JPH0518554B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a novel β-mannanase and a method for producing the same. More specifically, it belongs to the genus Bacillus,
Obtained by culturing a new alkaliphilic microorganism that has an alkaline optimal pH for growth, it has an optimal pH for enzyme reactions.
The present invention relates to an extracellular β-mannanase having an alkaline pH and a method for producing 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-1,4-D-mannan. Other known β-1,4-mannan-containing plants include Phoenicus canariensis and Orchis maquillata, both of 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 Chemistry (Agric.Biol.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. Problems to be Solved by the Invention Effective use of β-D-mannan, which exists in large quantities as a renewable resource in nature, and in particular, the use of enzymatic hydrolysis of the substance to produce mannooligosaccharides, mannose, etc.
In order to efficiently recover and utilize sugars such as glucose and galactose, we have developed a method that simplifies the neutralization process because it has excellent heat resistance and extraction operations for β-mannan from various plants are mainly carried out under alkaline conditions. In addition, in order to simplify the decomposition process, it is preferable to have the optimum pH for the enzymatic reaction on the alkaline side. Furthermore, by carrying out enzymatic reactions at high temperatures, it is expected that spoilage will be prevented, the enzyme reaction rate will be increased, and the mass production of products will be improved.
It is desirable that the optimum temperature is also high. However, as already mentioned, the conventional β-
All mannanases have drawbacks such as insufficient temperature stability and the need for long culture times to produce the enzymes. It has been difficult or unsatisfactory in terms of cost to utilize it to obtain hydrolysis products of β-D-mannan. Therefore, the object of the present invention is to develop a novel β-mannanase that satisfies the various requirements for enzymatic reactions as described above and that enables hydrolysis of β-D-mannan to be carried out economically and on an industrial scale. It is about providing. Another object of the present invention is to provide a method for producing the above-mentioned novel β-mannanase. 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 some bacteria that have PH and belong to the genus Bacillus produce enzymes that meet the above requirements, and that they can be produced in large quantities with good productivity. That is, the present invention first provides a novel β-mannanase, which has the following physical and chemical properties. (b) Action: β-1,4-D of mannan, glucomannan, galactomannan, galactoglucomannan
-Non-specifically hydrolyzes the mannopyranoside linkage 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℃ under heating conditions of PH8.0 and 30 minutes. (e) Range of optimum temperature for action: The optimum temperature for action is around 70°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: Inhibited by mercuric chloride, silver nitrate, disodium ethylenediaminetetraacetate (EDTANa ₂ ), urea, sodium dodecyl sulfate, and sodium dodecylbenzenesulfonate. (h) Isoelectric point by chromatofocusing method: 5.3-5.4 (li) Molecular weight by gel filtration method: 37000±3000 The present invention further relates to a method for producing the above-mentioned novel β-mannanase, and provides a method for producing the novel β-mannanase. According to the above, the β-mannanase belongs to the genus Bacillus, and can be obtained by culturing a microorganism that produces the β-mannanase extracellularly, producing and accumulating the enzyme in the culture solution, and then separating and purifying the enzyme. be able to. Novel extracellular β- used in the method of the present invention
The mannanase-producing bacterial strain was newly discovered and isolated from the natural world by the present inventors. These strains were listed in Bergey's Manual of Determinative Bacteriology.
Mannual of Determinative Bacteriology), 8th edition and The Genus Bacillus
Genus Bacillus, United States, Department of Agriculture (Dept. of Agriculture) edition]
Accordingly, all of them are aerobic spore-forming bacilli, motile, have pericytium, positive or variable Gram staining, and positive catalase test, so they belong to the genus Bacillus. However, 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 bacteria. Table 1 below shows various mycological properties of the isolated extracellular β-mannanase producing bacteria.

【table】

[Table] +: Growing or positive -: Not growing or negative The above bacteria have been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under deposit number FERM P-8858 (AM-044). The novel method for producing extracellular β-mannanase of the present invention will be explained in more detail. The above-mentioned extracellular β-mannanase-producing bacteria were inoculated into an appropriate medium, and incubated at 30 to 40°C for 48°C from the perspective of the growth temperature of the bacteria.
Incubate aerobically for ~72 hours, but the medium is a carbon source,
In addition to a nitrogen source, it contains inorganic salts, micronutrients, etc. as necessary. 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. can be exemplified as being inexpensive and easily available. It goes without saying that the organic nitrogen source also 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. be. 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 may be a liquid medium containing. In addition, the growth pH of the microorganism used in the method of the present invention
Since it 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 hydrogen carbonate can be cited as a typical example, but alkaline reagents such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium phosphate, and calcium hydroxide can also be used without being limited thereto. Since the bacteria used in the method of the present invention produce β-mannanase extracellularly, the produced β-mannanase is released into the culture solution and accumulated there. These bacteria can be cultured either batchwise or continuously, and the enzymes produced can be separated and purified, 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. For this purpose, purification can be carried out by, for example, salting out using ammonium sulfate, solvent precipitation using ethanol, acetone, isopropanol, etc., ultrafiltration, gel filtration, or general enzyme purification using ion exchange resins. Below, one example of a preferred purification method for β-mannanase of the present invention will be explained. The AM-044 strain, which belongs to the alkaliphilic Bacillus genus, is inoculated into a medium such as the one described above, and cultured aerobically at 37°C for 72 hours.
% (w/v) of cetabron (cetyltrimethylammonium bromide) was added, and after standing for 30 minutes, the cells were centrifuged at 7000 rpm and 0° C. for 20 minutes to remove the bacterial cells to obtain the supernatant liquid of 3. Next, ammonium sulfate was added to the supernatant to make it 75% saturated, and the mixture was heated at 4°C.
Leave it overnight. 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 supernatant was adsorbed onto DEAE-Toyopearl 650M equilibrated with the above buffer, and the enzyme was eluted using the concentration gradient method of the above buffer containing 0.1 to 0.5 M NaCl. do. The eluted active fractions are collected, dialyzed overnight at 4°C against the above buffer, and then adsorbed onto hydroxyapatite equilibrated with the above buffer. 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. Concentrated enzymes include columns for protein separation and purification for high-performance liquid chromatography, SHODEX, and protein (SHODEX protein) WS-
2003, filled with 10mM phosphate buffer (PH7.0)
Elute using 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 active fraction obtained was concentrated and subjected to polyacrylamide gel disk electrophoresis [Annals of the New York Academy of Science (ANN.NYAcad.
Sci., 1964, 121 , 404], 18 mg of a homogeneous enzyme preparation was obtained, and the activity yield was 23%. 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, quench in an ice bath, add 1.0 ml of Nelson's solution, stir well , and add water. Make it 10ml. 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. The molecular weight of the enzyme obtained from the above strain AM-044 is 37000±3000. Note that this molecular weight was determined by gel filtration method. Table 2 shows a comparison of the physicochemical properties and enzymatic chemical properties of the β-mannanase of the present invention and conventionally known β-mannanase derived from microorganisms.

【table】

[Table] Effect β-D-mannan, which exists in large quantities in nature,
Like starch, it is widely used in various fields such as foods, fibers, concentrated medicines, and sweetened sugar products. by the way,
In order to effectively utilize this β-D-mannan,
It is necessary to develop an enzyme (β-mannanase) that can efficiently hydrolyze this. That is, in order to obtain an enzyme that can hydrolyze β-D-mannan with high efficiency, it is necessary to decompose it into useful sugars such as mannooligosaccharides, mannose, glucose, and galactose, and to recover and utilize them. β-D-
This is important for purposes such as decomposing and removing mannan after use as mannan itself. In such uses, it is extremely desirable from the viewpoint of industrial application that β-mannanase has high temperature stability and has an optimum pH for enzyme reaction on the alkaline side. For this purpose, mannan hydrolases of various origins have been discovered and utilized, but none of them have been fully satisfactory from an industrial standpoint. That is, all the enzymes that have been studied and put into practical use have had poor high-temperature stability, or the culture time of enzyme-producing microorganisms has been extremely long. Therefore, the present inventors conducted various searches and found that bacteria belonging to the alkaliphilic Bacillus genus produce effective β-mannanase at a high production rate. This enzyme satisfies all of the requirements for the hydrolysis reaction of β-D-mannan,
All of the problems with previously known enzymes have been solved. That is, first of all, since the β-mannanase enzyme of the present invention is produced extracellularly by the above-mentioned microorganism, the separation and purification method is extremely simple, and a significant improvement can be expected in terms of labor and production costs. Furthermore, it has excellent high-temperature stability and has the optimum pH for enzymatic reactions on the alkaline side, so it can be used without neutralization after extracting β-mannan from various plants under alkaline conditions. Since it is possible to subject the enzyme to the enzymatic decomposition reaction as it is, the work is significantly simplified, and since the use of extra reagents becomes unnecessary, the production cost of the decomposition product can also be reduced. Thus, the novel enzyme of the present invention enables the decomposition and utilization of β-D-mannan on an industrial scale. It is also extremely advantageous in terms of cost performance. Hereinafter, the present invention will be explained in more detail with reference to Examples. Example Alkaliophilic bacterium Bacillus No.-AM-044 strain (FERM P-8858) was grown in a 500 ml Erlenmeyer flask with 2% palm oil residue, 1% soybean residue, and KNO ₃ 0.2
%, 100 ml of culture solution containing _0.1 % _Na2HPO4 , _0.02 % _MgSO4.7H2O and 0.5% soda carbonate (PH1.00)
The cells were inoculated and cultured at 40°C for 50 hours with shaking at 250 rpm. Next, the β-mannanase activity in the culture supernatant was measured in the same manner as above, and the result was 30 units/ml.
It was hot. Effects of the Invention As described in detail above, the novel β-
Mannanase has an optimal pH for enzymatic reaction on the alkaline side and has excellent high temperature stability. Therefore, β
- The enzymatic decomposition reaction of mannan can be carried out on the alkaline side, which makes it possible to carry out the enzymatic decomposition reaction immediately after the mannan extraction step. Furthermore, since the enzymatic reaction can be carried out at high temperatures, the reaction rate can be greatly increased. Thus, according to the β-mannanase of the present invention, it is possible to industrially advantageously decompose and dispose of unnecessary β-D-mannan and to produce its decomposition products, resulting in high decomposition efficiency and decomposition products. Product productivity can be achieved and manufacturing costs can be reduced. Furthermore, since the β-mannanase of the present invention can be obtained from an alkaliphilic microorganism belonging to the genus Bacillus that is produced extracellularly, it can be easily isolated and purified, and therefore can be mass-produced at low cost.

Claims (1)

[Claims] 1. A novel β-mannanase having the following physical and chemical properties: (a) Action: β-1,4-D of mannan, glucomannan, galactomannan, galactoglucomannan
-Non-specifically hydrolyzes the mannopyranoside linkage 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 70°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: Inhibited by mercuric chloride, silver nitrate, disodium ethylenediaminetetraacetate (EDTANa ₂ ), urea, sodium dodecyl sulfate, and sodium dodecylbenzenesulfonate. (H) Isoelectric point determined by chromatofocusing method: 5.3-5.4 (L) Molecular weight determined by gel filtration method: 37000±3000 2 Physical and chemical properties of the following: (B) Action: Mannan, glucomannan, galactomannan, galactogluco β-1,4-D of mannan
-Non-specifically hydrolyzes the mannopyranoside linkage 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 70°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: Inhibited by mercuric chloride, silver nitrate, disodium ethylenediaminetetraacetate (EDTANa ₂ ), urea, sodium dodecyl sulfate, and sodium dodecylbenzenesulfonate. (h) Isoelectric point determined by chromatofocusing method: 5.3 to 5.4 (li) Molecular weight determined by gel filtration method: 37000±3000 A Bacillus genus that has the ability to produce β-mannanase and has an alkaline optimum pH for growth. The method for producing the above-mentioned novel extracellular β-mannanase, which comprises culturing a microorganism belonging to the above-mentioned species, producing and accumulating the β-mannanase in a culture solution, and collecting the same. 3 The above microorganisms capable of producing β-mannanase have been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology
The second claim is characterized in that it is a strain deposited as FERM P-8858 (AM-044).
The method described in section. 4. The method for producing extracellular β-mannanase according to claim 2 or 3, wherein the culturing is carried out aerobically at a temperature within the range of 30 to 50°C. 5. The method for producing extracellular β-mannanase according to any one of claims 2 to 4, wherein the pH of the culture solution is within the range of 7.5 to 11.5.