JPH0370471B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a novel β-mannosidase and a method for producing the same. More specifically, the microbial β-
This article concerns mannosidase and its production method. Conventional technology β-mannosidase acts on low-molecular β-D-mannans (mannan glucomannan, galactomannan, galactoglucomannan) that have β-mannosidic bonds in the molecule, and sequentially removes the mannoside bonds from the non-reducing terminal site. It is an enzyme that hydrolyzes and produces mannose. Ivory nuts (scientific name: Phytelehus macrocarpa) and corozo are well known as those containing the above-mentioned β-D-mannan. others,
Known plants containing β-1,4-mannan include Phoenicus canariensis and Orchis maquiulata, which belong to the palm family. Typical galactomannans include various mucilage substances contained in carob and guar seeds, locust bean gum, and guar gum, and these two types of galactomannans are industrially produced as they are or after chemical modification. , 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, Gladeitssia 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. Glucomannan is obtained from these plants 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-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. 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 also has great significance in recovering and utilizing decomposition products (mannose, etc.). Therefore, the first object of the present invention is to provide a novel enzyme, β-mannosidase, that satisfies the above various requirements. Another object of the present invention is to use a novel microorganism to obtain the enzyme with ease and high yield.
An object of the present invention is to provide a method for producing mannosidase. Means for Solving the Problems The present inventors extensively searched the natural world to obtain microorganisms capable of producing enzymes having the above-mentioned properties that β-mannosidase for industrial use should have. As a result, some bacteria belonging to the genus Bacillus, which have an optimal pH for growth in alkaline conditions, have the ability to produce β-mannosidase that meets the above requirements.
It has been discovered that this can be produced with good productivity, and the present invention has been completed. The novel β provided by the first aspect of the present invention
-Mannosidase has the following physical and chemical properties: (a) Action: Hydrolyzes β-mannosidic bonds sequentially from the non-reducing end to produce mannose. (b) Substrate specificity: Completely decomposes β-methyl (methyl)-D-mannoside, and acts on β-bonded mannose-containing oligosaccharides to liberate mannose. β-D-mannoside of p-nitro-phenyl-glycoside can be used as a substrate, but α-D-mannoside, α-D-glucoside, α-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 40°C. (F) Inactivation conditions: 40℃, 30 minutes treatment condition: PH5.0 and
Completely deactivated at 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: 68000±3000. The novel β-mannosidase described above can be obtained by the method for its production provided by the second aspect of the present invention. That is, the β-mannosidase can be obtained by a method characterized by culturing a microorganism that intracellularly produces the β-mannosidase belonging to the genus Bacillus, collecting the bacteria, and then isolating and purifying the microorganism. . Novel intracellular β- used in the method of the present invention
The mannosidase-producing bacterial strain was newly discovered and isolated from the natural world by the present inventors. This strain was published in Bergey's Manual of Determinative Bacteriology (Bergey'S
Mannual of Determinative Bacteriology), 8th edition and The Genus Bacillus
Genus Bacillus] Identified according to the U.S. Department of Agriculture edition, it is an aerobic sporulating bacillus;
It was clear that it belonged to Bacillus because it was motile, had periflagella, and had a positive or variable Gram stain, and a positive catalase test. Because it grew, 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 intracellular β-mannosidase producing bacteria. [Table] [Table] (Note) +: Growing or positive -: Not growing or negative The above bacteria has been deposited as FERMP-8859 (AS-420) at the Institute of Microbial Technology, Agency of Industrial Science and Technology. Next, the novel method for producing intracellular β-mannosidase of the present invention will be explained in more detail. In the present invention, the above-mentioned intracellular β-mannosidase-producing bacteria are inoculated into a suitable medium and cultured aerobically for 48 to 72 hours at 30 to 40°C from the viewpoint of the growth temperature of the bacteria. . 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; organic nitrogen such as yeast extract, peptone, meat extract, cornstarch liquor, amino acid solution, and soybean meal, or inorganic nitrogen such as ammonium sulfate, ammonium nitrate, and ammonium chloride are inexpensive and available. This can be exemplified as one that is 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. 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 bicarbonate can be cited as a typical example, but alkaline reagents such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium phosphate, and potassium hydroxide can also be used without being limited thereto. All bacteria used in the method of the present invention are β
-Mannosidase is produced within the bacterial body 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 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 described. The above AS- belonging to the alkalophilic Bacillus genus
420 strain, for example, inoculated into a medium as described above,
The culture solution obtained by culturing aerobically at 37° C. for 48 hours is centrifuged at 12,000 rpm and 0° C. for 30 minutes to collect bacterial cells to obtain bacterial cells with a wet weight of 10 g. Next, the bacterial cells were cooled in permanent water and added to 10mM phosphate buffer (PH).
7.0) and subjected to ultrasonic disruption several times, totaling 3 times.
Do this for about a minute. Then, at 12000rpm, 0℃ for 30
Centrifuge for 1 minute to remove the residue and obtain 50 ml of supernatant. Then, ammonium acid was added to the supernatant.
Bring to 75% saturation and leave 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 adsorbed onto DEAD-Toyopearl 650M equilibrated with the above buffer, and then absorbed using the concentration gradient method of the above buffer containing 0.1 to 0.5 M NaCl. Elute the enzyme. The eluted active fractions were collected and incubated overnight in the same buffer.
After dialysis at 4°C, it is adsorbed onto hydroxyapatite equilibrated with the same buffer. Then,
Elute the enzyme with 0.4M phosphate buffer (PH8.0),
The active fractions are collected and concentrated using an ultrafiltration membrane with an average molecular weight cut off of 10,000. The concentrated enzyme is SHODEX protein WS-, a column for protein separation and purification for high-performance liquid chromatography.
Fill in 2003 and elute using 10mM phosphate buffer (PH7.0). After concentrating the active fraction thus obtained, it was 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 New York Academic Sciences (ANN .NYAcad.Sci.), 121, 404
(1964)] to obtain 15 mg of a homogeneous enzyme preparation. The activity yield was 18%. The method for measuring β-mannosidase activity and the method for expressing the activity are as follows. That is, 0.2ml of 0.2M phosphate buffer (PH7.0) and
Mix 0.1 ml of enzyme solution with 0.2 ml of 8 mM p-nitrophenyl-β-D-mannopyranoside aqueous solution,
After reacting at ℃ for 10 minutes, the enzyme was deactivated by adding 1.0 ml of 0.5 M sodium carbonate aqueous solution.
Add water to make 3ml. The degree of coloration is determined by ultraviolet light (wavelength
420 nm) with 1 μmol/ml p-nitrophenol as standard. The unit of enzyme activity is 1 minute under the above conditions.
The amount of enzyme that liberates 1 μmol of p-nitrophenol is expressed as 1 unit. The molecular weight of β-mannosidase obtained by the method of the present invention is 68000±3000. Note that this molecular weight was determined by gel filtration method. Table 2 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. [Table] Effect β-D-mannan is contained in relatively large amounts in various plants, and is used as it is in various fields.
Or after chemical modification treatment, it is used industrially as a paste, thickener, or food material. When this β-D-mannan is used, for example, as a thickening agent in the textile industry, it is removed after a certain processing process is completed, and in that case, its degrading enzyme, β-mannosidase, etc. are generally used. This type of enzyme is also used when β-D-mannan is hydrolyzed and the resulting decomposition product is used. However, all of the conventionally known β-mannosidases have low productivity, and many require complicated culture and purification methods. Therefore, it is expensive;
Large-scale industrial use as described above has been difficult. Furthermore, the extraction process for β-D-mannan is generally carried out in an alkaline environment;
It is advantageous to use enzymes with a high That is,
With conventional enzymes, which have an optimal pH on the acidic side, it is necessary to adjust the pH of the extract using a neutralizing agent before performing the decomposition reaction, which not only complicates the process but also increases costs. . Therefore, it is necessary to develop a method that can be mass-produced.
It is also necessary to develop enzymes with a higher optimal pH than known enzymes. According to the specific microorganism discovered by the present inventors, it is possible to obtain a large amount of an enzyme that satisfies all of the above requirements for the decomposition of β-D-mannan.
Various conventional problems can be solved all at once. That is, the problems of mass production and cost can be overcome by using the parent strain newly discovered by the present invention because it can be obtained in large quantities and at low cost by a simple method. Therefore, large-scale industrial use becomes possible. Furthermore, since the optimal pH for the mannan decomposition reaction of the resulting enzyme is near neutrality, it is possible to immediately proceed to the next decomposition reaction after making a slight pH adjustment after the mannan extraction process. Therefore, the disassembly operation is simplified and economically advantageous. Hereinafter, the present invention will be explained in more detail with reference to Examples. Example 1 FERMP-
Alkaliphilic bacterium Bacillus deposited as 8859
Strain AS-420 in a 500ml Erlenmeyer flask containing 0.5% guar gum, 5% cornstarch liquor, 0.1% ammonium sulfate, 0.1% K ₂ HPO ₄ , 0.02% MgSO ₄ 7H ₂ O
and 100 ml of culture solution containing 0.25% soda carbonate (PH
9.5) and cultured at 37°C for 48 hours with shaking at 200 rpm. Then, the culture solution was spun at 12000 rpm.
Centrifuge for 30 minutes at 0°C to collect bacterial cells, and add 5ml
After suspending the cells in 10mM phosphate buffer, the cells were disrupted using an ultrasonic disruptor. Next, this bacterial cell suspension was
After centrifugation at 12,000 rpm for 30 minutes at 0°C, the β-mannosidase activity of the resulting supernatant was measured and found to be 16 units/ml. Effects of the Invention As described in detail above, the present invention provides one of the β-D-mannan hydrolyzing enzymes, namely β-mannosidase, which has the optimum pH for enzymatic reaction on the alkaline side. By using it, β-D-mannan can be decomposed with high efficiency in a simple and economical process, unnecessary mannan can be quickly removed, or target decomposition products can be mass-produced. Moreover, according to the method for producing β-mannosidase of the present invention, the enzyme can be easily obtained with high productivity. Therefore, it becomes possible to utilize the enzyme at low cost on an industrial scale.

Claims (1)

[Claims] 1. A novel β-mannosidase having the following physical and chemical properties: (a) Action: Hydrolyzes β-mannosidic bonds sequentially from the non-reducing end to produce mannose. (b) Substrate specificity: Completely decomposes β-methyl (ethyl)-D-mannoside, and also acts on β-linked mannose-containing oligosaccharides to liberate mannose. β-D-mannoside of p-nitro-phenyl-glycoside 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 40°C. (F) Inactivation conditions: 40℃, 30 minutes treatment condition: PH5.0 and
Completely deactivated at 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: 68000±3000 2 FERMP to the Institute of Microbial Technology, Agency of Industrial Science and Technology
The β-mannosidase according to claim 1, which is produced by a bacterium deposited as -8859 (AS-420). 3 The following physical and chemical properties: (a) Action: Hydrolyzes β-mannoside bonds sequentially from the non-reducing end to produce mannose. (b) Substrate specificity: Completely decomposes β-methyl (ethyl)-D-mannoside, and also acts on β-linked mannose-containing oligosaccharides to liberate mannose. β-D-mannoside of p-nitro-phenyl-glycoside 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 40°C. (F) Inactivation conditions: 40℃, 30 minutes treatment condition: PH5.0 and
Completely deactivated at 10. Furthermore, when treated at pH 6.5 for 30 minutes, it is completely inactivated at 55°C. (g) A microorganism belonging to the genus Bacillus that has the ability to produce β-mannosidase with a molecular weight of 68000±3000 by gel filtration method and has an optimal pH for growth on the alkaline side is cultured, and the β-mannosidase is introduced into the bacterial body. A method for producing a novel intracellular β-mannosidase, which comprises producing and accumulating the product, and collecting the product. 4. The method for producing intracellular β-mannosidase according to claim 3, wherein the culture is carried out aerobically at a temperature within the range of 30 to 45°C. 5. The method for producing intracellular β-mannosidase according to claim 3 or 4, wherein the pH of the culture solution is within the range of 7.5 to 11.5. 6. Claim 3, wherein the microorganism is a bacterium deposited with the Institute of Biological Technology, Agency of Industrial Science and Technology as FERMP-8859 (AS-420).
6. The method for producing β-mannosidase according to any one of Items 5 to 5.