JPH0567279B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a new method for producing carbohydrates containing fructooligosaccharides, and more specifically,
A new extracellular fructosyltransferase is produced and accumulated in the culture solution by culturing an alkalophilic microorganism that belongs to the genus and has an optimal pH for growth between neutral and alkaline. The present invention relates to a method for producing a saccharide containing a fructo-oligosaccharide, which is characterized by allowing extracellular fructosyltransferase to act on a saccharide substrate having a β-D-fructofuranoside bond. Fructosyltransferase as used herein refers to a substrate that has a β-(2→1) bond between fructose and glucose, such as sucrose, and after cleaving the bond, converts the fructose into sucrose, 1-kestose, or nystose. It is an enzyme that has the effect of transferring to fructose residues such as and producing oligofructan or fructan with a higher molecular weight.
In plant tissues such as asparagus and Jerusalem artichoke, or Aureobasidium pullulans, Aspergillus.
It is known to exist as an intracellular enzyme in bacteria such as Aspergillus niger and yeast (Enzyme Handbook, p. 806, Asakura Shoten; U.S. Patent No.
No. 4309505; Japanese Patent Publication No. 166981/1983). When such fructosyltransferase acts on, for example, sucrose, as a result of the transglycosylation reaction, in addition to by-produced glucose and fructose, 1-kestose, nystose, fructosylnystose, etc., in which several molecules of fructose are transferred to sucrose, are produced. Not only can a sweet composition containing fructooligosaccharide and unreacted sucrose be obtained, but also a high fructose-containing product can be easily produced from sucrose by using the transferase and glucose isomerase in combination with sucrose. is known (US Pat. No. 4317.880). In addition, fructooligosaccharide, in which a few molecules of fructose are transferred to seuucrose, does not serve as a substrate for dextran eucrase produced by the caries-causing bacterium Streptococcus miutans, so it is not only non-cariogenic and does not cause dental caries. ,
It is also known as a substance that promotes the growth of Bifidobacterium, a useful intestinal bacterium, and is considered to be a very useful substance industrially. (Special Publication No. 59-53834). Conventionally, these fructooligosaccharides have been obtained mainly by aerobically cultivating Aureobasidium pullulans and allowing fructosyltransferase, which is produced and accumulated within the bacterial cells, to act on a high concentration of sucrose. (BIO
INDUSTRY, Volume 1, Issue 6, Pages 5-13, 1984
Since the enzyme is an intracellular enzyme, not only is the extraction and purification procedure from the microbial cells complicated, but also the productivity of the enzyme cannot be said to be very high. There is also a method of using the bacterial body itself as an enzyme source without extracting the enzyme from the bacterial body, but secondary reactions occur due to other enzyme groups present within the bacterial body.
In addition to reducing the yield of the desired fructooligosaccharides, the added microbial cells (enzymes) may cause clogging in the filtration and purification process of the transfer reaction product, making it virtually difficult to purify the sugar solution. I was getting used to it. Therefore, when using intracellular enzymes for the production of fructooligosaccharides, it was necessary to immobilize the cells using the entrapment method. The present inventors conducted an intensive search for microorganisms that have the ability to produce and accumulate fructosyltransferase in culture filtrate, which is stable to pH and temperature and is easy to purify.
It has been shown that some alkalophilic bacteria belonging to the genus Bacillus, which have an optimal pH for growth between neutral and alkaline conditions, produce and accumulate a significant amount of novel extracellular fructosyltransferase in the culture solution. They discovered that a significant amount of fructooligosaccharides could be produced by allowing the enzyme to act on sucrose, and completed the present invention. The novel extracellular fructosyltransferase used in the present invention can be obtained by culturing several new strains belonging to the alkalophilic Bacillus genus, which were newly searched and isolated from the natural world by the present inventors. It is something that is generated and stored inside. These new strains have been deposited as FERM P-8254, P-8255, and P-8256, respectively, at the Institute of Microbial Technology, Agency of Industrial Science and Technology. Table 1 shows the mycological properties of these strains.

【table】

_[ Table] ₊ : Grows -: Does not grow
Using a liquid medium containing _0.02 % MgSO4.7H2O and ₁ % _Na2CO3 , fructosyltransferase is produced in the culture supernatant by culturing aerobically at _35-55 ℃ for 20-50 hours. Produced and accumulated inside (outside the bacterial body). The medium used in this medium is not limited to the above-mentioned medium, and it is also possible to use cornstap liquor, amino acid solution, or inorganic substances that are substantially inexpensively available. Furthermore, the PH adjuster for the medium is not limited to soda carbonate; cultivation is possible with any substance that can substantially adjust the PH of the medium from neutral to alkaline, such as bicarbonate of soda, ammonium carbonate, potassium carbonate, and caustic soda. be. It is economical to use the supernatant (crude enzyme solution) obtained by removing the bacterial cells in the main culture solution by centrifugation or filtration for the transglycosylation reaction. Enzymes purified by common enzyme purification methods such as solvent precipitation, ultrafiltration, gel filtration, and ion exchange resins can also be used. Furthermore, immobilized enzymes prepared by various methods such as adsorption and entrapment methods can also be used. Next, the general characteristics of the novel extracellular fructosyltransferase used in the present invention will be described. (b) Action: Cutting the β-D-fructofuranoside bond of sugars having a β-D-fructofuranoside bond such as seuculose, 1-kestose, nystose, and raffinose, and converting the generated fructose into seuucrose, 1-kestose, and nystose. , transfers to sugars such as raffinose. (b) Substrate specificity It is often used for sucrose, 1-kestose, nystose, and ruffinose, but does not act on turanose, maltulose, and trehalose. (c) Optimal PH and stable PH range ●Optimum PH S-432 bacteria: 5.5 to 6.5 S-442 bacteria: 5.0 to 6.0 S-452 bacteria: 5.5 to 6.5 ●Stable PH range (processed at 55℃ for 30 minutes) S-432 bacteria S-442 bacteria S-452 bacteria 5.0-8.5 (d) Range of suitable temperature for action S-432 bacteria: 50-60℃ S-442 bacteria: 50-60℃ S-452 bacteria: 45-55℃ ( e) Conditions for inactivation depending on PH, temperature, etc. Under treatment conditions of 55℃ and 30 minutes, PH3.5 and 10.5
becomes completely inactive. It is completely inactivated at 70°C for 15 minutes at pH 5.5. (f) Inhibitors and stabilizers Inactivated by mercury, copper, and EDTA. Stabilized by calcium. (e) Molecular weight (gel filtration method) S-432 bacteria: 80000±10000 S-442 bacteria: 150000±10000 S-452 bacteria: 115000±10000 A comparison with transferases is shown in Table 2.

[Table] The method for measuring and displaying the activity of fructosyltransferase is as follows. 5% dissolved in 0.1M acetate buffer (PH5.5)
Enzyme solution in 0.5ml of sucrose solution (W/V)
Mix 0.1 ml, react at 55°C for 10 minutes, and then heat in a boiling water bath for 10 minutes to inactivate the enzyme. Reducing saccharides produced by transglycosylation reactions are processed using the Somogyi-Nelson method [Method in Carbohydrate Chemistry].
Chemistry, Volume 1, Page 386, 1962]. Here, the unit of enzyme is 1 minute under the above conditions.
The amount of enzyme that produces reducing sugar equivalent to 1 μmole of glucose is expressed as 1 unit. Furthermore, the fructooligosaccharide obtained by allowing the enzyme to act on sucrose was prepared using a Sensyu Pack NH ₂ -1251(H) column (4.6 x 250 mm) (manufactured by Sensyu Kagaku Co., Ltd.) in a mixture of acetonitrile:water (75:25). High performance liquid chromatography (HPLC) method using solvent system or TSK gel G2000PW
It was quantified by HPLC using a column (21.5 x 600 mm) (manufactured by Toyo Soda) and using water as a developing solvent. Next, for the production of fructooligosaccharides by the method of the present invention, it is preferable to have a high substrate concentration from the viewpoint of enzyme specificity and economy, and the concentration is 50 to 70% (W/
V), preferably around 6% (W/V). Further, the reaction temperature is preferably 50° C. or higher in order to prevent microbial contamination during the process. In addition, fructooligosaccharides produced by transglycosylation reactions are commonly used by those skilled in the art for the separation and purification of sugars, such as Diaion FRK-01 and Diaion FRK-
It can be easily isolated by column chromatography using a cation exchange resin such as 131 (manufactured by Mitsubishi Chemical Industries, Ltd.) or a gel filtration resin such as Toyo Pearl HW-40 (manufactured by Toyo Soda). The present invention will be explained in more detail below with reference to Examples. Example 1 Alkaliphilic bacterium Bacillus No.-S-432 strain (FERM P-8254) was grown in a 1-volume Erlenmeyer flask with 5% sucrose, 0.5% yeast extract, 0.5% polypeptone, 0.1% K ₂ HPO ₄ , MgSO ₄・
300 ml medium containing 0.02% 7H ₂ O and 1% soda carbonate
(PH10.3), cultured at 37℃ for 48 hours with rotational shaking at 250 rpm, and centrifuged the culture solution to yield 27.5 units/
ml of crude enzyme was obtained. 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 280 units/ml of enzyme. The fructosyltransferase thus obtained was dissolved in 60% (W/V) sucrose solution.
Two units per 1 g of sucrose were added to 10 ml (sucrose: 6 g), and the mixture was reacted at 55° C. and pH 6.0 for 48 hours. As a result of quantifying the obtained carbohydrates by HPLC method, fructose was 17.4% and glucose was 32.5%.
%, Syutalose 6.3%, Fructooligosaccharide
It was 43.8%. Example 2 The alkaliphilic bacterium Bacillus No.-S-442 strain (FERM) was grown in the medium composition described in Example 1, using medium 300 containing 0.25% sodium bicarbonate instead of sodium carbonate.
ml (PH8.6) and cultured with shaking under the same conditions as in Example 1 to obtain a crude enzyme solution of 20.3 units/ml. The enzyme was concentrated twice to obtain 200 units/ml of enzyme. 0.5 ml (100 units) of the fructosyltransferase thus obtained was equilibrated with 10 M acetate buffer (PH5.5) and 5 g (wet weight) of anion exchange resin Diaion 1RA904 (Mitsubishi Chemical Industries, Ltd.) made)
was adsorbed to. Next, the same resin was packed into a column kept at 55°C, and thoroughly washed with the same buffer solution.
The adsorption rate of the enzyme to the resin was approximately 90%, and the activity expression rate was approximately 65%. Then, 50% (W/
V) A sucrose solution (PH5.5) was passed through at SV0.2, and the resulting sugar solution was measured by HPLC, and the result was 18.1% fructose, 35.9% glucose, 7.5% sucrose, and 38.5% fructooligosaccharide. . Example 3 The alkaliphilic bacterium Bacillus No.-S-452 strain (FERM P-8256) was inoculated into the medium composition described in Example 1, but the pH was adjusted to 7.5 with caustic soda instead of sodium carbonate. A shaking culture was performed under the same conditions as in Example 1 to obtain a crude enzyme solution containing 24.8 units/ml. Five times the amount of cooled acetone was added to the crude enzyme solution, and the mixture was left in a cold room overnight. The resulting precipitate was collected by centrifugation and dried under reduced pressure to obtain 2500 units/g of oxygen powder. Add 1 g of the above enzyme powder (approximately 4.2 units/g sucrose) to 50% (W/V) sucrose solution 1 (sucrose: 600 g) adjusted to pH 6.0.
was added and kept at 50°C. The reaction mixture was batch-filtered at 50° C. for 5 hours in an ultrafiltration device (Pellicon Lab Cassette, manufactured by Nippon Millipore Limited) equipped with an ultrafiltration membrane having an average molecular weight cut off of 30,000.
Then, the sucrose solution was charged at a flow rate of 10 ml/min, and at the same time the filtrate (permeate) was added at the same rate.
was extracted and its sugar composition was measured by HPLC method, and the result was 17.9% fructose, 34.2% glucose, 8.5% sucrose, and 39.4% fructooligosaccharide.

Claims (1)

[Scope of Claims] 1. Extracellular fructosyltransferase obtained by culturing an alkalophilic microorganism belonging to the genus Bacillus and having an optimum pH for growth from neutral to alkaline, 1. A method for producing a saccharide containing a fructooligosaccharide, which is characterized by acting on a saccharide substrate having a sid bond. 2. The method for producing a saccharide containing a fructooligosaccharide according to claim 1, wherein the substrate is sucrose.