JPH0523183A - New alpha-galactosidase and production of sugar using the same - Google Patents

Abstract

PURPOSE:To provide the subject new enzyme which is an extracellular secretion enzyme produced by Bacillus stearothermophilus JD-72 strain and capable of producing sugars such as mannooligo-saccharide and mannose from galactomannan in high efficiency. CONSTITUTION:The objective new enzyme is produced by culturing Bacillus stearothermophilus JD-72 strain (FERM P-12274), collecting the product secreted from the cell into the cultured product and purifying the substance. The enzyme has the following properties. Specifically hydrolyzing the galactopyranoside bond on the side chain of a heteropolysaccharide having galactosyl group on the side chain to produce galactose, having an optimum pH of 6.0, stable at pH6-8 under the heating condition of 45 deg.C and 20min, having an optimum work temperature of about 55 deg.C, completely inactivated at pH3.0 and 10.0 under the condition of 45 deg.C and 20min, completely inactivated at 70 deg.C by the treatment at pH7.0 for 20min, having an isoelectric point of 5.6 and a molecular weight of about 80,000 (SDS-PAGE method) and capable of producing sugars such as mannose and galactose from galactomannan.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel α-galactosidase and a method for producing saccharides such as mannooligosaccharides, mannose and galactose from galactomannan using this enzyme.

[0002]

2. Description of the Related Art In recent years, various oligosaccharides have been attracting attention as functional foods having a low cariogenic property and having functions such as improving the intestinal flora. Among these, β-galacto-oligosaccharides produced by utilizing the transfer reaction of β-galactosidase are already on the market. This is because β-galactosidase can be industrially easily, efficiently, and inexpensively produced.

On the other hand, galactomannan is obtained as a mucilage contained in the seeds of carob and guar, that is, locust bean gum, guar gum, etc., and is present in a relatively large amount in the natural world. This galactomannan is used as a starch, a thickener, a food material, etc., as is the case with starch, or after being subjected to a chemical modification treatment, such as fiber, cosmetics,
It is widely used in various fields such as food and agricultural chemicals.

By the way, if an enzyme that efficiently hydrolyzes galactomannan is obtained, it can be decomposed into sugars such as mannooligosaccharides, mannose, and galactose, and these sugars can be used for products with higher added value. Yes, and after using it as the galactomannan itself,
It can also be applied to the field of disassembling and removing this.

In order to meet such a purpose, in recent years, β-mannanase and β-mannosidase have been developed and are about to be industrialized (JP-A-63-56289, JP-A-63-3).
6779, JP-A-63-49093).

However, in galactomannan, mannose is attached to the main chain by β-1,4, and glassose is α-1,4.
As it has a structure with attached side chains, β
-When mannanase or β-mannosidase is allowed to act, the reaction is interrupted in the vicinity of the side chain, resulting in degradation efficiency,
It was not satisfactory from an economical point of view.

From this point of view, the inventors of the present invention, if galactomannan is allowed to act with an enzyme capable of releasing galactose in the side chain without substantially degrading the main chain of mannan, the mannooligosaccharides. We thought that sugars, mannose, galactose and other sugars could be efficiently collected and used.

As such an enzyme, α-galactosidase can be considered. α-Galactosidase is an enzyme that acts on α-D-galactoside such as melibiose, raffinose, stachyose, and hydrolyzes it to release galactose.

The α-galactosidases known so far are derived from plants, bacteria, yeast and higher animal organs. Of these, α-galactosidase derived from filamentous fungi, actinomycetes, and bacteria has been conventionally produced in-house in the sugar beet sugar industry and used for decomposing raffinose contained in the sugar solution extracted from sugar beet.

Further, as will be described later, some reports have been made on strains belonging to Bacillus stearothermophilus and having the ability to produce α-galactosidase.

[0011]

However, all of the above-mentioned α-galactosidases have low α-D-galactosidase titers, and many of them are complicated in culture method and purification method, and should be produced industrially at low cost. Has the problem that it is difficult to use, and therefore its use has only been used for research purposes outside of the beet sugar industry.

Most of the strains belonging to Bacillus stearothermophilus and capable of producing α-galactosidase are those which produce α-galactosidase in the cells, and the enzyme can be easily produced from the culture. It could not be separated and purified and was not suitable for industrial use.

Therefore, an object of the present invention is to produce a novel extracellular secretory type α-galactosidase which can be industrially advantageously produced, and to efficiently use this enzyme to efficiently produce saccharides such as mannooligosaccharides, mannose and galactose from galactomannan. To provide a manufacturing method.

[0014]

In order to achieve the above-mentioned object, the present inventors have widely used the natural world to obtain microorganisms which produce industrially advantageous exocrine secretory and highly potent α-galactosidase. As a result of the search, it was found that a certain microorganism belonging to the genus Bacillus collected from the soil near the source of Hatage Onsen, Kannan Town, Shizuoka Prefecture, produces a novel α-galactosidase with the above requirements in a mass-producible manner. The invention was completed.

That is, the novel α-galactosidase of the present invention is characterized by having the following physicochemical properties.

(A) Action: An oligosaccharide such as melibiose, raffinose and stachyose, and a galactopyranoside bond present in the side chain of a heteropolysaccharide having a galactosyl group in the side chain such as galactomannan are specifically hydrolyzed, Produces galactose.

(B) Substrate specificity: It completely decomposes melibiose and acts on oligosaccharides containing galactose at the non-reducing end such as raffinose and stachyose to release galactose. α-D-galactopyranoside of p-nitrophenyl-glucoside can be used as a substrate, but β-D-galactopyranoside, α-L-arabinopyranoside, α- and β-D-glucoside, α- and β-D-xyloside, α
-And β-L-fucoside, β-D-fucoside, α- and β-D-mannoside cannot serve as substrates.

(C) Optimum pH and stable pH range: The optimum pH is 6.
It is 0, and it is stable within the range of pH 6 to 8 under the heating condition of 45 ° C. for 20 minutes.

(D) Stability to temperature: It is stable up to 45 ° C. under heating conditions of pH 7.0 and 20 minutes.

(E) Optimum working temperature range: The optimum working temperature is around 55 ° C.

(F) Deactivation condition: Under the treatment condition of 45 ° C. for 20 minutes, it is completely deactivated at pH 3.0 and 10.0. Also, pH 7.
It is completely inactivated at 70 ° C for 0 or 20 minutes.

(G) Inhibition and activation: Inhibited by mercuric chloride and lead, zinc, copper, ferrous iron, p-chloromercuribenzoate, N-bromosuccinimide.

(H) Isoelectric point Isoelectric point by electrophoresis: 5.
6

(I) Molecular weight by SDS-polyacrylamide gel electrophoresis: about 80,000

The method for producing saccharides of the present invention is characterized in that galactomannan is caused to act with the above-mentioned α-galactosidase and β-mannanase and / or β-mannosidase.

The present invention will be described in more detail below with reference to preferred embodiments.

The α-galactosidase of the present invention is a novel microorganism newly isolated from the soil near the source of Hatage Onsen, Kannan Town, Shizuoka Prefecture by the present inventors, Bacillus
Produced by Stearothermophilus strain JD-72.
This strain has the ability to produce α-galactosidase extracellularly.

This Bacillus stearothermophilus J
The mycological properties of the D-72 strain are as follows.

[0029]   1. Morphological properties     Shape bacillus     Size 0.8-1.2 x 3.0-4.0     Motile (periflagellated)     Gram stain positive     Sporangia not swelling or slightly swelling     Spore shape Elliptical and terminal     Spore size 0.8-1.0 x 1.7-2.0

[0030]   2. Growth on various media     Meat broth agar plate culture It appears white and forms a circular colony. table                                       The ridge of the surface is flat. The periphery is filamentous.     Meat broth agar slope culture Grows in spread form     Broth gelatin puncture culture not liquefied     Glucose broth liquid medium     Anaerobic growth −     Generation of Anaerobic Gas from Nitrate −     5.0% salt broth liquid medium −     7.5% Salt broth liquid medium −

3. Biochemical properties Hydrolysis of gelatin and casein − Hydrolysis of starch + Use of citric acid − Reduction of nitrate + VP-Test- Generation of indole − Hydrogen sulfide + Use of inorganic nitrogen source ＋ Dye formation − Urease + Oxidase Catalase + Attitude toward oxygen

4. Growth pH and temperature Growth pH 5.0-8.0 Growth temperature 30-70 ℃

5. Utilization of sugar L-arabinose, L-xylose, D-glucose,
It assimilates D-mannose, D-fructose, D-galactose, maltose, sucrose, lactose, melibiose, raffinose, starch and glycerin. In addition, L-arabinose, L-xylose, D-glucose, D-mannose, D-fructose, and D-galactose produce an acid, and no gas is produced.

[0034] 6. GC content 52.4%

In the above items 2 and 3, + means growth or positive, and − means no growth or negative.

This strain was prepared by the Burgers Manual.
Of systematic bacteriology (Bergey's M
annual of Systematic Bacteriology), First Edition, and the Genus Bacillus: Published by the US Department of Agriculture.
It is an aerobic spore-forming bacterium, has motility, has periflagellates, and is positive for Gram stain.
llus sp.). In addition, since it is positive for catalase and negative for VP reaction and grows at 65 ° C or higher, it can be used for Bacillus stearothermophilus (Bacillus).
stearothermophilus).

On the other hand, as the Bacillus stearothermophilus capable of producing α-galactosidase, the following have been reported so far. SBTF strain "J. Delente et al, Biotech. Bioeng., 16, 1227-1243
(1974) "AT-7 strain" DMPederson et al, Canadian J. Microbiol., 26, 9 "
78-984 (1980) "KVE39 strain" C. Ganter et al, J. Biotechnol., 8, 301-310 (1988)
(ATCC) strain "G. Talbot et al, Appl. Environ. Microbiol., 56, 3
505-3510 (1990) ''

However, the above-mentioned strains are α-
While galactosidase is produced intracellularly, the strains isolated by the present inventors are different in that α-galactosidase is produced extracellularly. The above strains also produce β-mannanase together with α-galactosidase, but the above strains differ in that they do not produce β-mannanase. Since the above strain produces β-mannanase together with α-galactosidase,
When used as a crude enzyme, it is difficult to control the amount used because both enzymes are present at the same time.

From the above examination results, it was determined that the above strain was a new strain belonging to Bacillus stearothermophilus. This strain has been deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as "Bacillus stearothermophilus JD-72", Microorganism Research Institute No. 12274.

The α-galactosidase of the present invention is prepared by culturing the above Bacillus stearothermophilus JD-72 strain,
It can be harvested from this culture.

The medium for culturing is preferably a medium containing a carbon source, a nitrogen source, and if necessary, inorganic salts, trace nutrients and the like.

As the carbon source, galactose, arabinose and lactose are preferable, and in addition to these, corn shrimp seed coat and its alkali extract, guar gum partial decomposition product, copra meal and the like can also be used.

As the nitrogen source, yeast extract, corn steep liquor, defatted soybean powder and the like are preferably used. Needless to say, the organic nitrogen source also serves as a carbon source.

Further, various commonly used inorganic salts, micronutrients and the like can be used as necessary, and examples thereof include magnesium salt, potassium salt, phosphate salt, iron salt and the like. Inorganic salts, vitamins, etc. can be used.

Specific examples of the medium containing the above components include 1% lactose, 2% defatted soybean flour, and 0.2%.
A liquid medium containing K ₂ HPO ₄ and 0.02% MgSO ₄ · 7H ₂ O can be used.

The culture can be carried out by either a batch method or a continuous method.

The medium as described above is adjusted to pH 5.0 to 7.5, preferably 6.5 to 7.0, and then inoculated with Bacillus stearothermophilus JD-72 strain at 37 to 60 ° C., preferably 45 ° C. It is preferable to cultivate aerobically at around temperature for about 72 hours. By culturing in this way, α-galactosidase is produced and accumulated in the culture solution as an extracellular secretory enzyme.

Separation and purification of the produced and accumulated α-galactosidase can be carried out, for example, as follows.

First, the cells in the culture solution are separated into the cells and the filtrate by a known means such as centrifugation or filtration.

The filtrate thus obtained contains α-galactosidase, and this filtrate can be used as it is for the hydrolysis reaction of mannooligosaccharides, the transfer reaction of galactooligosaccharides, etc., which is economical. Is advantageous.

Further, α-galactosidase can be further purified from the above-mentioned filtrate. Examples of the method include salting out with ammonium sulfate, solvent precipitation with ethanol, acetone, isopropanol, etc., ultrafiltration and gel filtration. A general oxygen purification method using an ion exchange resin or the like is adopted.

More specifically, the α-galactosidase of the present invention can be produced by the following method.

Bacillus stearothermophilus JD-72
The strain was inoculated into the above liquid medium and aerobically cultivated at 45 ° C for 72 hours to obtain a culture broth at 4 ° C of 10,000 g × 20
The cells are removed by centrifugation for a minute to obtain a supernatant. Next, ammonium sulphate is added to the supernatant to make it 90% saturated, and the mixture is allowed to stand overnight at 4 ° C. for salting out. The precipitate formed is collected by centrifugation, dissolved in 20 mM phosphate buffer pH 7.0 and dialyzed against the same buffer as above at 4 ° C. overnight.

The precipitate produced by dialysis was removed by centrifugation, and the supernatant was adsorbed on "DEAE-Toyopearl 650M" (trade name, manufactured by Tosoh Corporation) equilibrated with the same phosphate buffer as above. After that, the enzyme is eluted by the same concentration gradient method of a phosphate buffer solution containing 0 to 0.5 M sodium chloride as described above.

The active fractions eluted as described above were collected, and this fraction was concentrated using an ultrafiltration membrane having an average molecular weight cut-off of 10,000,
"Sephacryl S-300HR" (trade name, manufactured by Pharmacia Co., Ltd.) equilibrated with the same phosphate buffer solution containing 0.2 M sodium chloride as described above is filled and eluted. The obtained active fraction was treated again with "DEAE-Toyopearl 650M" and "Sephacryl S-300HR", and then the active fraction was concentrated and then subjected to polyacrylamide gel electrophoresis to form a uniform band. Obtain purified enzyme.

In the present invention, the method for measuring the α-galactosidase activity and the method for displaying the activity are as follows.

1.0 mL of 50 mM phosphate buffer, pH 6.0, containing 2 mM p-nitrophenyl-α-D-galactopyranoside
Mix 50 μL (microliter) of enzyme solution with (mL), react at 50 ° C for 10 minutes, and add 1.0 mL of 0.2M sodium carbonate aqueous solution to inactivate the enzyme.
Stop the reaction. The degree of coloring of the obtained solution is 1 μmol / mL
Using p-nitrophenol as a standard, it is measured by ultraviolet light having a wavelength of 420 nm.

The unit of enzyme activity is:
The amount of enzyme that releases 1 μmol of p-nitrophenol in 1 minute is shown as 1 unit.

The α-galactosidase of the present invention thus obtained has the physicochemical properties as described above,
It was judged to be a novel enzyme, unlike any of the α-galactosidases reported so far. Incidentally, the α-galactosidase of the present invention and the above-mentioned (AT
Table 1 shows a comparison with α-galactosidase produced by the (CC) strain.

[0060]

[Table 1] (In the table, pNP-α-Gal is p-nitrophenyl-α
-D-galactopyranoside is meant. )

Next, in the method for producing saccharides of the present invention, galactomannan is reacted with the above α-galactosidase and β-mannanase and / or mannosidase to produce saccharides such as mannooligosaccharides, mannose and galactose. Is the way to do it.

By allowing α-galactosidase to act on galactomannan, it is possible to release the glassose, which is the side chain of mannose. When β-mannanase is allowed to act, the main chain of mannan is randomly decomposed into several molecular weight units, and mannose is separated from 2 to 9
A molecularly linked mannooligosaccharide is produced. Also, β
-When mannosidase is allowed to act, the main chain of mannan is decomposed one molecule from the end to produce mannose.

Therefore, when it is desired to obtain mannooligosaccharide, it is preferable to allow α-galactosidase and mannanase to act, and to obtain mannose,
It is preferable that α-galactosidase and mannosidase act, or α-galactosidase, mannosidase and mannanase act. All of these enzymes may be added to the reaction system at the same time for reaction, but α-galactosidase may be reacted first, and then β-mannanase and / or mannosidase may be reacted.

In the present invention, as β-mannanase, the enzyme disclosed in JP-A-63-56289 is preferably used. Further, as β-mannosidase, there is disclosed in JP-A-63-63
The enzyme disclosed in No. -36779 is preferably used. The method for preparing these enzymes is described in detail in the above publication, and therefore the description thereof is omitted.

As the substrate galactomannan, various plant-derived substances such as locust bean gum and guar gum can be used. The substrate concentration in the reaction solution is not particularly limited, but it is preferably 1 to 100 mg / mL.

The concentration of each enzyme in the reaction solution was 10 to 1000 U (unit) for α-galactosidase, 100 to 10,000 U for β-mannanase, and β to 1 g of the substrate.
For mannosidases, 100-10000 U is preferred.

The pH of the reaction solution is preferably 5 to 7, the reaction temperature is preferably 40 to 60 ° C., and the reaction time is preferably 1 to 72 hours.

After the enzymatic reaction is carried out in this manner, the mannooligosaccharides can be treated by a conventional method such as decolorization, desalting, fractionation by various columns, concentration and drying.
Sugars such as mannose and galactose can be obtained individually or as a mixture in the form of a concentrate, a powder or the like.

[0069]

Since the α-galactosidase of the present invention is produced outside the cells, it is extremely easy to separate and purify the enzyme.
It is industrially very advantageous in terms of labor and manufacturing cost.
Further, this enzyme is excellent in high temperature stability and has an optimum pH for the enzyme reaction in a substantially neutral region. Therefore, after slightly adjusting the pH of the galactomannan solution extracted with a dilute alkali, It is possible to shift to a decomposition reaction. Furthermore, this enzyme has a high α-D-galactosidase titer, and oligosaccharides such as melibiose, raffinose and stachyose,
Further, since galactopyranoside bond existing in the side chain of a heteropolysaccharide having a galactosyl group in the side chain such as galactomannan is specifically hydrolyzed to produce galactose, it can be applied to the production of various saccharides.

According to the method for producing saccharides of the present invention,
By allowing α-galactosidase to act on galactomannan, galactose in the side chain of galactomannan can be released. Then, together with α-galactosidase, when β-mannanase is allowed to act, the main chain of mannan is randomly decomposed into several molecular weight units to produce manno-oligosaccharide, and when β-mannosidase is allowed to act, mannan The main chain is decomposed from the end one molecule at a time to produce mannose. In this case, α
-Since galactose which is a side chain is released by galactosidase, galactomannan can be efficiently decomposed without steric hindrance of β-mannanase and β-mannosidase.

[0071]

【Example】

Example 1 250 mL of a liquid medium containing lactose 1.0%, yeast extract 1.0%, dipotassium phosphate (anhydrous salt) 0.1%, and magnesium sulfate (heptahydrate) 0.02% was placed in a 2 L (liter) Erlenmeyer flask, and the Bacillus Stearothermophilus JD-72 strain was inoculated and cultured at 45 ° C. for 72 hours at 180 rpm with rotary shaking.

Next, this culture solution was subjected to 10,000 g at 4 ° C.
The cells were removed by centrifugation for 20 minutes, and the culture supernatant was collected. The activity of α-galactosidase in the obtained supernatant was measured and found to be 14.6 units / mL.

Ammonium sulfate was added to the supernatant to 90% saturation, and the mixture was allowed to stand overnight at 4 ° C. to cause precipitation. The precipitate was collected by centrifugation, dissolved in 20 mM phosphate buffer pH 7.0 and dialyzed against the same buffer as above at 4 ° C. overnight.

The precipitate generated by dialysis was removed by centrifugation, and the supernatant was adsorbed on "DEAE-Toyopearl 650M" (trade name, manufactured by Tosoh Corporation) equilibrated with the same phosphate buffer as above. After that, the enzyme was eluted by the same concentration gradient method of a phosphate buffer solution containing 0 to 0.5 M sodium chloride as described above.

The active fractions eluted above were collected, concentrated using an ultrafiltration membrane having an average molecular weight cut off of 10,000, and equilibrated with the same phosphate buffer solution containing 0.2 M sodium chloride as "Sephacryl S". -300HR "(trade name, manufactured by Pharmacia Co., Ltd.) was filled and eluted. The obtained active fractions were again used for "DEAE-Toyopearl 650M" and "Sephacryl S-30".
After treatment with "0 HR", the active fraction was concentrated and then, by polyacrylamide gel electrophoresis, 0.624 mg of an enzyme preparation having a uniform band was obtained. The activity yield was 4.8%.

Example 2 Using the enzyme preparation obtained in Example 1, an experiment on its action was carried out. (1) 1.0% aqueous solution of various polysaccharides (pH 6.0) with different origins of action on various polysaccharides
The purified α-galactosidase obtained in Example 1 was added to 1 mL of 3.
Add 3 units, react at 50 ℃ for 24 hours, and then at 100 ℃
The galactose liberated by heating for 3 minutes was quantified using a lactose / galactose analysis F kit (Boehringer Mannheim). The results are shown in Table 2.

[0077]

[Table 2]

From the results shown in Table 2, this enzyme is capable of specifically hydrolyzing the galactopyranoside bond present in the side chain of a heteropolysaccharide having a galactosyl group in the side chain such as galactomannan to produce galactose. I understand.

(2) Substrate specificity 1 mL of 50 mM sodium phosphate buffer (pH 6.0) containing 2 mM of various p-nitrophenyl derivatives or 10 mM of various sugar solutions
0.16 units of α-galactosidase was added to the
Let react for minutes. After the reaction, the released p-nitrophenol or galactose was quantified and compared. Table 3 shows the results of measurement of various synthetic substrates. In addition, Table 4 shows the results of measuring the reaction rates of those that could be substrates and natural oligosaccharides.

[0080]

[Table 3]

[0081]

[Table 4]

(3) Optimum pH and stable pH range 50 μL of the purified enzyme solution of the present invention was mixed with 2 mM of pNP-α-D-galactopyranoside dissolved in 50 mM buffer of each pH,
The activity was measured by the method described above, and the relative activity with the maximum activity being 100 was determined. The results are shown in the left column of FIG. The buffers used were sodium acetate buffer (~ pH 6.0), sodium phosphate buffer (pH 6.0 ~ 8.0), glycine-NaCl.
-NaOH buffer (pH 8.0-).

Further, the above-mentioned buffer solution was used in the range of pH 4.0 to 10.0, and heat treatment was carried out at 45 ° C. for 20 minutes at each pH, and the residual activity was measured. The relative activity was calculated. The results are shown in the right column of FIG.

(4) Action Stability against temperature range and temperature The activity of the purified enzyme was measured at pH 6.0 at each temperature. The results are shown in the left column of FIG.

The purified enzyme was added at pH of 40 to 80 ° C. to pH
The residual activity after heat treatment for 7.0 and 20 minutes was measured. The results are shown in the right column of FIG.

(5) Inhibition and activation To the purified enzyme solution dissolved in 20 mM MOPS buffer (pH 7.0), chlorides of various metals were added so that the final concentration was 0.01 to 1 mM, and the mixture was kept at 20 ° C. for 20 minutes. After the treatment, the activity was measured, and the relative activity was determined by setting the activity without addition as 100. The results are shown in Table 5.

Similarly, Table 6 shows the measurement results of various enzyme inhibitors.

[0088]

[Table 5]

[0089]

[Table 6]

(6) Isoelectric point The isoelectric point of the α-galactosidase of the present invention was determined by the isoelectric focusing method using Cervalite Precoat (PI 3-10) manufactured by Selva. The result is as shown in FIG. 3, which was 5.6.

(7) SDS-PAG plate manufactured by Daiichi Pure Chemicals (4/20)
The molecular weight was determined by SDS-polyacrylamide gel electrophoresis. The result was as shown in FIG. 4, and was about 80,000.

Example 3 A 1% galactomannan solution (pH 6.0) was added to a commercially available industrial mannanase (Novo, trade name "Gamanase") 5,00.
0 VHCU / g-ds and α-galactosidase of the present invention 100 U /
g-ds was added, and the mixture was reacted at 50 ° C for 48 hours. A small amount of activated carbon was added to the reaction solution, which was boiled and filtered through a 0.45 μm membrane filter to obtain a sample. The sugar composition of the sample was analyzed using a column for high performance liquid chromatography manufactured by Bio-Rad (trade name "Aminex HPX42A").

Locust bean (carob) gum and guar gum are used as the galactomannans, and the results of comparison with and without addition of the α-galactosidase of the present invention are shown in Tables 7 and 8.

[0094]

[Table 7]

[0095]

[Table 8]

As shown in Tables 7 and 8, when the α-galactosidase of the present invention was added to galactomannan together with mannanase, galactose, mannose, and the number of molecules were 2 as compared with the case where α-galactosidase was added.
It can be seen that the production amount of sugars such as mannooligosaccharides of ~ 4 increases.

[0097]

As described above, since the α-galactosidase of the present invention is an extracellular secretory enzyme, it is extremely easy to separate and purify the enzyme and is suitable for industrial production. In addition, this enzyme has high α-D-galactosidase titer, excellent stability at high temperature, and has an optimum pH for enzyme reaction in a nearly neutral region. Thus, it has excellent properties such as being able to shift to the next decomposition reaction after slightly adjusting the pH. Further, according to the method for producing a saccharide of the present invention, galactose, which is a side chain of galactomannan, is released by α-galactosidase, and since mannanase and mannosidase are allowed to act, galactomannan is efficiently decomposed, and a manno-oligosaccharide. The production amount of sugars such as mannose, mannose and galactose can be increased.

[Brief description of drawings]

FIG. 1 is a diagram showing the optimum pH and stable pH range of α-galactosidase of the present invention.

FIG. 2 is a graph showing the stability of the α-galactosidase of the present invention with respect to the optimum working temperature range and temperature.

FIG. 3 is a diagram showing the results of isoelectric point measurement of α-galactosidase of the present invention by an isoelectric focusing method.

FIG. 4 is a diagram showing the results of molecular weight measurement of α-galactosidase of the present invention by SDS-polyacrylamide gel electrophoresis.

Claims (3)

[Claims] 1. A novel α-galactosidase having the following physicochemical properties. (A) Action: The galactopyranoside bond present in the side chain of the heteropolysaccharide having a galactosyl group in the side chain such as oligosaccharides such as melibiose, raffinose, stachyose and galactomannan is specifically hydrolyzed to produce galactose. To do. (B) Substrate specificity: It completely decomposes melibiose and acts on oligosaccharides containing galactose at the non-reducing end such as raffinose and stachyose to release galactose. p-
The α-D-galactopyranoside of nitrophenyl-glucoside can be used as a substrate, but β-D-galactopyranoside, α-L-arabinopyranoside, α- and β-D-glucoside, α- And β-D-xyloside, α- and β-
L-fucoside, β-D-fucoside, α- and β-D-mannoside cannot serve as substrates. (C) Optimum pH and stable pH range: The optimum pH is 6.0,
It is stable in the pH range of 6 to 8 under heating conditions of 20 ° C. and 20 minutes. (D) Stability against temperature: It is stable up to 45 ° C under heating conditions of pH 7.0 and 20 minutes. (E) Optimal working temperature range: Optimal working temperature is around 55 ° C. (F) Deactivation condition: pH 3. under treatment conditions of 45 ° C and 20 minutes.
Complete deactivation at 0 and 10.0. When treated at pH 7.0 for 20 minutes, it is completely inactivated at 70 ° C. (G) Inhibition and activation: mercuric chloride and lead, zinc,
Copper, ferrous iron, p-chloromercuribenzoate, N-
Inhibited by bromosuccinimide. (H) Isoelectric point by isoelectric focusing: 5.6 (b) Molecular weight by SDS-polyacrylamide gel electrophoresis: about 80,000 2. Bacillus stearothermophilus JD-
The novel α-galactosidase according to claim 1, which is an extracellular secretory enzyme produced by strain 72 (Bacillus stearothermophilus JD-72, Microtechnology Research Institute, No. 12274). 3. The galactomannan has the α according to claim 1.
-Galactosidase and β-mannanase and / or β
-A method for producing a saccharide, which comprises reacting with mannosidase.