JPH0412707B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a novel microorganism. For more details,
The present invention relates to a new alkaliphilic microorganism that has the ability to produce β-mannanase and β-mannosidase and has an optimal pH for growth on the alkaline side, and a method for using the same. Conventional technology β-mannanase contains β-1,4-D-
The β-1,4-D-mannopyranoside bond, which is the main skeleton of mannan, glucomannan, galactomannan, galactoglucomannan, etc., which are homo and hetero β-D-mannans with a mannopyranosidic bond, is optionally hydrolyzed to increase the viscosity. It is an enzyme that simultaneously lowers the mannooligosaccharides and produces a series of mannooligosaccharides. First, ivory nuts (scientific name: Phytelehus macrocarpa) and corozo are well known as those containing β-D-mannan. Other β
-1,4-mannan-containing plants include Phoenicus canariensis and Orchis maquillata, which belong to the palm family. Typical examples of galactomannan are the mucilage contained in carob and guar seeds, locust bean gum, and guar gum. widely used. Galactomannan is also found in large amounts in leguminous plants such as soybeans, coffee beans, alfalfa, red clover, and fenugreek. Other galactomannan-containing plants include Genista scoparia, Gleditsia fuerox,
Known examples include Leucaena glauca. The most famous glucomannan-containing substance is konjac (scientific name: Amorphopharas konjac), but other sources include arum roots of the Araceae family, jack pine of the Pinus genus, bulbs of the Orchidaceae family, and plants of the spruce genus, such as Picea abies and Pinus genus. Are known. Other known glucomannan-containing plants include Asparagus oficinalis, Eremulus fuscus, Eremulus legeri, Eremulus spectabilis, and Phaceolus aureus. These are generally obtained by an alkali extraction method or the like. Further, these β-D-mannans are consumed in large quantities industrially in the food industry and the textile industry as thickeners or thickeners. β-mannanase, which is known as an enzyme that arbitrarily hydrolyzes β-D-mannan, has been the subject of research by many researchers, and a large number of microbial-derived enzymes have been investigated. .
For example, [Advances in Carbohydrate Chemistry and Biochemistry]
and Biochemistry), 1976, 32 , 299-316] In particular, filamentous fungi [Acta.Chem.Scand., 1968, 22 , 1924;
1969, 43 , 317; Biochem.J., 1984, 219, 857], Actinobacteria [Agricultural and Biological Chem., 1984, 48 ,
2189], Bacteria [J.Biochem., 1982, 91 , 1181;
No. 57-65182] and other enzymes have been well studied. However, many of these enzymes have poor temperature stability or require a long period of time for culturing, which poses difficulties in using the enzymes industrially at low cost. In addition, β-mannosidase is a low-molecular β-D-mannan (mannan, glucomannan, galactomannan,
It is an enzyme that acts on galactoglucomannan) and hydrolyzes mannoside bonds sequentially from the non-reducing end to produce mannose. β-mannosidase, which is conventionally known as an enzyme that hydrolyzes β-D-mannan into mannose units from the non-reducing end, has been used in animals [Biochemistry, 1972 , 11 , 1493-
1501: Biochim.Biophys.Acta, 1973, 268 , 488~
496: Biochim.Biophys.Acta, 1973, 315 , 123~
127], Plants [J.Biol.Chem., 1964 , 239 ,
990-992], Microorganisms [Biochim.Biophys.Acta],
1978, 522, 521-530: JP-A No. 51-38486)] and other enzymes have been well studied. However, all of these enzymes have low productivity and many require complicated culture and purification methods, leaving difficulties in using the enzymes industrially at low cost. Problems to be Solved by the Invention Effective use of β-D-mannan, which exists in large amounts as a renewable resource in nature, particularly mannooligosaccharides and mannose produced by enzymatic hydrolysis of the substance.
In order to efficiently recover and utilize sugars such as glucose and galactose, it is preferable that the enzyme has excellent stability and is easy to purify. However, as mentioned above, the previously proposed β-mannosidases, which have various origins such as animals, plants, and microorganisms, are insufficient in terms of productivity, and their production and purification methods are also complicated. However, it was still unsatisfactory for practical use. Furthermore, for the reasons mentioned above, β-mannanase is insufficient in its physical and chemical properties, especially its temperature stability and optimum pH, for industrial large-scale use, and it is also disadvantageous in terms of economic efficiency. It was hot. Therefore, the development of a new enzyme of this type that is easy to produce and purify as described above and has high stability is an effective way to degrade recyclable β-mannan, which exists in large quantities in nature along with starch. It is of great significance in recovering and utilizing decomposition products (mannose, galactose, glucose, mannooligosaccharides, etc.). Therefore, the first object of the present invention is to provide a new microorganism capable of producing novel enzymes, β-mannosidase and β-mannanase, with high production efficiency, which satisfy the above various requirements. Moreover, a method utilizing the above-mentioned novel microorganisms to obtain these enzymes easily and in high yield constitutes another object of the present invention. Means for Solving the Problems The present inventors have extensively searched the natural world in order to obtain microorganisms capable of producing enzymes having these properties that β-mannanase for industrial use should have. As a result, growth is optimal in alkaline conditions.
The present invention was completed by discovering that a certain type of microorganism that has a pH and belongs to the genus Bacillus produces an enzyme that meets the above requirements, and that it can be produced in large quantities with good productivity. That is, the present invention first provides a novel microorganism that produces β-mannanase and β-mannosidase as described above;
FERM P-8856 is a new microorganism belonging to the genus Bacillus that has the ability to produce mannanase and β-mannosidase and has an optimal pH for growth on the alkaline side. The novel strain of the present invention was newly searched and isolated from the natural world by the present inventors, and was published in Bergey's Manual of Determinative Bacteriology.
of Determinative Becteriology), 8th edition and The Genus Bacillus
Bacillus: Identified according to the U.S. Department of Agriculture edition], it is an aerobic sporulating bacillus, motile, with periflagella, and Gram staining variable. Therefore, it was clear that it belonged to the genus Bacillus, but since it grows well in an alkaline pH of 7.5 to 11.5, it was considered to be a new strain that is taxonomically different from known Bacillus sp. . Table 1 below shows various mycological properties of the isolated β-mannanase and β-mannosidase producing bacteria.

【table】

【table】

[Table] +: Growing or positive -: Not growing or negative The above bacterial strain has been deposited with the Institute of Microbiology, Agency of Industrial Science and Technology as FERM P-8856. In addition, the novel strain of the present invention was screened as follows. First, the sample was collected from old rotted manure in a field in Yaho, Kunitachi City. The compost residue was suspended in water, and one drop of the supernatant was spread on an agar medium having the following composition. The agar medium used was 2% agar, 0.5% NaHCO ₃ , 1% coconut oil extraction residue,
0.5% polypeptone, 0.5% yeast extract, 0.1%
of _{K2HPO4 and} 0.02% _{MgSO4.7H2O .} Thus, each colony that appeared on the plate was obtained by culturing aerobically at 37°C on an agar plate.
Each colony was further cultured aerobically at 30 to 40°C for 48 to 72 hours in a liquid medium having the same composition as above except for the agar. Next, 12000g of each culture solution
The cells were centrifuged at 4°C for 10 minutes to separate the bacterial cells and the supernatant. The supernatant thus obtained and the suspension obtained by further suspending the bacterial cells in 0.1M phosphate buffer (PH7.0) were tested for β-mannanase, respectively, by the activity assay method described below. and β-mannosidase activity were selected. the result,
A strain with the mycological characteristics described above was isolated. The new strain of the present invention has the ability to produce β-mannanase outside the bacterial cells and to produce β-mannosidase inside the bacterial cells. Therefore, these enzymes can be advantageously produced by utilizing the properties of this substance. That is, the present invention also provides a method for utilizing the above-mentioned new bacterial strain, and the method comprises culturing the new bacterial strain and culturing β.
- This method is characterized by producing and accumulating mannanase in a culture medium and within β-mannosidase cells, and collecting the resulting product. β-mannanase and β-mannosidase obtained by this method each have the following physicochemical properties. () β-mannanase (a) Action: β-1,4 of mannan, glucomannan, galactomannan, galactoglucomannan
-D-mannopyranoside bonds are non-specifically hydrolyzed to produce mannooligosaccharides. (b) Substrate specificity: Acts specifically on β-mannan and does not act on α-mannan. It acts on mannooligosaccharides with a molecular weight greater than β-1,4-D-mannotetraose and hydrolyzes them. (c) Optimal PH and stable PH range: The optimal PH is 8 to 10, and is stable within the PH range of 6 to 10 under heating conditions at 60° C. for 30 minutes. (d) Stability against temperature: Stable up to 65°C under heating conditions of PH8.0 and 30 minutes. (e) Range of optimum temperature for action: The optimum temperature for action is around 65°C. (f) Inactivation conditions: PH5.0 and
It becomes completely inactive at 12.5. Furthermore, when treated at pH 8.0 for 30 minutes, it is completely inactivated at 80°C. (g) Inhibition and activation: mercuric chloride, silver nitrate, disodium ethylenediaminetetraacetate (EDTANa ₂ ), urea,
Sodium dodecyl sulfate (SDS), sodium dodecylbenzenesulfonate (DBS)
is inhibited by (h) Isoelectric point by chromatofocusing method:
5.0-5.4 (li) Molecular weight by SDS-polyacrylamide electrophoresis 43000±3000 and 57000±3000 () β-mannosidase (a) Action: Hydrolyzes β-mannosidic bonds sequentially from the non-reducing end to generate mannose . (b) Substrate specificity: Completely decomposes β-methyl (ethyl)-D-mannoside, and also acts on β-linked mannose-containing oligosaccharides to liberate mannose. β- of p-nitrophenyl-glycoside
D-mannoside can be used as a substrate, but α-D
-Mannoside, α-D-glucoside, β-D
-Glucoside, α-D-galactoside, β-
D-galactoside, β-D-xyloside, α
-L-fucoside and β-D-glucuronide cannot be used as substrates. (c) Optimal PH and stable PH range: The optimal PH is 6 to 7, and is stable within the PH range of 6 to 9 under heating conditions at 40° C. for 30 minutes. (d) Stability against temperature: Stable up to 45°C under heating conditions of PH6.5 and 30 minutes. (e) Range of optimum temperature for action: The optimum temperature for action is around 50°C. (f) Inactivation conditions: Under the treatment conditions of 40°C and 30 minutes, it is completely inactivated at pH 5.0 and 10. Furthermore, when treated at pH 6.5 for 30 minutes, it is completely inactivated at 55°C. (g) Molecular weight as determined by gel filtration method: 63000±3000 The method of using the novel strain of the present invention will be explained in more detail. The β-mannanase and β-mannosidase producing bacteria described above were inoculated into an appropriate medium, and from the viewpoint of the growth temperature of the bacterial cells, the bacteria were incubated at 30 to 40°C.
Incubate aerobically for 48-72 hours. Here, the medium contains inorganic salts and micronutrients as necessary in addition to a carbon source and a nitrogen source. First, various conventionally known materials can be used as the carbon source, and typical examples include konjac flour, locust bean gum, canalob gum, guar gum, and plants containing these. There are also no particular restrictions on nitrogen sources, including organic nitrogen such as yeast extract, peptone, meat extract, cornstarch liquor, amino acid solution, and soybean meal, or inorganic nitrogen such as ammonium sulfate, urea, ammonium nitrate, and ammonium chloride. This can be exemplified as something that is inexpensive and easily available. It goes without saying that the organic nitrogen source serves as a carbon source. Furthermore, in addition to such carbon sources and nitrogen sources, it is also possible to add various commonly used salts, such as inorganic salts such as magnesium salts, potassium salts, phosphates, and iron salts, and vitamins. . A suitable medium for use in the method _of the invention includes, for example, 1% konjac flour, 2% polypeptone, 0.2% yeast extract, 0.1% _K2HPO4 and 0.2 _{% MgSO4.7H2O} . It can be a liquid medium containing. Furthermore, since the growth pH of the microorganism in the method of the present invention is within the basic range, it is necessary to adjust the pH value of the medium using an appropriate alkali. For this purpose, 0.5% sodium bicarbonate can be used as a typical example, but alkaline reagents such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium phosphate, and calcium hydroxide can also be used. In the method of use of the present invention, the above-mentioned bacterium first produces β-mannosidase within the bacterium and accumulates it there.
Cultivation of this bacterium can be carried out either batchwise or continuously, and the enzyme produced can be separated and
Purification can be carried out, for example, as follows. That is, it is possible to first collect the bacterial cells in the culture solution by known means such as centrifugation or filtration, and then use the obtained bacterial cells as they are for the hydrolysis reaction of mannooligosaccharides, which is economical. It is advantageous. Of course, it can also be further purified and used. For this purpose, for example, after crushing and extracting the bacterial cells, purification can be performed by salting out with ammonium sulfate, solvent precipitation with ethanol, acetone, isopropanol, etc., ultrafiltration, gel filtration, general enzyme purification using ion exchange resin, etc. I can do it. Below, one example of a preferred purification method for β-mannosidase of the present invention will be explained. The AM-001 strain belonging to the alkaliphilic Bacillus genus is inoculated into a medium such as the one described above, and the culture solution obtained by culturing it aerobically at 37°C for 48 hours is incubated at 12000 rpm and 0°C. The cells were collected by centrifugation for 30 minutes to obtain cells weighing 10 g wet. Next, the bacterial cells are suspended in 10 mM phosphate buffer (PH7.0) while being cooled in ice water, and subjected to ultrasonic disruption several times for a total of about 3 minutes. Next, the mixture is centrifuged at 12,000 rpm and 0° C. for 30 minutes to remove the residue and obtain 50 ml of supernatant. Ammonium sulfate was then added to the supernatant to make it 75% saturated, and the mixture was allowed to stand overnight at 4°C. The resulting precipitate is filtered off, dissolved in 10 mM phosphate buffer (PH7.0), and dialyzed against the same buffer overnight at 4°C. The resulting precipitate was removed by centrifugation, and the resulting supernatant was equilibrated with the same buffer as DEAE-Toyopearl.
The enzyme is adsorbed to 650M and eluted by a concentration gradient method using the same buffer containing 0.1-0.5M NaCl. The eluted active fractions are collected and dialyzed against the above buffer solution overnight at 4°C, and then adsorbed onto hydroxyapatite equilibrated with the above buffer solution. Then, the enzyme is eluted with 0.4M phosphate buffer (PH8.0), active fractions are collected, and concentrated using an ultrafiltration membrane with an average molecular weight cutoff of 10,000. The concentrated enzyme is SHODEX protein, a column for protein separation and purification for high-performance liquid chromatography.
Fill WS-2003 and add 10mM phosphate buffer (PH
7.0). The active fraction thus obtained was concentrated and then subjected to chromatography again under the same conditions using the same column as above, and the resulting active fraction was concentrated and subjected to polyacrylamide gel disc electrophoresis [Annals New York Academic Sciences (ANN. NYAcad.Sci.), 121,
404 (1964)], the activity yield of 11 mg of homogeneous enzyme preparation was 17%. The method for measuring β-mannosidase activity and the method for displaying the activity are as follows. That is, 0.2 ml of 0.2 M phosphate buffer (PH7.0) and 8 m
Mix 0.1 ml of enzyme solution with 0.2 ml of p-nitrophenyl-β-D-mannopyranoside aqueous solution of M, and incubate at 40℃.
After reacting for 10 minutes, add 1.0 ml of 0.5 M sodium carbonate aqueous solution to inactivate the enzyme, and then add water to make up to 3 ml. Coloring degree with ultraviolet light (wavelength 420n)
m) with 1 μmol/ml p-nitrophenol as a standard. The unit of enzyme activity is per minute under the above conditions.
The amount of enzyme that liberates 1 μmol of p-nitrophenol is expressed as 1 unit.

【table】

【table】

[Table] Furthermore, since the above-mentioned bacterial strain of the present invention produces β-mannanase extracellularly, the produced β-mannanase is released into the culture solution and accumulated there. Cultivation of this bacterium can be carried out in the same manner as described above, and separation and purification of the purified enzyme can be carried out, for example, as follows. That is, it is possible to first remove the bacterial cells in the culture solution by centrifugation, filtration, etc., and then apply the resulting supernatant (crude enzyme solution) as it is to the hydrolysis reaction of β-mannan. Economically advantageous.
It can also be used after further purification. The purification method can be carried out in the same manner as for β-mannosidase. Below, one example of a preferred purification method for β-mannanase of the present invention will be explained. The AM-001 strain of the present invention, which belongs to the alkaliphilic Bacillus genus, is inoculated into, for example, the above-mentioned medium and cultured aerobically at 37°C for 72 hours.
Add cetabron (cetyltrimethylammonium bromide) from v) and leave it for 30 minutes, then add 7000r.
pm, centrifuge for 20 minutes at 0°C to remove bacterial cells.
Obtain the supernatant liquid of 3. Then, ammonium sulfate was added to the supernatant to make it 75% saturated, and the mixture was left overnight at 4°C. The resulting precipitate is filtered off, dissolved in 10 mM phosphate buffer (PH7.0), and dialyzed against the same buffer overnight at 4°C. The resulting precipitate was removed by centrifugation, and the resulting supernatant was equilibrated with the same buffer as DEAE-Toyopearl.
The enzyme is adsorbed to 650M and eluted by a concentration gradient method using the same buffer containing 0.1-0.5M NaCl. The eluted active fractions are collected, dialyzed once against the above buffer at 4°C, and then adsorbed onto hydroxyapatite equilibrated with the above buffer. Then
When the enzyme was eluted using a concentration gradient method of 0.01-0.4M phosphate buffer (PH7.0), two fractions (F-A and F-B) with β-mannanase activity were obtained. The combined activity yield of the two fractions was 29%. Next, each fraction was determined to have an average molecular weight cutoff.
Concentrate the enzyme using a 10,000 ultrafiltration membrane, and convert the concentrated enzyme into a column system for protein preparative purification for high performance liquid chromatography (SHODEX).
Protein) WS-2003 and elute using 10mM phosphate buffer (PH7.0). After the active fraction thus obtained is concentrated, it is subjected to chromatography again under the same conditions using the same column as above, and the obtained active fraction is concentrated to obtain a purified enzyme. Then,
These purified enzymes were subjected to SDS-polyacrylamide disc electrophoresis [Biochem.Biophys.Res.Commun., 1967 , 28 ,
815] and polyacrylamide disc electrophoresis [Annals New York Academy]
Science (ANN.NYAcad.Sci.), 121 , 404
1964)], as a result of examining their homogeneity, the molecular weights of both the FA and FB fractions were 57,000 ± 3,000 and 43,000 ± 3,000, respectively, using SDS-polyacrylamide disc electrophoresis.
However, in polyacrylamide disc electrophoresis, two bands with β-mannanase activity were detected in the F-A fraction, while in the F-B fraction, a uniform band was detected. Uniform bands were also detected by this electrophoresis method. These three β-mannanases had almost the same enzymatic chemical properties except for their different molecular weights. The method for measuring β-mannanase activity and the method for displaying the activity are as follows. That is, 0.4 ml of 0.1 M glycine-NaOH-NaCl buffer (PH9.0) and 0.5 ml of 1% (w/v) konjac mannan aqueous solution.
ml and 0.1 ml of enzyme solution, reacted at 50°C for 10 minutes, and then added to the Somogyi Journal of Biological Chemistry (J.Biol.
Chem.), 1952, 195, 19] to inactivate the enzyme, and then heat in a boiling water bath. After 10 minutes, rapidly cool in an ice bath, add 1.0 ml of Nelson's solution, stir well , and then add 10 ml of water. Make it. The degree of coloration is measured using ultraviolet light (wavelength: 660 nm) using 100 μg/ml mannose as a standard. The unit of enzyme activity is the amount of enzyme required to produce reducing sugar equivalent to 1 μmol of mannose per minute under the above conditions.
Display as a unit. Effects β-D-mannan, which exists in relatively large amounts in nature, can be used as it is, like starch, or after chemical modification, as a paste, thickener, food material, etc. in fibers, cosmetics, and foods. It is widely used in various fields such as agricultural chemicals. By the way, this β-D
- To utilize mannan effectively, it is necessary to obtain enzymes (β-mannanase, β-mannosidase, etc.) that can efficiently hydrolyze it. That is, β−
To obtain an enzyme that can hydrolyze D-mannan with high efficiency, it is necessary to decompose it and recover and use it as useful saccharides such as mannooligosaccharides, mannose, glucose, and galactose. It is extremely important for purposes such as decomposing and removing mannan after its use. In such applications, it is extremely desirable from the viewpoint of industrial application that β-mannanase and β-mannosidase have high temperature stability and have an optimal pH for enzyme reaction in the neutral to alkaline range. . However, although mannan-degrading enzymes of various origins have been discovered and used for this purpose, for example, β-mannanase has poor high-temperature stability, and the culture time of enzyme-producing microorganisms is extremely long. long, economical, β-D-
This was not desirable from the viewpoint of mannan decomposition efficiency. Furthermore, β-mannosidase has low productivity, requires complicated culture and purification methods, is expensive, and is difficult to use on a large scale industrially. Therefore, the present inventors conducted various searches and found that certain microorganisms belonging to the alkaliphilic Bacillus genus were found to have useful β-β properties.
- It has been found that mannanase and β-mannosidase can be simultaneously produced at a high production rate. It has been found that all of these enzymes satisfy the various requirements for the above-mentioned β-D-mannan hydrolysis reaction, and that they solve all of the problems of the conventionally known enzymes. That is, first, since the novel microorganism of the present invention produces β-mannanase extracellularly, it is extremely easy to separate and purify the enzyme, and a significant improvement can be expected in terms of labor and production costs. Furthermore, this enzyme has excellent high-temperature stability and has an optimal pH for enzyme reaction on the alkaline side (PH8-10), so it can be used after extracting β-mannan from various plants under alkaline conditions. , it is possible to directly proceed to the enzymatic decomposition reaction without performing operations to make acidic conditions as in the past, and a significant improvement can be expected in terms of workability and economic efficiency. Furthermore, the microorganism of the present invention intracellularly produces β-mannosidase. Since this β-mannosidase has an optimum pH for enzymatic reaction in a nearly neutral region, it is possible to proceed to the next decomposition reaction after performing a slight pH adjustment after the extraction operation as described above.
In addition, this enzyme can be used as it is or after simple separation and purification of the bacterial cells obtained during the separation of the culture supernatant containing β-mannanase, which is advantageous in terms of labor, mass production, and economy. be. Thus, according to the novel microorganism of the present invention, β-
Efficient production of β-mannanase and β-mannosidase that are useful for hydrolyzing D-mannan and effectively utilizing the decomposition products, or for decomposing and removing β-D-mannan itself after use as a thickening agent, etc. However, since these enzymes are excellent in PH stability, high temperature stability, etc., industrial large-scale utilization of these enzymes becomes possible. Examples Hereinafter, the present invention will be explained in more detail with reference to Examples. Example 1 Alkaliphilic bacterium Bacillus AM-001 strain (FERM P-8856) was mixed with 0.5% guar gum, 5% corn staple liquor, 0.1% ammonium sulfate, 0.1% K ₂ HPO ₄ , in a 500 ml Erlenmeyer flask. _MgSO4・
Inoculate 100 ml of culture solution (PH9.5) containing 0.02% 7H ₂ O and 0.25% soda carbonate, and incubate at 37 degrees for 48 hours at 200 rpm.
Cultured with shaking at pm. Then, the culture solution
The cells and culture supernatant were collected by centrifugation at 12,000 rpm and 0° C. for 30 minutes. First, the collected cells were suspended in 5 ml of 10 mM phosphate buffer, and then the cells were disrupted using an ultrasonic disruptor. Next, this bacterial cell disruption solution was
After centrifugation at 12000 rpm for 30 minutes at 0°C, the β-mannosidase activity of the resulting supernatant was measured and found to be 14 units/ml. Then, the β-mannanase activity in the culture supernatant was measured as described above, and the result was 53 units/ml. Effects of the Invention As described in detail above, according to the present invention, β-
A novel microorganism belonging to the alkalophilic Bacillus genus is provided that simultaneously produces β-mannanase and β-mannosidase that efficiently hydrolyze D-mannan. Both enzymes produced by this new microorganism satisfy all the conditions required for the hydrolysis reaction of β-D-mannan, decompose it with high efficiency, and effectively decompose and remove it after use, as well as effectively decompose the decomposed products. This greatly facilitates the use and can also guarantee significant economic improvements.

Claims (1)

[Claims] 1. A microorganism belonging to the genus Bacillus that has the ability to produce β-mannanase and β-mannosidase and has an alkaline optimum pH for growth, deposited in Microtechnical Research Institute No. 8856.