JPS6229965A - Novel microorganism - Google Patents

Abstract

NEW MATERIAL:A microbial strain having the following bacteriological properties. Shape and size of the cell, bacillus of (0.4-0.6)mumX(4.0-5.0)mum in size: polymorphism of cell, none: mobility, positive; sporulation capability, positive; shape of spore, ellipsoid or sphere of 0.8-1.0mum in diameter and positioned at the terminal; Gram-dyeability, negative; acid resistance, negative; etc. EXAMPLE:Clostridium sp. KB-10 (FERM P-8317). USE:A thermophilic xylase-producing strain belonging to Clostridium genus and capable of producing an enzyme effective to decompose an agricultural waste containing large amount of xylan such as rice straw, in high efficiency. PREPARATION:Clostridium sp. KB-10 separated from the feces of animal is inoculated in PY-X medium and cultured in an anaerobic atmosphere for 15-72hr.

Description

[Detailed description of the invention] Take-”↑7　jf　’IP The present invention relates to a novel microorganism.

Background of the Invention: Xylan is a strong polysaccharide that is used to construct the cell walls of plants such as rice straw, the outer structure of trees, and the fibers of trees. Cellulose is a substance that is known to be different from cellulose. The xylan is partially decomposed by various enzymes including cellulose-degrading enzymes such as cellulase, but not completely. Therefore, in order to effectively utilize various raw materials containing xylan, it is necessary to use highly specific enzymes, especially xylanase, to act on xylan. Only medium-temperature products with a limit of about 45 to 50°C are known.

Invention 41') f Mother-1-j (In view of the above-mentioned actual situation (62), the present inventors have devised a method for efficiently decomposing and producing cylan from agricultural wastes containing a large amount of xylan such as rice straw. We searched the natural world for microorganisms that can produce 4 enzymes. As a result, we found a new thermostable xylanase-producing bacterium belonging to the genus Cross 1 Helidium in special animal waste waste, and hereby completed the present invention. reached.

That is, the present invention relates to a novel microorganism that belongs to the genus Clostridium and has the following mycological properties.

△ Morphological characteristics (a) Cell shape and size: Bacillus, size 0.4~
The size is 0.6 μm×4.0 to 5.0 μm.

(b) Cell pleomorphism: None.

(c) Motility: Yes.

(d) Spore forming ability: Yes. The spores are oval to spherical in shape, located at the ends, and have a diameter of 0.8 to 1.0 μm.

(e) Gram staining; negative.

(f) Anti-acidity: None.

B Growth status in the following medium (anaerobic conditions) (a)
1. Mass milk: acidic, coagulating.

C Physiological properties (a) VP test: Negative.

(b) Gelatin liquefaction ability: Negative.

(c) Generation of hydrogen sulfide: Negative.

(d) Starch hydrolysis: Positive.

(e) Casein resolution: Negative.

(f) Utilization of inorganic nitrogen sources: negative.

(g) Formation of phyllodes: Negative.

(h) Catalase production: Negative.

(i) Growth range: pH 4-8, temperature 35-65°C.

(j) Attitude towards oxygen: Disgust +! llQ).

(k) O-F test; aerobically, acid is produced more than glucose.

(l) Sugars that produce acid and gas from the following sugars:
Acid production Gas-generated arabinose + xylose → + D-glucose + + 6 D-mannose + → D-galac 1~-s + maltose + + sucrose ”-lactose
＋ + 1-Levalose → ``[]-Sorhitsu l---- D-Mannitol 10'' Inoshira i---- G1 Nohirin -- Starch Shishijoki Morphological retention, growth condition, and physiological properties The microorganism of the present invention, which is characterized by No species to which this microbe was found.The closest match is Clostridium remolyticum
thermosaccharolyticum
), but the xylan-degrading ability of Clostridium genus bacteria, including this strain, is unknown. The only Clostridium bacterium whose xylan-degrading ability has been described is
Fu-Etlum (c, thermocellum) A
There is TCC27405, but this strain is essentially different from the present strain in that it only produces xylanase in the presence of cellbiose and is a strong cellulase producer. Clostridium thermocellum (V 1ljoen, Fred
and Peterson, 1926°7) is shown in Table 1 below.

Table 1: As shown in Table 1 above, this strain is clearly distinguished from Clostridium thermocellum in that it is negative for pigment production and produces butyric acid.

In addition, as mentioned above, there is no species corresponding to this strain in the genus Clostridium, so the present inventors recognized this strain as a new species belonging to the genus Clostridium, and identified it as a new species belonging to the genus Clostridium. J Suby, KB-10 (
Clostridium sp, KB-10). This strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology with the accession number 8317 (FERM P-
8317).

The microorganisms of the present invention also include ordinary mutant strains of the KB-10 strain, that is, mutant strains that have been mutated by conventional methods such as ultraviolet or X-ray irradiation, treatment with a variable inducer, etc. This includes seasonal information.

The microorganism of the present invention, such as the above-mentioned KB-10 strain, is also characterized in that it produces two types of novel xylanases. One of the xylanases is mainly produced outside the microorganism by culturing the above-mentioned microorganism (hereinafter this enzyme will be referred to as KB-
10-I).

The other one is mainly produced within the bacterial body (hereinafter referred to as KB-10-II). The characteristics of each xylanase are shown below.

<Xylanase KB-10-I> ■Substrate specificity Decomposes xylan well. Cellulose does not decompose.

■Optimal pH This enzyme is brought into contact with the substrate solution at 65°C in the range of IIH3.0 to 7.0, and the amount of reducing sugar in the reaction solution is measured to determine the enzyme activity (relative value). The results are shown in Table 2 below.

In addition, as a substrate solution, phosphate buffer (0.2M, l'l
Equal volumes of a solution prepared by suspending 1% xylan (wood or rice straw) and heating and dissolving it in a microwave oven and an enzyme solution were used.

In addition, the enzyme activity is defined as the amount of enzyme that generates reducing sugar equivalent to 1 μmol of Pheasant rose per minute at 55°C, and the relative value is the highest activity value (when pH = 4.5) is 10
It is expressed as a percentage relative to 0%.

Table 2 (Condition Relative activity (%L 3.0' 71 3, 5 80 4, 0 90 4.5 100 5.0 9F5 5, 5 90 6, 0 80 6.5 60°7, 0 54 From Table 2 above, it can be seen that the optimum pH of this enzyme is around 4.5.

■ Optimum flow rate This enzyme was brought into contact with the substrate solution at r) H5.0 and 0 to 100°C in the same manner as in (■) above, and the amount of reducing sugar in the reaction solution was measured to determine the enzyme activity (relative value). ) was sought. The results are shown in Part 3 below.
Shown in the table.

Table 3 Temperature (℃) Relative activity (%) 80 9013 - Temperature (℃) Relative activity (%) From Table 3 above, it can be seen that the optimum temperature for this enzyme (well, it is around 70℃) .

■Temperature stability This enzyme was brought into contact with the substrate solution at 0 to 100℃ for 1 hour at PT 15.0 in the same manner as in ■ above, and the amount of reducing sugar in the reaction solution was measured to determine the enzyme activity. , and its residual activity (%) was determined. The results are shown in Table 4 below.

Table 4 Temperature (℃ and Residual-Shield-JLL Desired 9-Temperature ('C)
) Residual activity (%) From Table 4 above, this enzyme has 100% residual activity when treated at 55°C for 1 hour (pl-(-5,0), and 100% residual activity when treated at 70°C for 1 hour (+18=5.0). It can be seen that 90% residual activity was exhibited even after treatment with .

② Xy5-decomposition pre-inflammation This enzyme was prepared using a phosphate buffer containing 0.5% of xylan powder as a substrate solution, pi-1 = 5.0. The results of determining the degree of xylan decomposition over time by acting at a temperature of 65° C. are shown in Table 5 below.

Table 5 Time (t + r) Decomposition rate (%) 0, 5 20 i, o 4゜2.0 55 3, 0 58 4, O65 5,065 From Table 5 above, it is clear that this enzyme ) at 65℃
It can be seen that 65% of the water can be decomposed by 65% of the water after 4 cycles at I'lH5.0.

■ Characteristics of substrate decomposition In the test described in (1) above, reaction over time) (1 was sampled and developed on TLC (silica gel, manufactured by Merli) (butanol:pyridine:water-8; i:i ) After spraying with sulfuric acid, oligosaccharides, xy[1-triose, xylobiose, and pheasant rose were used as martnos, and each decomposition product was detected by comparing with the spots of each marker.

The results are shown in Table 6 below. In addition, the display for each decomposition product in the table indicates the following.

川・・・・・・・・・人文、廿・・・・・・・・・Middle+・・・・・・・・・Elementary, −・・・・・・・・・No Table 6 Decomposition product Action time (minutes) After 60 minutes After 120 minutes Oligosaccharide Multiplyed xylotriose +-1-■ It can be seen that this shows a mode of action.

xylanase KB-10-n> ■Substrate specificity Decomposes xylan well. Cellulose does not decompose.

■Optimal pH: Add this enzyme to a pH range of 3.0 to 7.0 at 65°C.
In the same manner as the xylanase KB-10-I test, the enzyme activity (relative value) was determined by contacting with the substrate solution and measuring the reduction pattern in the reaction solution. The results are shown in Table 7 below.

Table 7 pH conditions Relative activity (%) 3.0 10 3, 5 20 4, 0 30 4, 5 40 5, 0 70 5, 5 90 6.0 100 6.5 30 7.0 10 Table 7 above The results show that the optimum pH of this enzyme is around 6.0.

■Optimal temperature This enzyme is brought into contact with the substrate solution at pH 6.0 and 0 to 100℃ in the same manner as in ■ above, and the amount of reducing sugar in the reaction solution is measured to determine the enzyme activity +1 < relative value). I asked for it.

The results are shown in Table 8 below.

Table 8 Temperature (°C) Relative Activity (%) Temperature ("C) Relative Activity (%) From Table 8 above, it can be seen that the optimum temperature of this enzyme is around 65°C.

■Temperature stability This enzyme was brought into contact with the substrate solution at pi-15.5 at 0 to 100°C for 10 days in the same manner as in (■) above, and the amount of reducing sugar in the reaction solution was measured to determine the enzyme activity. ↑4 was determined, and its residual activity (%) was determined. The results are shown in Table 9 below.

Table 9 Temperature (℃-) Residual milk - Processing temperature (℃)
Residual activity (%) From Table 9 above, this enzyme was treated at 55°C for 1 hour (pH
= 5.5) and 100% treatment at 60°C for 1 hour (p
It can be seen that even H-5,5) shows a residual activity of 50%.

■Xylan Degradation A phosphate buffer solution containing 0.5% xylan powder is mixed with a substrate solution in 1. Then, the enzyme was 1) activated at H=5.0 and temperature of 65°C. The results of determining the degree of xylan decomposition over time are shown in Table 10 below.

-21 = Table 10 Time (1-(r) Degradation rate (%) 0, 5 25 1, 0 35 2, 0 40 3.0 42 4, 0 45 5, 0 46 From Table 10 above, this enzyme is xylan (wood) at 65°
It can be seen that 46% of the product can be hydrolyzed after 5 hours of action at pH 5.0.

■ Specificity of substrate decomposition In the test described in (■) above, the reaction solution was sampled over time and developed on a 1°LC (silica gel, manufactured by Merck & Co., Ltd.) (butanol:pyridine:water - 8:1:1) L After spraying with sulfuric acid, oligosaccharides, xylotriose, xylobiose, and pheasant rose were used as markers, and each decomposition product was detected by comparing the spots of each marker. The results are shown in Table 11 below. In addition, the display for each decomposition product in the table indicates the following.

1 ((......human, sweet...
・Medium+・・・・・・・・・Small, −・・・・・・・・・・
・No Table 11 Decomposed product Action time (minutes) after 60 minutes 12
0 minutes later oligosaccharide −− xylotriose − − xylobiose −− husband si ``]: zu ＋
+1+-1111 From Table 11 above, it can be seen that this enzyme exhibits an exo-type mode of action.

From the above various test results, the enzymes (xylanases) produced by the microorganisms of the present invention are characterized by particularly excellent heat resistance, and in this respect, they are novel and different from conventional xylanases. It is confirmed. That is,
All conventionally known xylanases are stable at moderate temperatures, and the upper limit of their optimal temperature for action is approximately 45 to 50°C.
However, the optimal operating temperatures of xylanase KB-10-I and KB-10-H produced by the microorganism of the present invention are 70°C and 65°C, respectively, which is unprecedented in the prior art. It turns out that it is a high temperature resistant enzyme. Therefore, by using the microorganism of the present invention, such heat-stable xylanase can be obtained, and this heat-stable xylanase can be used particularly for the effective use of various raw materials containing xylan, such as agricultural waste such as rice straw. It is very useful for separating and obtaining pheasant rose from such raw materials, and has the advantage that the hydrolysis can be carried out efficiently.

The above-mentioned xylanase can be harvested from the microorganism of the present invention by culturing the microorganism in the same manner as the usual culture of this type of anaerobic bacteria. The medium used for the above culture may be any medium containing the nutrient sources used by the microorganism of the present invention, including various carbon sources, nitrogen It may contain a suitable amount of salt, inorganic metal salt, etc. A particularly preferred medium is, for example, a PY-X medium containing the following various nutritional sources and others.

(PY-X medium) Xylan 0.5 (+ polypeptone
1.0g yeast extract 1. Og Inorganic salt solution"' 4. Ow & hemin aqueous solution x 2 1. Om (1 cysteine hydrochloride
0.05 (1 ion exchange water 100 mQ pH 7.0 The above inorganic salt solution x 1 has the following composition.

Ca CC' 2 0.02%M (+80
4 782 0 2 HP to 0.048%
O40.1% KH2POa 0. 1%Na
HCO30.1% Also, hemin in the hemin aqueous solution '2 is 0.1% in the medium.
Contained at a ratio of 5m(+/100mG).

As shown in the above-mentioned medium PY-X, it is particularly preferable to add xylan to the culture medium for the microorganism of the present invention, thereby increasing the production base of the target enzyme. It is also preferable to add mahemin and cysteine to the medium.

The culture conditions for culturing the microorganism of the present invention using the above-mentioned medium are not particularly limited, but it is preferable to adopt the optimal temperature and pH conditions for the microorganism commonly used. The culture can be carried out by various conventional culture methods, but static culture and liquid culture are particularly preferred, and it is preferable to maintain an anaerobic atmosphere, such as a nitrogen atmosphere or a carbon dioxide atmosphere, during the culture. .

The desired xylanase can be produced in the culture through the above cultivation. It is best to collect the enzyme from the culture when a significant amount of the target enzyme has accumulated in the culture, usually around 24 hours after the start of the culture, but it can also be collected after about 15 to 72 hours. The above enzyme can be collected according to a conventional method. That is,
In the case of xylanase KB-10-T (exocellular enzyme), for example, the supernatant obtained by centrifuging the culture, particularly preferably the culture solution, is purified by a method such as dialysis to obtain a crude enzyme solution. I can do it. Also, xylanase KB-10-T
I (intracellular enzyme) is obtained by eluting the enzyme from the bacterial cells that are killed in the same manner as above using conventional means such as ultrasonic disruption or Lysodeme treatment, and then centrifuging the cells and collecting the supernatant to extract the bacterial cells. It can be used as a crude enzyme solution.

Each crude enzyme solution obtained as described above can be used as it is as an enzyme solution, or it can be further purified by conventional purification means and used as an enzyme preparation. Examples of the purification means include resin adsorption, salting out, extraction, and column chromatography.

EXAMPLES Examples will be given below to explain the present invention in more detail.

Example 1 Clostridium 1 sp. K E'l -10
(Miken Bacteria No. 8317) was added to PY-X medium (11t
-17,0) 1x105 to 1x10 in 100mC1
Sow seeds at a concentration of 6 seeds/region, and place them in small vials (
After replacing the gaseous air with 90% nitrogen gas, 5% charcoal gas, and 5% hydrogen gas, it was heated at 55°C for 24 hours and 48 hours. and static culture for 72 hours.

After the above culture, centrifugation (8000 rl 1 m, 10
The supernatant and the bacterial cells were separated by 1 minute).

The above supernatant was mixed with phosphate buffer (0.02M, 1) l-47,
0) with 0.5 mG of 2-mercaptoethanol! /3Q
Dialysis was carried out all day and night against 50 μg of the solution added at a ratio of
A crude enzyme solution was obtained as a dialysis fluid.

The results of examining the relationship between the obtained crude enzyme solution and its culture time are shown in Table 12 below.

Table 12 Culture time (Hr) Xylanase KB-10-I enzyme activity (units/mQ) 24 0.12 48 0.12 72 0.12 Example 2 The bacterial cells isolated using the centrifugal car 1 in Example 1 were ,
After washing twice with the same buffer (100%), the same buffer (0,0
2M, 11H6,O) before 100% suspension, ultrasonic disruption (5 minutes), and then centrifugation (8000pp
m for 10 minutes) to obtain a bacterial cell extract crude enzyme solution as a supernatant.

The results of examining the relationship between the obtained crude enzyme solution and its culture time are shown in Table 13 below.

Table 13 Culture time Bacteria weight (dry xylanase KB-(Hr
) Weight, +++g/mff 10-10-T
l active culture solution) (unit/person) 24 0.92 0.1448 0
．． 84 0.09 (from -L)

Claims (1)

[Claims] (1) A new microorganism that belongs to the genus Clostridium and has the following mycological properties. A Morphological characteristics (a) Cell shape and size: Bacillus, size 0.4~
The size is 0.6 μm×4.0 to 5.0 μm. (b) Cell pleomorphism: None. (c) Motility: Yes. (d) Spore forming ability: Yes. The spores are oval to spherical in shape, located at the ends, and have a diameter of 0.8 to 1.0 μm. (e) Gram staining: negative. (f) Anti-acidity: None. B Growth status in the following medium (anaerobic conditions) (a) Litmus milk: acidic, coagulated. C Physiological properties (a) VP test: negative. (b) Gelatin liquefaction ability: Negative. (c) Generation of hydrogen sulfide: negative. (d) Starch hydrolysis: Positive. (e) Casein resolution: negative. (f) Utilization of inorganic nitrogen sources: negative. (g) Pigment formation: negative. (h) Catalase production: negative. (i) Growth range: pH 4-8, temperature 35-65°C. (j) Attitude towards oxygen: anaerobic (obligate). (k) O-F test: No acid is produced than glucose under aerobic conditions. (l) Presence or absence of acid and gas production from the following sugars Sugars Acid production Gas production L-arabinose + + D-xylose + + D-glucose + + D-mannose + + D-fructose + + D-galactose + + Maltose + + Cane sugar + + Lactose + + Trehalose + + D-sorbitol − − D-mannitol + + Inositol − − Glycerin − − Starch + +