utilizing the microorganism. More specifically, the present invention relates to Bacillus (Bacill), which is a microorganism that produces thermostable alkaline xylanase.
us) genus Bacillus sp. BX-1 (Microtech Lab. No. 13052), Bacillus sp. BX-2 (Microtech Lab. No. 13053), Bacillus sp. BX-
3 (Microtech Lab. No. 13054), Bacillus SP BX-4 (Microtech Lab. No. 13055), and a method for producing a thermostable alkaline xylanase by the thermostable alkaline xylanase-producing bacterium, and a thermostable alkaline xylanase The present invention relates to a method for bleaching pulp with a heat-resistant alkaline xylanase produced by a producing microorganism and / or the microorganism.

[0002] Xylanase is an enzyme that decomposes xylan in hemicellulose, but its effective utilization has not been developed so far. However, when kraft pulp is treated with xylanase, the bleaching property is improved, chemicals such as chlorine used in the bleaching process of pulp and AOX in the drainage can be reduced, and since the usefulness of functional oligosaccharides was clarified, Xylase is an enzyme that has come to the fore.

[0003]

2. Description of the Related Art In the pulp manufacturing process, many treatments are carried out under alkaline conditions and high temperature conditions such as kraft cooking. Therefore, in order to efficiently carry out enzyme treatment 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, the chemicals for adjusting the pH can be reduced, and since the reaction can be carried out under the conditions of high temperature and alkali, propagation of various bacteria can be prevented.

As a microorganism producing xylase, a review of xylanase (Microbiological Reviews Sept., 3
05,1988), etc. Aspergillus (Aspergillus
) Genus, Trichoderma genus, Aureobasidium genus, Streptomyces genus, Schizophyllum co
mmune) and many other microorganisms such as bacteria such as Bacillus and Clostridium are known, but most produce acidic or neutral active xylanase. For the xylanase-producing microorganisms, Bacillus
us) Genus (System.Appl.Microbiol., 8,152,1986, Appl.Mic
robiol.and Biotechnol., 19,335,1984), Aeromonas
(Aeromonas) genus (Agric.Biol.Chem., 40,3037,1985),
Streptomyces genus (Biotechnol.L
et., 12,225,1990) and only some strains are known.

Also, some attempts to reduce the bleaching chemicals and AOX by making xylanase act on pulp have been reported, for example, Viikali et.al Bleachi.
ng with enzymes, 3rd international conference on
biotechnology in the pulpand paper industry p.67
1988, Japanese Unexamined Patent Publication No. 2110086, Japanese Unexamined Patent Publication No. 2-2
There are 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, Pulpzyme HB manufactured by Novo Co. is available.
derson et.al "Bleach boosting of kraft pulp using
alkaline hemicellulase ", Production of bleached ch
emical pulp in the futureinternational pulp bleach
ing conference 1991, Vol.2 p.107 and Paper and Pulp Technology Times, 5, 20-25, 1992.
This enzyme has an optimum pH of neutral to weak alkali, and has almost no activity at pH 9.

Another example of 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 a softwood oxygen-exposed kraft pulp is treated with alkaline xylanase having an optimum pH of 9 and an optimum temperature of 65 ° C., a kappa number of 17.
It is shown that the value drops from 1 to 15.6.

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 is pH
By treating alkaline xylanase from Bacillus stearothermophilus at a temperature of 5 to 8 ° C. at a temperature of 5 to 80 ° C. for treating hardwood unbleached kraft pulp, chlorine gas in chemical bleaching was reduced to 2
It shows that it can be reduced by 5%.

Generally, xylanase has the highest productivity when xylan is used as a carbon source, and although the productivity is reduced, agricultural waste such as wheat bran containing xylan, plant fiber such as pulp and bagasse, etc. It can also be produced by using the carbon source. However, xylan-free glucose or the like hardly produces it.

[0010]

In the paper pulp manufacturing process where there are many alkaline and high temperature conditions, when using acidic xylanase, the temperature and pH are lowered to a range where an enzymatic reaction is possible, and the temperature during the enzymatic reaction and The pH needs to be controlled. In addition, depending on the process, it may be necessary to add an alkali after the completion of the enzymatic reaction to return to the conventional pH, or to raise the temperature, so a great deal of chemicals and energy,
And equipment is required.

If a thermostable alkaline xylanase can be used, it is possible to efficiently incorporate xylanase treatment into a conventional production line, since adjustment and control of temperature and pH are unnecessary. However, currently readily available xylanase is an acidic to neutral mesophilic xylanase. Since these xylanases are extremely unstable in alkalinity and high temperature, they cannot be used for reaction in alkali and high temperature.

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

From the above, the present invention aims to provide a novel microorganism which produces thermostable alkaline xylanase.

[0014]

[Means for Solving the Problems] The inventors of the present invention have sought a xylanase-producing bacterium having heat resistance and alkali resistance, and have conducted extensive and extensive screening. As a result, the heat resistance and alkali resistance of the soil in a pulp mill are excellent. We found a strain belonging to the genus Bacillus, which produces thermostable alkaline xylanase with different properties.

The present invention is directed to Bacillus sp. BX-2 and Bacillus sp. BX-2 which belong to the genus Bacillus which are thermostable alkaline xylanase producing microorganisms. ), Bacillus sp. BX-3 (Bacillus sp. BX-3), Bacillus sp. BX-4 (Bacillus sp. BX-4), and a method for producing thermostable alkaline xylanase by the thermostable alkaline xylanase-producing bacterium, and the microorganism. To a method for bleaching pulp with thermostable alkaline xylanase.

The present strain of the present invention is an alkaline medium (glucose or xylan) containing 1% sodium carbonate.
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 below. ) 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. Bacillus sp. BX-3 and Bacillus sp. BX-4 are all bacilli in the form, and the size of Bacillus sp. BX-1 is 2.5 to 5 x 0.3 to 0.7 [mu].
m, Bacillus SP BX-2 is 2.5-5 × 0.4
~ 0.7 μm, Bacillus SP BX-3 is 2.5 ~
5.5 × 0.4-0.7 μm, Bacillus SP BX-
4 is 2.5 to 5 × 0.4 to 0.7 μm. (2) Bacillus SP BX-1, Bacillus SP BX-
2. Bacillus sp. BX-3 and Bacillus sp. BX-4 are all oval type of endospores (1.0 ~
2.5 × 0.4-1.0 μm), and has (3) mobility. (4) Bacillus sp. BX-1, Bacillus sp. BX-2, and Bacillus sp. BX-3 are single cells, but may be in the form of several chains. Although Bacillus sp. BX-3 is single cell, it may have several tens of linkages.

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

(B) The growth conditions and the like in various media are as follows (the numerical values showing the component composition in the media are% by weight). (1) Meat broth agar plate (Difco nutrient broth 0.8,
Agar 1.5, Na ₂ CO ₃ 1.0, pH 10.3) Bacillus sp. BX-1, Bacillus sp. BX-2, Bacillus
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 it grows, and the periphery becomes smooth but irregular as it grows. Colors are Bacillus SP BX-1, Bacillus SP BX-
2. Bacillus SP BX-4 is a pale ocher opaque and slightly dull luster. Bacillus SP BX-3 is slightly white, transparent and glossy.

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

(3) Meat broth agar plate medium containing 12% of 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 also have poor growth.

(4) Meat broth 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 do not grow well.

(5) Difco beef ext
ract 1.0, peptone 1.0, NaCl 0.5, gelatin 12.0
, Na ₂ CO ₃ 1.0, pH 10.3) Bacillus sp. B
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] Table 1 SP SP SP SP SP BX-1 BX-2 BX-3 BX-4 (1) Reduction of nitrate +-+-(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 sugars Arabinose + + + + Galactose + + + + Glucose + + + + Maltose + + + +-Mannitol + + + + Mannose + + + + -Sucrose + + + +-+ Xylose +-+-+-

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

(10) The temperature range of growth of Bacillus sp. BX-1, Bacillus sp. BX-2, Bacillus sp. BX-3, and Bacillus sp. BX-4 is 20 to
The temperature is 55 ° C, and the optimum temperature for growth is 40 to 50 ° C.

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

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

Regarding the above-mentioned mycological properties, Bergy's Manual
As a result of examination with reference to Systematic Bacteriology, these 4 strains are spore-forming aerobic Gram-positive bacilli, and since they are catalase-positive, it is clear that they belong to the genus Bacillus. There was no corresponding substance, and these strains were judged to be new strains.

These strains were designated Bacillus sp. BX-
1. Bacillus sp. BX-2, Bacillus sp. BX-3 and Bacillus sp. BX-4 were named and deposited at the Institute of Microbiology and Technology. Accession number is Bacillus
SP-BX-1 (Bacillus sp. BX-1) is the first microbiological research host
No. 3052, Bacillus sp. BX-2 (Bacillus sp.
BX-2) is Microbiology Research Institute No. 13053, and Bacillus sp. BX-3 (Bacillus sp. BX-3) is Microbiology Research Institute No. 1305.
No. 4, Bacillus sp. BX-4 (Bacillus sp. BX-4)
Is Microbiology Research Institute No. 13055.

The known alkaline xylanase-producing bacterium of the genus Bacillus C-125 (System. Appl. Mic
robiol.8,152,1986) and the present invention are compared to C-125.
Is negative for VP test, whereas Bacillus sp. BX-1, Bacillus sp. BX-2, Bacillus sp. BX-3, and Bacillus sp. BX-4 are all positive, and C-125 has alkaline xylanase production. 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 distinct strains.

Further, a known thermostable alkaline xylanase-producing bacterium of the genus Bacillus (Appl. Microbio
l. and Biotechnol., 19,335,1984) W1, W2, W
Compared with 3, W4, W2 and W4 are negative for oxidase, whereas Bacillus sp. BX-1, Bacillus sp. BX-2, Bacillus sp. BX-3,
Bacillus sp. BX-4 are all oxidase positive.

12% NaC for W1, W2, W3 and W4
While Bacillus sp. BX-1, Bacillus sp. BX-2, Bacillus sp. BX-3, Bacillus sp.

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

From the above, Bacillus sp. BX-1, Bacillus sp. BX-2, Bacillus of the present invention
SP BX-3, Bacillus SP BX-4 is a known alkaline xylanase-producing bacterium Bacillus (Baci).
llus), which is a strain distinct from W1, W2, W3, and 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 to produce thermostable alkaline xylanase. For example, as the carbon source, agricultural waste such as xylan or wheat bran containing xylan, plant fiber such as pulp or bagasse, and monosaccharides such as molasses, starch or glucose containing no xylan are used. be able to.
For example, nitrogen compounds such as yeast extract, peptone, various amino acids, soybean, corn steep liquor and various inorganic nitrogens can be used as the nitrogen source. Further, various salts, vitamins, minerals and the like can be used.

The culture temperature and pH may be in any range as long as the cells 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. .5. After culturing the microorganism of the present invention, cells can be separated and the culture filtrate can be used as it is as a crude thermostable alkaline xylanase enzyme solution. The thermostable alkaline xylanase thus obtained has an optimum reaction temperature of 50 to 70 ° C. and an optimum pH of 7-1.
It is 1.5.

The crude enzyme can be concentrated or solidified by means such as dialysis, salting out, ultrafiltration and freeze-drying. 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 carried out by subjecting pulp to the heat-resistant alkaline xylanase treatment of the present invention. Before and after the bleaching treatment with the xylase, pulp bleaching can be performed by further incorporating chemical bleaching or alkali extraction. The alkaline xylanase treatment can also be performed twice or more.

Examples will be shown below, but the present invention is not limited thereto.

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 into the liquid medium of No. 3, respectively, and cultured at 45.degree. C. for 24 hours. After culturing, centrifuge (10,000 rpm
X 10 min.) And the culture supernatant was separated 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 200 μl of enzyme solution to 50 μl,
React at 70 ° C. for 10 minutes. 500μ of DNS reagent
Immediately after adding 1 and boiling for 5 minutes, ice-cooling is performed, 4 ml of distilled water is added, and the absorbance at 500 nm is measured. The calibration curve is prepared using xylose of known concentration. For the xylanase activity, the amount of enzyme that produces 1 μmol of reducing sugar per minute under the above conditions is defined as 1 Unit. As a result, the thermostable alkaline xylanase activity in the culture supernatant was 51 U / ml for Bacillus sp. BX-1, 37 U / ml Bacillus sp. BX- for Bacillus sp. BX-2.
3 is 34 U / ml, Bacillus SP BX-4 is 31
It was U / ml.

[0042]

Example 2 Glucose 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 10.3 liquid medium was inoculated with Bacillus sp. BX-1, Bacillus sp. BX-2, Bacillus sp. BX-3, Bacillus sp. BX-4, respectively, at 45 ° C. It was 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, it was found that 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 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 thermostable alkaline xylanase in the culture supernatant in the same manner as in Example 1, 33 U of Bacillus sp.
/ Ml, Bacillus SP BX-2 is 23 U / 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 liquid medium in Bacillus spb BX-1,
Bacillus SP BX-2, Bacillus SP BX
-3, Bacillus sp. BX-4 were inoculated respectively,
The cells were cultured at 45 ° C for 24 hours. After culturing, centrifuge (1
The culture supernatant was separated at 0,000 rpm × 10 min.) To obtain a thermostable alkaline xylanase. The thermostable alkaline xylanase in the culture supernatant was measured in the same manner as in Example 1. As a result, Bacillus sp. BX-1 was 30 U / ml, Bacillus sp. BX-2 was 20 U / ml, and Bacillus sp. B.
X-3 was 17 U / ml and Bacillus sp. BX-4 was 15 U / ml.

[0045]

Example 5 <Improving Pulp Bleachability by Treatment with Heat-Resistant Alkaline Xylanase> To each of the hardwood unbleached kraft pulps, 10 U of the heat-resistant alkaline xylanase obtained in Example 1 was added, based on the absolute dry weight of the pulp. pH 9, 60 ° C
React for 4 hours. After the completion of the reaction, bleaching was carried out in the order of chlorine-alkali extraction-hypochlorite-chlorine dioxide by a conventional method.

As a result, the amount of chlorine was 26% for Bacillus sp. BX-1 thermostable alkaline xylanase and 24% for Bacillus sp. , Bacillus SP BX-3 with 26% of thermostable alkaline xylanase, Bacillus SP B
With the thermostable alkaline xylanase of X-4, it could be reduced by 25%.

[0047]

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

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Claims (2)

[Claims] 1. A strain of Bacillus sp. BX-1 and Bacillus sp. BX- belonging to the genus Bacillus, which is a microorganism that produces thermostable alkaline xylanase.
2, Bacillus SP BX-3, Bacillus SP BX-4. 2. A method for producing a thermostable alkaline xylanase, which comprises culturing the thermostable alkaline xylanase-producing bacterium according to claim 1 and collecting the thermostable alkaline xylanase from the culture.