JPH0248231B2 - SHINKIKISHIRANAAZEOYOBISONOSEIZOHO - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel xylanase W and a method for producing the same, and more specifically, culturing a novel xylanase W-producing bacterium belonging to the genus Bacillus,
The present invention relates to a method for producing a novel xylanase W, which is characterized by separating and collecting the novel xylanase W from the culture. Xylanase is an enzyme that hydrolyzes the polysaccharide xylan, which mainly consists of β-1,4-linked xylose, to produce xylose and low-molecular-weight xylooligosaccharides, which are its constituent components, and is widely present in plants and animals. Some microorganisms also produce xylanase, and so far, yeast, mold, bacteria,
Enzymes such as actinomycetes have been studied. On the other hand, in recent years, the usefulness of xylanase using biomass has been attracting attention. That is, xylan exists in wood and grasses, and its content is second only to cellulose, and xylose, a hydrolyzed product, has a wide range of uses as a raw material for foods and medicines. Enzymes that can be used for such purposes are desired to be produced in large quantities from inexpensive raw materials and to be stable against acids, alkalis, and heat. However, to date no xylanase has been found that satisfies all of these conditions. As a result of intensive research into a method for producing the enzyme xylanase with properties that meet these conditions, the present inventors discovered that a new microorganism belonging to the genus Bacillus produces a new xylanase with characteristic physical and chemical properties that were previously unknown. They obtained the knowledge to produce the product and completed the present invention. The present invention belongs to the genus Bacillus and has a pH of 8
-11 strong alkaline medium is used to culture alkaliphilic thermophilic bacteria that can grow at high temperatures of 45° to 55°C, and the culture has a stable pH range of 4.5 to 10.0 and a stable temperature range of up to 60°C, alkaline and heat resistant. The present invention relates to a method for collecting sex xylanase. The bacterial species used in the xylanase production method is PH8
-Can only grow on strongly alkaline media with a pH of 11.
9-10 showed the maximum growth amount and enzyme production,
It can grow at temperatures up to 55°C, especially at 45°-50°C.
This is a newly isolated and identified bacterium of the genus Bacillus, which is alkalophilic and thermophilic and exhibits maximum growth and enzyme production at ℃. Further, as a medium for producing the enzyme using the bacteria, a strongly alkaline medium containing about 4% of wheat as a carbon source can be used. Conventionally, there has been no known method of culturing thermophilic alkaliphilic bacteria of the genus Bacillus in a strongly alkaline medium at high temperatures, as in the present invention. Under such culture conditions, bacterial growth is extremely fast, culture time can be shortened, and contamination with various bacteria is prevented, which is advantageous in terms of culture management. The present invention will be explained in detail below. First, the microorganism used in the present invention is Bacillus, which has the ability to produce xylanase W.
Bacillus sp. No.W1 and No.W3 are examples of Bacillus sp.
(hereinafter simply referred to as W1 and W3, respectively). These microorganisms will be explained in detail below. (1) Method for isolating alkaliphilic thermophilic bacteria W1 and W3 The above two bacterial species used in the method of the present invention were isolated by the following method. First, soil samples collected from various parts of Japan were added to an alkaline basic medium with the composition shown in Table 1, containing 15.0 [g/] of silane and 10.0 [g/] of xylan.
Using a solid medium to which 40.0 [g/] of ] or wheat was added, culture in a thermostat at 55°C for 2 to 3 days, and isolate the bacterial colonies that appear. Next, the isolated bacteria were cultured for 2 days in a constant temperature shaker at 47°C using a liquid medium prepared by adding xylan 10.0 [g/] to the basic medium shown in Table 1.
The xylanase activity of the culture was measured by the method described below, and those with strong activity were selected. The two titled bacteria were isolated and purified by this method. Table 1 Basic medium composition Composition g/ yeast extract 5.0 Polypeptone 5.0 K ₂ HPO ₄ 1.0 MgSO ₄・7H ₂ O 0.2 PH 10.0 (Adjusted with Na ₂ CO ₃ ) Xylanase activity was determined using 0.5% xylan as a substrate at pH 8. Suspended in 0.0 pine kilbene buffer (0.1 M citric acid - 0.2 M disodium phosphate),
Add 0.05 ml of the diluted culture filtrate to 0.5 ml of the homogeneously dispersed product using ultrasound, and heat at 60℃.
The reaction was allowed to proceed for 10 minutes, and the reducing sugar produced was quantified by the 3,5-dinitrosalicylic acid reagent method, and the amount of enzyme that produced 1 μmol of xylose per minute was defined as 1 unit (U). Bacteria isolated, selected and purified in this way
The two types, W1 and W3, were identified as bacteria of the genus Bacillus as described below, and were deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on September 7, 1981. Its microorganism deposit number is W1, number 7220.
No. W3 is No. 7222. (2) Mycological properties of W1 and W3 The mycological properties of both species were determined by growing them in an alkaline medium with a pH of 10 at 47°C.
This was carried out based on the method described in "Determinative Bacteriology" (1974) and "The Genus Bacillus" (1973). [a] Morphology Both bacterial species were rod-shaped, and the size (0.3~
0.5) x (2.0~5.0) μm, single or chained. The cells are Gram-positive, nonacid-fast, motile, and form oval spores. [b] Growth status in culture medium a) Broth agar plate culture: The colonies of both bacterial species are circular, flattened or raised in a convex shape, the periphery is complete, and the surface is smooth or translucent. b) Internal juice agar slant culture: Both bacterial species grow well and grow in a filamentous or warty shape. The surface is smooth and translucent. Neither produces pigment in the medium. c) Meat juice liquid medium: Both bacterial species become somewhat cloudy as the bacteria grow, producing a viscous and soft sediment. Fungal rings are not formed. d) Meat juice gelatin culture: Both bacterial species liquefy gelatin. e) Litmus milk: converts milk into peptone. [C] Physiological properties A) Nitrate reduction: Positive for both bacterial species B) Denitrification reaction: Negative for both bacterial species C) VP test: Positive for both bacterial species D) Indole production: Negative for both bacterial species ) Production of hydrogen sulfide: Negative for both bacterial species F) Hydrolysis of starch: Negative for both bacterial species G) Hydrolysis of casein
: Both bacterial species are positive H) Utilization of inorganic nitrogen: Both bacterial species can utilize ammonium salts and nitrates. li) Pigment production: Negative for both bacterial species Urease: Negative for both bacterial species Oxidase: Positive for both bacterial species O) Catalase: Positive for both bacterial species Wa) Attitude toward oxygen
:Both species are aerobic.) Utilization of citric acid: Grows on Simon's medium and Christensen's medium, but Koser's
Will not grow on culture medium. However, only W1
Does not grow on Simon's medium. Yo) O-F test
: Generates acid both aerobically and anaerobically) Salt tolerance : Grows in 10% NaCl. L) Growth range (PH and temperature): PH8-
11; Optimal PH9-10. Temperature 28-55℃; Optimal temperature 45-50℃. e) Utilization of carbon sources: Both bacterial species produce acids aerobically and anaerobically from D-glucose, D-mannose, D-fructose, sucrose, and trehalose. However, no gas is generated. T) Nucleic acid (DNA) base composition: DNA GC
The content [mol%] is W1, 42.2%; W3,
42.9%. Based on the above mycological properties, two
A search for bacterial species W1 and W3 revealed that they belong to the genus Bacillus, but both species are clearly different from known bacterial species in that they can only grow in the alkaline range of PH8-11. It is also different from the alkalophilic bacteria of the genus Bacillus described in the literature in that xylanase production reaches its maximum at high temperatures of 45° C. or higher, as described below.
Therefore, both bacterial species do not match any known bacterial species,
It was concluded that it is appropriate to designate these as new bacterial species of the genus Bacillus. (3) Method for producing xylanase [a] Effect of PH on enzyme production As mentioned above, the two bacterial species W1 and W3 are
It can grow between 8 and 11. Therefore, xylan 10.0 [g/] was added to the basic medium shown in Table 1.
Prepare a liquid medium and adjust its pH from 7 to
The cells prepared in step 11 were cultured and the amount of xylanase produced was examined. The second result is
Shown in the table.

[Table] For culturing, dispense 10ml of each medium with a pH of 7-11 into test tubes, inoculate each with the two bacterial species, and heat at 45°C.
The mixture was shaken for 47 hours. The enzyme activity of the supernatant obtained by removing bacterial cells from the culture using a centrifuge was measured. The method for measuring enzyme activity is as described above, but here, instead of pH8.0,
A pine kilbene buffer with a pH of 6.0 was used (the same applies in subsequent studies). As shown in Table 2, enzyme production reaches its maximum at pH 10 for both bacterial species. It has thus been revealed that in order to produce xylanase using two bacterial species, it is sufficient to adjust the initial pH of the culture medium to 8 to 10. [b] Effect of temperature on enzyme production Next, we will discuss the effect of temperature on enzyme production. The liquid medium in the previous section [a] adjusted to pH 10 was dispensed into test tubes, cultured with shaking at a temperature of 30-51°C for 47 hours, and xylanase production was compared. The results are shown in Table 3. In addition,
The method for measuring enzyme activity is as described in the previous section [a].

【table】

[Table] As is clear from Table 3, enzyme production is maximum when both bacterial species are cultured at a temperature of 42° to 48°C. In this way, for xylanase production by two bacterial species, the culture temperature is set at 42° to 48°C.
It was revealed that it is sufficient to set it to . From the results of [a] and [b] above, the new bacterial species Bacillus sp. No.
It was found that the optimal culture conditions for xylanase production by W1 and No. W3 are pH 8 to 10 and temperature 42° to 48°C. [c] Effect of carbon source in the medium We will discuss the effect of the carbon source in the medium on enzyme production. The basic medium shown in Table 1 was cultured with various carbon sources (saccharides and wheat), and the enzyme activity of the culture was measured.
Shown in the table. Based on the results in the previous section, the culture conditions were adjusted to PH10 and the culture temperature to 45℃.
Shaking culture was carried out for 47 hours using a test tube set at .

[Table] Wheat = 10 18 36
40 38 43
^(*) Wheat〓/Water 40 3 5

As is clear from Table 4, both bacterial species showed remarkable enzyme production when 1.0% xylan was used as the carbon source in the culture medium, and there was almost no enzyme production when glucose was used as the carbon source. Furthermore, when 4% wheat was added, enzyme production was found to be equivalent to or higher than when xylan or xylose was added, demonstrating that wheat can be effectively used as an inexpensive carbon source. The results shown at the bottom of Table 4 (marked with an asterisk) are similar to those obtained by using a medium containing only 4% wheat and water instead of the basic medium shown in Table 1. This figure shows enzyme production when cultured under the following culture conditions. That is, medium composition: Wheat 40g Water 1000ml PH 10 Culture conditions: Shaking culture at 45°C for 47 hours. Based on the results of the above studies, the most effective medium composition, PH, and culture temperature to be used for the production of alkaline-resistant and thermostable xylanase are set as follows. Medium composition: Carbon source xylan 10.0 [g] (or wheat 40.0) Yeast extract 5.0 Polypeptone 5.0 K ₂ HPO ₄ 1.0 MgSO ₄・7H ₂ O 0.2 Water 1000 ml Medium PH: 10.0 (adjusted with Na ₂ CO ₃ ) Culture temperature : 45°C Culture method: Shaking culture or aerated agitation culture (4) Enzyme purification method and properties The method for separating and purifying the enzyme from the culture obtained by the methods described in the previous section and the properties of the enzyme will be explained. The bacterial cells are removed from the culture, ammonium sulfate is added to the clear solution to precipitate the enzyme, and this is dehydrated and dried to obtain a crude enzyme powder. Dissolve the crude enzyme powder in 0.05M Tris-HCl buffer with pH 7.2.
The xylanase fraction is separated using DEAE-cellulose and then purified by filtration using Sephadex G-100. This purified enzyme is concentrated and freeze-dried to obtain the target enzyme xylanase W. Thus, the two types of xylanase of the present invention obtained from the two bacterial species W1 and W3, W1 and W3, respectively.
and W3 have the following physical and chemical properties. Action and substrate specificity Acts specifically on xylanase and xylooligosaccharides, cleaving their β-1,4-xyloside bonds to produce xylose and xylobiose. Optimum PH and stable PH range The optimal PH is PH6.0 (see Figure 1), and the stable PH range is PH4.5 to 10.0 (in the case of 60 minutes treatment at 45℃, the third
(see figure). Measurement method for titer Suspend 0.5% xylan as a substrate in pine kilbene buffer (0.1M citric acid - 0.2M disodium phosphate) at pH 8.0, disperse it uniformly by ultrasonication, add xylanase W to 0.5ml of the suspension, and add xylanase W
Add 0.05 ml of sample containing
The amount of enzyme that produces 1 μmol of xylose is 1 unit (U). Range of suitable temperature for action: 55 to 70℃ at pH 6.0 to 7.0 (second
(see figure). Thermal stability: Stable up to 60°C when treated for 10 minutes. Purification method Cells are removed from the xylanase W-producing bacterial culture, and ammonium sulfate is added to the clarified liquid to precipitate the enzyme, which is dehydrated and dried to obtain a crude enzyme powder. Crude enzyme powder with pH7.2
Dissolve DEAE in 0.05M Tris-HCl buffer.
It is separated using cellulose and further purified by filtration using Sephadex G-100. Molecular weight: Molecular weight W1 by ultrafiltration method is 49500, W3
is 48000. Inhibition, activation Inhibited by silver ions and copper ions. The enzyme according to the present invention has a stable PH range on the acidic side (PH
4.5) to alkaline side (PH10.0),
It differs from conventional enzymes in that its optimal action temperature is between 60°C and 70°C, and the reaction products are mainly xylose and xylobiose. The method of the present invention will be explained below using examples. Example 1 (Medium composition) Xylan 1.0g Yeast extract 0.5 Polypeptone 0.5 K ₂ HPO ₄ 0.1 MgSO ₄・7H ₂ O 0.02 Water 100ml PH 10.0 (Adjusted with Na ₂ CO ₃ ) 100ml of the medium with the above composition was placed in a 300ml flask. After steam sterilizing at 121°C for 10 minutes, Bacillus bacteria No.
No.) and No. W3 (Feikoken Bacterium No. 7222) were inoculated into separate flasks and cultured with shaking at 45°C for 48 hours. When the xylanase activity of the clear liquid obtained by removing the bacterial cells from the culture was measured, the following results were obtained. Bacteria Seed Enzyme Production Amount [U/ml] W1 92 W3 115 Ammonium sulfate is added to each of the clarified liquids to precipitate the enzymes, which are dehydrated and dried to obtain coarse powders of xylanase W1 and W3, respectively. Dissolve this in 0.05M Tris-HCl buffer of PH7.2,
Separation using DEAE-cellulose, further filtration and purification using Sephadex G-100, concentration and lyophilization yield purified xylanase W1 and W3, respectively.

[Brief explanation of drawings]

FIG. 1 is a graph showing the optimum pH range, FIG. 2 is a graph showing the optimum temperature range for action, and FIG. 3 is a graph showing the stable PH range for the two types of xylanases of the present invention.

Claims (1)

[Claims] 1. A xylanase having the following physicochemical properties:
Xylanase W selected from the group consisting of W1 and W3. Action and substrate specificity It acts specifically on xylan and xylooligosaccharides, cleaving their β-1,4-xyloside bonds to produce xylose and xylobiose. Optimal PH and stable PH range Optimum PH is PH6.0, stable PH range is PH4.5 to 10.0 (45
°C, 60 min treatment). Measurement method for titer: Suspend 0.5% xylan as a substrate in pine kilbene buffer (0.1M citric acid - 0.2M disodium phosphate) at pH 8.0, and add xylanase W to 0.5ml of the suspension, which is homogeneously dispersed by ultrasound. Add 0.05 ml of the sample containing the enzyme, react at 60°C for 10 minutes, quantify the reducing sugar produced using the 3,5-dinitrosalicylic acid reagent method, and calculate the amount of enzyme that produces 1 μmol of xylose per minute as 1 unit (U). do. Suitable temperature range for action: 55-70°C at pH 6.0-7.0. Thermal stability: Stable up to 60°C when treated for 10 minutes. Purification method Cells are removed from the xylanase W-producing bacterial culture, and ammonium sulfate is added to the clarified liquid to precipitate the enzyme, which is dehydrated and dried to obtain a crude enzyme powder. The crude enzyme powder is dissolved in 0.05M Tris-HCl buffer at pH 7.2, separated using DEAE-cellulose, and further purified by filtration using Sephadex G-100. Molecular weight The molecular weight by ultrafiltration method is 49500 for W1, W3
is 48000. Inhibition, activation Inhibited by silver ions and copper ions. 2. A xylanase W characterized by culturing a xylanase W-producing bacterium belonging to the genus Bacillus, and separating and collecting xylanase W from the culture.
manufacturing method. 3 The xylanase W-producing bacterium belonging to the genus Bacillus is Bacillus sp. No.W1.
and No.W3.