JPS6336779A - Beta-mannosidase and production thereof - Google Patents

Abstract

PURPOSE:To produce novel beta-mannosidase having optimum pH of enzymatic reaction at alkaline side, by culturing a microorganism belonging to Bacillus genus. CONSTITUTION:A novel microbial strain belonging to Bacillus genus and capable of producing a novel beta-mannosidase having optimum pH of enzymatic reaction at alkaline side, e.g. AS-420 strain (FERM P-8859) is inoculated in a proper medium and cultured at 7.5-11.5pH and 30-45 deg.C for about 48-72hr under aerobic condition. The objective novel beta-mannosidase can be separated from the cultured cell.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a novel β-mannosidase and a method for producing the same. More specifically, it is obtained by culturing a new alkaliphilic microorganism that belongs to the genus Bacillus and has an optimum pH for growth on the alkaline side, and has an intracellular β-mannosidase and an optimum pH for enzyme reaction near neutrality. Regarding its manufacturing method.

Conventional technology β-mannosidase is a low-molecular β-D-mannan (mannan, glucomannan, galactomannan, galactoglucomannan) that has a β-mannosidic bond in the molecule.
It is an enzyme that hydrolyzes mannoside bonds sequentially from the non-reducing end site to produce mannose.

First, as a substance containing β-D-mannan, Ipoly-
Natsutsu (scientific name: Huiterephas macrocarba) and corozo are well known. Other plants containing β-1,4-mannan include palm trees Phoenicus canariensis and Orchis maculata.

Typical examples of galactomannans are 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 known galactomannan-containing plants include Genista scoparia, Daledeiracha fuerolas, and Leucaena glauca.

The most well-known glucomannan-containing substance is konjac (scientific name: Niamorphopharus konjac), but other substances include arum roots of the Cytomoaceae family, jackpines of the Pinus family, bulbs of the Orchidaceae family, and plants of the Spruce family, such as rainbow pine and holly fir. It has been known. Other known glucomannan-containing plants include Asparagus officinalis, Eremulus fuscus, Eremulus legeri, Eremulus spectabilis, and Phaseolus 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 has been known as an enzyme that hydrolyzes β-D-mannan into mannose units from the non-reducing end, has been developed in animals [Biochemistry (Bio'
chemistry), 1972.11, 1493
~1501: Biochim, Biophys, Acta,
1973.268, 488-496: Biosimica
Biophysics Acta (Biochim, Bi
phys, Acta), 1973, 315.12:
3-127), plants [Journal of Biological Chemistry - (J, Biol, Chem
), 1964. 239. 990-9921,
Microorganisms [Biosimichae biophysicaacta (Bi
ochim, Biophys, Acta), 1978
, 522.521-530; JP-A No. 51-38486)] 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 β-D- exists in large quantities as a renewable resource in the natural world.
In order to effectively utilize mannan, especially to efficiently recover and utilize mannooligosaccharides and sugars such as mannose, glucose, and galactose through enzymatic hydrolysis of mannan, stability must be compromised and enzyme purification must be easy. is preferred.

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. It was still unsatisfactory for practical use.

Therefore, it is important to newly develop this type of enzyme that is easy to produce and purify and has high stability as described above.
-It is of great significance in decomposing mannan or 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 a method for producing β-mannosidase using a novel microorganism, which allows the enzyme to be obtained easily and in high yield.

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. result,
The present invention was completed based on the discovery that some bacteria belonging to the genus Bacillus, which have an alkaline optimum pH for growth, can produce β-mannosidase that meets the above requirements and can produce it with good productivity. It is something.

That is, the present invention first provides a novel β-mannosidase, which has the following physical and chemical properties.

(a) Action: Hydrolyzes β-mannoside 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-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 nifcoside, 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 conditions of pH 6, 5, and heating for 30 minutes.

(e) Range of suitable temperature for action: It has an optimum operating temperature around 50°C.

(f) Inactivation conditions: pH 5.0 and 1 under treatment conditions of 40°C and 30 minutes.
At 0, it is completely inactivated. Further, in the treatment at pH 6, 5, and 30 minutes, the activity is completely inactivated at 55°C.

(g) Molecule destruction by gel filtration method: 63,000 to 68,000 The present invention also relates to a method for producing the above-mentioned novel β-mannosidase, wherein the β-mannosidase belongs to the genus Bacillus and the β-mannosidase is Z can be obtained by a method characterized by culturing microorganisms produced within cells, collecting them, and separating and purifying them.

The novel intracellular β-mannosidase producing strain used in the method of the present invention was newly searched for and isolated from the natural world by the present inventors. These strains were listed in Bergey's Manual of Determinative Bacteriology.
eterminative Bacteriology
), 8th edition and The Genus Bacillus [:The
GenusBacillus United States, Department of Agriculture (Dept, of Ag)
When identified according to the Bacillus (Ricalture) version, they are all aerobic sporobacilli, are motile, have periflagella, and are positive for Durham staining or positive for barrier pull and catalase tests.
It was clear that it belonged to the genus s), but the pH was 7.5~
Since it grows well in alkaline conditions of 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 two isolated intracellular β-mannosidase producing bacteria.

The above bacteria were sent to the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERMP-8859 (AS-420) and FERMP-8860 (AS-440), respectively.
It has been deposited as.

The novel method for producing intracellular β-mannosidase of the present invention will be explained in more detail. 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, carob 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 something that is inexpensive and easy to handle.

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. .

Suitable media for use in the method of the invention include, for example, 1% konjac flour, 2% polypeptone, 0.2%
yeast extract, 0.1% of 28P04 and 0.2%
It is a liquid medium containing Mg5O<・7H20.

Furthermore, since the growth pH of the microorganisms used 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. 0.5% for that
Sodium hydrogen carbonate can be mentioned 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.

All of the bacteria used in the method of the present invention produce β-mannosidase within their cells and accumulate it there. Cultivation of these bacteria can be carried out either batchwise or continuously, and the produced enzymes 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, salting out with ammonium sulfate,
It can be purified by a solvent precipitation method using ethanol, acetone, isopropanol, etc., an ultrafiltration method, a gel filtration method, a general enzyme purification method using an ion exchange resin, etc.

Below, one example of a preferred purification method for β-mannosidase of the present invention will be explained. For example, an 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 12.00 Or, p, m , 30 at 0℃
The bacterial cells were collected by centrifugation for a minute to obtain bacterial cells with a wet weight of 10 g. Then, the bacterial cells were cooled in ice water.
It is suspended in IA phosphate buffer (pH 7,0) and subjected to ultrasonic disruption several times for a total of about 3 minutes. Then, 12.00O
r, p, m Centrifuge at 0°C for 30 minutes to remove the residue to obtain 50 ml of supernatant. Ammonium sulfate was then added to the supernatant to achieve 75% saturation, and the mixture was allowed to stand overnight at 4°C. The resulting precipitate was filtered and added to 10mM phosphate buffer (pH 7,
0) and dialyzed against the same buffer at 4°C overnight.

The resulting precipitate was removed by centrifugation, and the resulting supernatant was adsorbed onto DEAD-)Yopal 650M equilibrated with the above buffer, followed by a concentration gradient of the above buffer containing 0.1 to 0.5 M NaCl. The enzyme is eluted by a method.

The eluted active fractions were collected and incubated against the same buffer overnight for 4 hours.
After dialysis at °C, it is adsorbed onto hydroxyapatite equilibrated with the same buffer. Then, the enzyme is eluted with 0.4 M'J acid buffer (18.0), and the active fractions are collected and concentrated using an ultrafiltration membrane with an average molecular weight cutoff of 10.000. The concentrated enzyme is Mujodex Protein (S) for high performance liquid chromatography.
tlOD[EX protein)WS-2003
and elute using 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 blade ram. N, Y
, Acad, Sci.), 121. 4
04 (1964): 15 mg of a homogeneous enzyme preparation was obtained with an activity yield of 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.

The method for measuring β-mannosidase activity and the method for displaying the activity are as follows.

That is, 0.2M phosphate buffer (pH 7.0)
l and 3m! , 0.1 ml of the enzyme solution was mixed with 0.2 ml of p-nitrophenyl-β-D-mannopyranoside aqueous solution of 1. After adding Oml to inactivate the enzyme, add water to make up to 3ml. The degree of coloration is measured using ultraviolet light (wavelength 4
20 nm) with 1 μmol/ml p-nitrophenol as a standard.

The m-position of enzyme activity is expressed as one unit, which is the amount of enzyme that liberates 1 μmol of p-nitrophenol per minute under the above-mentioned conditions.

In addition to the above, the physicochemical and enzymatic properties of β-mannosidase obtained by the method of the present invention are as follows:
The molecular weights of the enzymes obtained from 20 and AS-440 are 68.000±3.000 and 63.000±, respectively.
It is 3.000. Incidentally, these molecular weights were 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 2 β-mannosidase action β-D-mannan is contained in relatively large amounts in various plants, and is used in various fields as a thickener and thickener, either as is or after chemical modification. is used industrially as a food material. By the way, when this β-D-mannan is used, for example, as a thickening agent in the textile industry, it is removed after a predetermined processing process, 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 and difficult to use on a large scale industrially as described above.

Furthermore, the extraction process of β-D-mannan is generally carried out in an alkaline environment, and in such cases it is advantageous to use an enzyme that is more stable than known enzymes in alkaline conditions and has a higher optimum pH. be. In other words, with conventional enzymes that have an optimum pH on the acidic side, the pH of the extract is reduced using a neutralizing agent before the decomposition reaction.
It is necessary to perform H adjustment, which not only complicates the process but also increases cost.

Therefore, there is a need to develop legacy methods, and there is a need to develop enzymes with a higher pH optimum than known enzymes.

According to the specific microorganisms discovered by the present inventors, the above β-
It is possible to obtain a large amount of an enzyme that satisfies all the requirements related to the decomposition of D-mannan, and various conventional problems can be solved at once.

That is, the problems of mass production and cost can be overcome by using the parent strain newly discovered according to the present invention, which can be obtained in large quantities by a simple method and is inexpensive. Therefore, large-scale industrial use becomes possible.

In addition, the optimal p in the mannan decomposition reaction of the obtained enzyme is
Since H is near neutrality, it is possible to immediately proceed to the next decomposition reaction after performing a slight pH adjustment after the mannan extraction process. Therefore, the disassembly operation is simplified and economically advantageous.

Example Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 Alkaliphilic bacterium Bacillus AS-420 strain (F[ERM
P-8859) in a 500ml Erlenmeyer flask,
Guar gum 0.5%, cornstarch liquor 5%, ammonium sulfate 0.1%, K2HP 0.0.1%, IgsO<・7
Inoculate 100ml of culture solution (pH 9.5) containing 0.02% 8200.02% and 25% soda skin, and incubate at 37℃ for 48 hours.
The cells were cultured with shaking for 20 hours, p, m, and 20 hours. Then, the culture solution was subjected to 12. ooor, p, ml, 0℃
Collect the bacterial cells by centrifugation for 30 minutes at
After suspending in +t+M phosphate buffer, the bacterial cells were disrupted using an ultrasonic disruptor. Next, this bacterial cell disruption solution was heated to 12.0OOr,
p, m.

Centrifugation was performed at 0°C for 30 minutes, and the resulting supernatant was β-
Mannosidase activity was measured and found to be 16 units/ml.

Example 2 To alkalophilic bacterium Bacillus strain S-044 (ORIJ
P-8860) in a 500ml Erlenmeyer flask, 2% palm oil residue, 1% soybean residue, 30.2% KNO, 2% N
a2 HP 040.1%, MgSO3・7H200,
100ml of culture solution containing 0.02% and 0.5% soda
1 (pH 10,0) and incubated at 40°C for 50 hours 25
The cells were cultured with shaking at 0r, pom. Next, this cell suspension was incubated at 12.0 OOr, p, m, at 0°C for 30 minutes.
The β-mannosidase activity of the supernatant obtained after centrifugation for one minute was measured and found to be 38 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 an 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 the target decomposition product 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 (4)

[Claims] (1) A novel β-mannosidase having the following physical and chemical properties. (a) Action: Hydrolyzes β-mannoside 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-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 conditions of pH 6.5 and heating for 30 minutes. (e) Range of optimum temperature for action: The optimum temperature for action is around 50°C. (f) Inactivation conditions: pH 5.0 and 1 under treatment conditions of 40°C and 30 minutes.
At 0, it is completely inactivated. Furthermore, when treated at pH 6.5 for 30 minutes, the activity is completely inactivated at 55°C. (g) Molecular weight by gel filtration method: 63,000 to 68,000 (2) The following physical and chemical properties: (a) Action: Hydrolyzes β-mannoside 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-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 conditions of pH 6.5 and heating for 30 minutes. (e) Range of optimum temperature for action: The optimum temperature for action is around 50°C. (f) Inactivation conditions: pH 5.0 and 1 under treatment conditions of 40°C and 30 minutes.
At 0, it is completely inactivated. Furthermore, when treated at pH 6.5 for 30 minutes, the activity 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 63,000 to 68,000 by gel filtration method and has an optimum pH for growth on the alkaline side is cultured, and the β-mannosidase is The above-described novel intracellular β
- A method for producing mannosidase. (3) The method for producing intracellular β-mannosidase according to claim 2, wherein the culturing is carried out aerobically at a temperature within the range of 30 to 45°C. (4) Claim 2 or 3, characterized in that the pH of the culture solution is within the range of 7.5 to 11.5.
A method for producing intracellular β-mannosidase as described in 2.