JPH0379991B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a novel method for producing carbohydrates containing mannosaccharides, and more specifically, the present invention relates to a novel method for producing carbohydrates containing mannosaccharides, and more specifically, a method for producing a novel β-mannanase and/or β-mannosidase.
The present invention relates to a method for producing a mannosaccharide-containing carbohydrate, which is characterized by acting on a saccharide substrate having a 4-D-mannopyranoside bond. 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 produces a series of mannooligosaccharides, and a large number of microorganisms and plants are known to produce this enzyme [Advances in Carbohydrate Chemistry & Biochemistry]
Chemistry and Biochemistry), 1976, 32 , 299
−316]. 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 terminal site to produce mannose. Similar to the β-mannanase mentioned above, a wide range of plants and microorganisms have the ability to produce this enzyme. [Biochim. Biophys. Acta, 1978, 522 , 521-
530; Japanese Patent Publication No. 51-38486]. Natural products that are hydrolyzed by β-mannanase and/or β-mannosidase to give useful saccharides include β-mannan, which is known to exist in large quantities in nature along with cellulose;
Examples include glucomannan, galactomannan, and galactoglucomannan. 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, which belong to the palm family. Typical examples of galactomannan are the mucilage contained in carob and guar seeds, locus 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
Known examples include Scoparia, Gleeditssia fuerox, and 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. Among these carbohydrates, mannose in particular has been obtained from glucose at a yield of around 20% by reacting at high temperatures using metal catalysts such as molybdenum (Japanese Patent Publication No. 1987-40700). However, as described above, this method requires a large number of steps such as purification of mannose, and is economically disadvantageous, so that the price of mannose is inevitably high. On the other hand, 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. It's here. In particular, filamentous fungi [Acta.Chem.Scand., 1968, 22 , 1924;
Noka, 1969, 43 , 317; Biochem.J., 1984, 219 , 857], actinomycetes [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. Furthermore, β-mannosidase, which is conventionally known as an enzyme that hydrolyzes β-D-mannan into mannose units from the non-reducing end, can be used in animals [Biochemistry, 1972, 11 ;
1493-1501: Biochim.Biophys.Acta, 1973, 315 ,
123-127], Plants [J.Biol.Chem., 1964,
239, 990-992], Microorganisms [Biochim.Biophyys.
Acta), 1978, 522 , 521-530: Japanese Patent Publication No. 51-38486
Enzymes such as [No.]] have been well studied. However, all of these enzymes similarly have low productivity, and many of them require complicated culture and purification methods, leaving problems 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, 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. Also, 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-
Both mannanase and β-mannosidase have drawbacks such as insufficient temperature stability and the need for long culture times to produce the enzymes, so these enzymes cannot be used industrially. It has been difficult or unsatisfactory in terms of cost to utilize it to obtain hydrolysis products of β-D-mannan on a commercial scale. Therefore, the purpose of the present invention is to satisfy various requirements in the above-mentioned enzymatic reactions, especially reactions for producing mannosaccharide-containing carbohydrates, and to carry out the hydrolysis of β-D-mannan economically and on an industrial scale. The object of the present invention is to provide a method for producing mannosaccharide-containing carbohydrates using novel β-mannanase and β-mannodase. 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 possess. As a result, growth is optimal in alkaline conditions.
The present invention was completed by discovering that some bacteria that have PH and belonging 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 provides novel β-mannanase and/or β-mannosidase, β-1,4-
The present invention provides a method for producing mannosaccharide-containing carbohydrates, which is characterized by acting on a saccharide substrate having a mannopyranoside bond as the main skeleton, and each of these enzymes has the following physical and chemical 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℃ 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 gel filtration method: 37,000-57,000 () β-mannosidase (b) Action: Hydrolyzes β-mannosidic bonds sequentially from the non-reducing end and produces 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-galctoside, 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 by gel filtration method: 63,000 to 68,000 The novel β-mannanase-producing strain and novel β-mannosidase-producing strain used in the method of the present invention were newly searched and isolated from the natural world by the present inventors. It is. These strains were collected in Bergey's Manual of Determinative Bacteriology.
Determinative Bacteriology), 8th edition and The Genus Bacillus,
According to the U.S. Department of Agriculture version], all of them are aerobic spore-forming bacilli, motile, have pericytial hairs, and are Gram-stain positive or variable. , it was clear that it belonged to the genus Bacillus because of its positive catalase test, but since it grows well in alkaline conditions of pH 7.5 to 11.5, it is a new species that is taxonomically different from the known genus Bacillus. I thought it was a bacterial strain. Tables 1 to 3 below show two types of extracellular β-mannanase producing strains and two types of intracellular β-mannanase producing strains isolated respectively.
- The mycological properties of a mannosidase-producing strain and a type of β-mannosidase and β-mannanase-producing strain are shown. [Table] [Table] +: Growing or positive -: Not growing or negative [Table] [Table] +: Growing or positive -: Not growing or negative [Table] [Table] +: Growing or positive -: No growth or negative The above bacteria were submitted to the Institute of Microbiology, Agency of Industrial Science and Technology under the FERM P-8857 (AM-024) and FERM respectively.
P-8858 (AM-044), FERM P-8859 (AS-
420), FERM P-8860 (AS-440) and
It has been deposited as FERM P-8856 (AM-001). The method for producing the novel extracellular β-mannanase etc. useful in the method of the present invention will be explained in more detail. For example, extracellular β-mannanase producing bacteria as described above are inoculated into a suitable medium and cultured aerobically at 30 to 45°C for 48 to 72 hours from the viewpoint of bacterial growth temperature. In addition to carbon sources and nitrogen sources, it contains inorganic salts, micronutrients, etc. as necessary. First, various conventionally known materials can be used as the carbon source, such as coconut oil extraction residue, konjac flour, locust bean gum, canalbum gum, guar gum, red pine hemicellulose, or locust bean powder containing β-mannan. Any plant can be used. 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. . Microbial media suitable for use in the method of the invention include, for example, 1% konjac mannan,
2% polypeptone, 0.2% yeast extract, 0.1%
of _K2HPO4 and 0.02 _{% MgSO4.7H2O and}
It can be _a liquid medium containing 0.5% _NaHCO3 or _Na2CO3 . 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. Enzyme-producing microorganisms useful in the methods of the invention
Since both AM-024 and AM-044 produce β-mannanase extracellularly, the produced β-mannanase is released into the culture medium 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 used in the present invention will be explained. For example, AM- belonging to the alkalophilic Bacillus genus
024 strain, for example, inoculated into a medium as described above,
0.8% (w/v) cetabron (cetyltrimethylammonium bromide) was added to the culture solution obtained by culturing aerobically at 37°C for 72 hours, and after standing for 30 minutes, the culture was incubated at 7000 rpm for 20 hours at 0°C. Centrifuge for a minute to remove the bacterial cells and obtain the supernatant liquid in step 3. Then, ammonium sulfate was added to the supernatant to make it 75% saturated, and the mixture was left overnight at 4°C. Filter out the resulting precipitate,
Dissolve in 10mM phosphate buffer (PH7.0) and incubate overnight.
Dialyze against the same buffer at °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 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. 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 subjected to chromatography again under the same conditions using the same column as above, and the obtained active fraction was concentrated and subjected to polyacrylamide gel disc 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%. Furthermore, AM-044 useful in the method of the present invention
Bacteria can also be purified in the same manner as the above method. The method for measuring β-mannanase activity and the method for displaying the activity are as follows. That is, 0.4 ml of 0.1M glycine-NaOH-NaCl buffer (PH9.0)
and 1% (w/v) konjac mannan aqueous solution
Mix 0.1 ml of enzyme solution with 0.5 ml and react at 50℃ for 10 minutes.
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. In addition to the physicochemical and enzymatic properties of μ-mannanase obtained by the method of the present invention, the molecular weight of the enzyme obtained from each strain AM-024 and AM-044 is 45,000 ± 3,000 and 37,000 ± 3,000, respectively. be. Incidentally, these molecular weights were determined by gel filtration method. Table 4 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. In addition, both AS-420 and AS-440 are β-
Mannosidase is produced within the bacterial body and accumulated there. This culture can be carried out in the same manner as for the β-mannanase producing strain, and the produced enzyme can be isolated and purified, for example, as follows. That is, it is possible to first collect bacteria in a culture solution by known means such as centrifugation or filtration, and then use the obtained bacteria as is 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, the bacterial cells can be disrupted by appropriate means and then purified by a general enzyme purification method similar to that used for β-mannanase. Below, one example of a preferred purification method for β-mannosidase of the present invention will be explained. For example, the AS-440 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.
Centrifuge for 30 minutes to collect bacterial cells and weigh 10 g wet.
Obtain bacterial cells. 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 following steps were performed in the same manner as above to perform polyacrylamide gel disk electrophoresis [ANN.NYAcad.Sci., 121 , 404]
(1964)], 15 mg of homogeneous enzyme preparation was obtained.
The activity yield was 18%. Note that the AS-420 strain useful in the method of the present invention can also be purified in the same manner as the above method. Furthermore, 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) using 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. In addition to the physicochemical and enzymatic properties of β-mannosidase obtained by the method of the present invention, the molecular weight of the enzyme obtained from each strain AS-420 and AS-440 is 68000±3000 and 63000±3000, respectively. It is. Incidentally, these molecular weights were determined by gel filtration method. Table 4 shows a comparison of the physicochemical and enzymatic properties of the β-mannosidase of the present invention and the conventionally known β-mannosidase derived from microorganisms. Furthermore, the AM-001 strain produces β-mannanase outside the bacterial cells and β-mannodase inside the bacterial cells, so in this case, after culturing in the same manner as the above-mentioned strains, the bacterial cells and culture solution are centrifuged. After separation by known means such as separation and filtration, β-mannanase and β-mannosidase can be recovered respectively according to operations similar to those described above. First, β-mannosidase can be purified according to the above method. Further, for β-mannanase, the same procedure is performed according to the above method until it is adsorbed to hydroxyapatite, and then the following procedure is performed. That is, when the enzyme is eluted by the concentration gradient method of 0.01 to 0.4M phosphate buffer (PH7.0), it is found that 2
Two fractions (FA, FB) were obtained, and the combined activity yield of the two fractions was 29%. Next, each fraction was determined to have an average fractional molecular weight.
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 disk 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 disk 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 types of β-mannanases had almost the same enzymatic chemical properties except for their different molecular weights. The methods for measuring and displaying the activities of β-mannosidase and β-mannanase obtained from the AM-001 strain are the same as described above. Similarly, Tables 4 and 4 below show the characteristics of each produced enzyme in comparison with conventional ones. [Table] [Table] [Table] [Table] Thus, in the method of the present invention, the novel β-mannanase and/or β-mannosidase produced by the above-mentioned novel strain is converted into β-1,4-D
- Mannosugar-containing carbohydrates are obtained by acting on a saccharide substrate having a mannopyranosidic bond in its main skeleton, and useful saccharide substrates in this method include mannans originating from the various plants mentioned above;
Typical examples include glucomannan, galactomannan, galactoglucomannan, and oligomers thereof. The enzymatic decomposition reaction of these saccharide substrates is carried out at the optimal pH (or stable
PH) at the optimum temperature (or stable temperature range). Since the extraction operation of the above-mentioned saccharide substrates from natural products is generally carried out in an alkaline region, it is preferable to use a combination of enzymes that have an alkaline optimum pH or stable pH, but in some cases, a pH regulator may be used. The enzymatic reaction can be carried out after making the solution neutral to weakly acidic. There is no particular restriction on the amount of enzyme for the sugar substrate, but considering economics such as reaction time and yield, β
Advantageously, both mannanase and β-mannosidase are each at least 10 units/g carbohydrate. Function β-D-mannan, which exists in large amounts in nature, is widely used along with starch in various fields such as foods, textiles, medicines, and cosmetics. Also, this β−
A series of mannooligosaccharides containing mannose or other sugars in the molecule can be produced by hydrolyzing D-mannan or mannooligosaccharides obtained by hydrolyzing these with acids or appropriate enzymes using a specific enzyme. Sugar and mannose are obtained, and these mannooligosaccharides, mannose, and their reduction products, such as manno sugar alcohols, can not only be used as sweetness sources, but also useful as medium components for microorganism identification in the field of clinical testing, or as pharmaceuticals. It is. Furthermore,
These carbohydrates are also known as substances that promote the growth of Bifidobacterium, which is a useful intestinal bacterium, and are considered to be extremely useful substances from an industrial standpoint. Conventionally, among these carbohydrates, mannose in particular has been obtained from glucose at a yield of around 20% by reacting at high temperatures using a metal catalyst such as molybdenum (Japanese Patent Publication No. 1983-40700). however,
This method requires a large number of steps such as the purification of mannose as described above, and is economically disadvantageous, so the price of mannose is inevitably high. Furthermore, even with the previously proposed β-D-mannan degrading enzymes, it has not been possible to use them industrially due to the production potency, the stability of the enzyme itself, the optimal pH, etc. It was difficult. In this regard, the β-mannanase and β-mannosidase used in the present invention satisfy all of the various conditions for the decomposition reaction described above, and the production titers of the microorganisms that produce them are also high, so they can be used on an industrial scale. , it becomes possible to significantly reduce the cost of decomposition products (carbohydrates) such as mannose. Both β-mannanase and β-mannosidase used in the present invention are suitable for alkaline to slightly acidic pH.
This is because it has high temperature stability and is also based on the high enzyme production titer of a strain belonging to the genus Alkaliphilic Bacillus, which was discovered as a result of a search by the present inventors. . In the method of the present invention, in the hydrolysis reaction of β-D-mannan, it is of course possible to immobilize each enzyme according to a conventionally known method and use it in a bioreactor, and it is possible to recover and reuse it. I can do it. In this case as well, properties such as high temperature stability are desirable from the perspective of maintaining enzyme activity, and sufficient activity can be expected to be maintained even after immobilization. EXAMPLES Hereinafter, the present invention will be explained in more detail based on Examples, but the scope of the present invention is not limited in any way by these Examples. Example 1 Alkaliphilic bacterium Bacillus No. AM-001 strain (FERMP-8856) was mixed with 1% konjac powder, 2% boripeptone, 0.2% yeast extract, 0.1% K ₂ HPO ₄ , MgSO ₄ in a 1-volume Erlenmeyer flask.・_7H2O0.02 %,
Alkaline medium containing 0.5% Na ₂ CO ₃ (separately sterilized)
Inoculate 100ml (PH9,0) and incubate at 37℃ for 40 hours.
Culture with shaking at 200 rpm, and centrifuge the culture solution.
94 ml of supernatant was obtained. This supernatant contained 50 units/ml of β.
- Contains mannanase. Ammonium sulfate was added to the β-mannanase solution thus obtained to make it 80% saturated, and the solution was stored overnight at 4°C. The resulting precipitate was filtered and 10 ml of 10
It was dissolved in mM glycine-NaOH-NaCl buffer (PH9.0) to obtain an enzyme solution containing 370 units/ml. Furthermore, the bacterial cells obtained by centrifugation were added to 10ml of 10mM.
Suspend in phosphate buffer (PH7.0), cool in ice water, and perform ultrasonic disruption several times for a total of about 3 minutes. Then, centrifuge at 12,000 r.pm for 30 minutes at 0°C to remove the residue and obtain 10 ml of supernatant. This was concentrated to 3 ml using an ultrafiltration membrane with an average molecular weight cut off of 10,000. This concentrate contained 9 units/ml of β-mannosidase. After previously dissolving the β-mannanase obtained as above in a sodium hydroxide solution,
Hydrolysis of carbohydrates obtained by adding 150 units per 1 g of mannan to 100 ml of 1% (w/v) elephant palm mannan solution (mannan: 1 g) adjusted to pH 9 and reacting at 65°C for 50 hours. As a result of measuring the rate 21%
As a result of analyzing this carbohydrate by high performance liquid chromatography, a series of mannooligosaccharides such as mannose, mannobiose, and mannotriose were detected. Furthermore, 20 units/g (solid matter) of β-mannosidase obtained by the above method was added to the carbohydrate, and the pH was 7.0.
As a result of reacting at 55℃ for 50 hours, the hydrolysis rate was 29%, and the result was a product mainly composed of mannose and containing mannooligosaccharides.
Carbohydrates were obtained. Example 2 Alkaliphilic bacterium Bacillus No. AM-024 strain (FERMP-8857) was cultured in the same manner as in Example 1, using coconut oil extraction residue instead of konjac powder in the medium composition, and cultured in 95 ml. 28 units/ml of β-
Contains mannanase. Next, the crude enzyme solution was concentrated about 10 times using an ultrafiltration membrane with an average molecular weight cutoff of 10,000 to obtain a concentrated enzyme solution with a concentration of 210 units/ml. The β-mannanase obtained in this way was
% (w/v) konjac mannan solution (100 ml per 1 g of mannan) was added to 100 units per 1 g of mannan and reacted at 65°C for 50 hours. The resulting carbohydrate hydrolysis rate was 20%. As a result of analyzing the carbohydrate by high performance liquid chromatography, mannose and a series of oligosaccharides containing mannose and glucose in the molecule were detected. Example 3 The alkaliphilic bacterium Bacillus AM-044 strain (FERMP-8858) was grown using guar gum instead of konjac flour and 5% cornstarch liquor instead of polypeptone in the culture medium composition of Example 1. 21 units/ml of β was added to the culture supernatant.
- 94 ml of crude enzyme solution with mannanase activity was obtained. Then, 4 times the amount of cold isopropanol (-40°C) was added to the enzyme, and the mixture was left in a cold room overnight. The resulting precipitate was filtered and dissolved in 10 ml of 10 mM glycine-NaOH-NaCl buffer (PH9.0).
A β-mannanase solution of units/ml was obtained. The β-mannanase obtained in this way was
After pre-dissolving in sodium hydroxide solution, pH9
1% coconut oil extraction residue mannan solution adjusted to 100%
110 per 1g of mannan in ml (1g of mannan)
The hydrolysis of carbohydrates obtained by adding a unit of sugar and reacting at 70°C for 50 hours was 21%. Furthermore, as a result of analyzing this carbohydrate by high-performance liquid chromatography, mannose and a series of oligosaccharides containing mannose and galactose in the molecule were detected. Example 4 Alkaliphilic bacterium Bacillus No. AS-420 strain (FERMP-8859) was inoculated into 300 ml of the same medium (PH9.0) used in Example 1, and shaken at 200 rpm for 24 hours at 37°C. It was cultivated for a long time. The cells were then collected by centrifugation and added to 5 ml of 10mM phosphate buffer (PH).
7.0), 0.5 ml of a 10 mg/ml lysozyme chloride solution was added, and the mixture was left at 37°C overnight. Next, add polyethyleneimine to 0.1%,
The eluted nucleic acids were allowed to stand for 30 minutes to aggregate, and then centrifuged to obtain 9.5 ml of supernatant. 8.5 in Honjosumi
unit/ml of β-mannosidase. The β-mannosidase solution thus obtained was added to the elephant palm mannan β obtained in Example 1.
- Adding 20 units per gram of mannanase to a hydrolyzate and allowing it to react at 40°C and pH 7.0 for 50 hours, the resulting carbohydrate hydrolysis rate was 28%. As a result of analysis by high performance liquid chromatography, mannose and a small amount of mannooligosaccharides were detected. Example 5 The alkaliphilic bacterium Bacillus No. AS-440 strain (FERMP-8860) was inoculated into 300 ml of the same medium (PH9.0) used in Example 2, and the same culture method and method as in Example 4 were carried out. By the method for obtaining β-mannosidase, 9.6 ml of 18 units/ml β-mannosidase solution was obtained. 1 g of mannan is added to 100 ml of a 10% (w/v) konjac mannan solution (10 g of mannan) in which the β-mannosidase solution thus obtained is liquefied by the action of the β-mannanase obtained in Example 1. 20 units per and β- obtained in Example 1
Add 120 units of mannanase and incubate at 50℃ and pH 8.0.
As a result of measuring the hydrolysis rate of the carbohydrate obtained by the time reaction, it was 29%, and as a result of analyzing the carbohydrate by high performance liquid chromatography, mannose and a small amount of mannooligosaccharide were detected. Example 6 Alkaliphilic bacterium Bacillus No.AS-440 (FERM
P-8860) was grown in 9.5 ml using the same culture method and enzyme obtaining method as in Example 4, using coffee extract grounds instead of konjac powder in the medium composition of Example 1.
A β-mannosidase solution of 12 units/ml was obtained. The thus obtained β-mannosidase solution was added to 100 ml of 5% (w/v) guar gum solution (5 g of mannan) which had been liquefied by the action of the β-mannanase obtained in Example 3 per 1 g of mannan.
The results of measuring the hydrolysis rate of carbohydrates obtained by adding 25 units and 150 units of β-mannanase obtained in Example 3 and reacting at 50°C and pH 8.0 for 50 hours.
It was %. Furthermore, as a result of analyzing this carbohydrate by high-performance liquid chromatography, mannose and mannooligosaccharides containing small amounts of mannose, glucose, and galactose were detected in the molecule. Effects of the Invention As explained in detail above, according to the method for producing a mannosaccharide-containing carbohydrate of the present invention, a novel β-mannanase that satisfies all the requirements for the enzymatic decomposition reaction of β-D-mannan is produced. By using β-mannosidase and β-mannosidase, all the conventional problems have been solved, and it has become possible to provide β-mannan hydrolysis products useful in various industrial fields at a high production rate on an industrial scale. It became possible. This fact also means that the above-mentioned β-mannanase and β-mannosidase can now be mass-produced using the novel microorganisms newly searched and isolated by the present inventors using simple operations and processes, and moreover, it has become cost-effective. This is due to a number of things.

Claims (1)

[Scope of Claims] 1 β-mannanase and/or β-mannosidase having the following physicochemical properties,
1. A method for producing a mannosugar-containing saccharide, which comprises acting on a saccharide substrate having a 1,4-mannopyranoside bond as a main skeleton. 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 gel filtration method: 37000-57000 β-mannosidase (b) Action: Hydrolyzes β-mannosidic bonds sequentially from the non-reducing end, Produces 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 by gel filtration method: 63,000 to 68,000 2. If the saccharide substrate is mannan, glucomannan,
2. The method for producing a carbohydrate according to claim 1, wherein the carbohydrate is at least one selected from the group consisting of galactomannan, galactoglucomannan, and oligomers thereof. 3. The β-mannanase belongs to the genus Bacillus,
Alkaliphilic microorganisms with an optimal pH for growth in alkaline conditions, 30
Cultured at a temperature of ~45℃ and under pH7.5~11.5,
Claim 1, characterized in that the enzyme is obtained by producing and accumulating the enzyme in a culture solution.
The method for producing carbohydrates according to item 1 or 2. 4 The above-mentioned mannosidase belongs to the genus Bacillus,
Alkaliphilic microorganisms with optimal pH for growth in alkaline conditions, Fiber Microorganisms No. 8859 or No. 8860.
Any one of claims 1 to 3, which is obtained by culturing at 30 to 45°C and PH7.5 to 11.5 to produce and accumulate the enzyme in the bacterial body.
The method for producing carbohydrates described in section. 5. The above β-annanase and β-mannosidase belong to the genus Bacillus, and are microorganisms that have an optimal pH for growth in alkaline conditions.
Cultivate at 30-45℃ under pH7.5-11.5,
The carbohydrate according to any one of claims 1 to 4, which is obtained by producing and accumulating mannanase outside the bacterial cell and β-mannosidase inside the bacterial cell. manufacturing method.