JPH0581235B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel extracellular fructosyltransferase and a method for producing the same, and more specifically, the present invention relates to a novel extracellular fructosyltransferase and a method for producing the same. Generates and accumulates extracellular enzymes in the culture solution,
The present invention relates to a novel fructosyltransferase obtained by collecting this and a method for producing the same. 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.
It is known to exist in plant tissues such as asparagus and Jerusalem artichoke, or as an enzyme within bacteria such as Aureobasidium pullulans (Enzyme Handbook, p. 806, Asakura Shoten; U.S. Patent No. 4309505) issue;
(Japanese Patent Publication No. 166981/1983). When such fructosyltransferase acts on sucrose, in addition to the glucose and fructose produced as by-products as a result of the transglycosylation reaction, fructo-oligosaccharides such as 1-kestose, nystose, and fructosylnystose, in which several molecules of fructose are transferred to sucrose, are produced. Not only can a sweet composition containing unreacted sucrose be obtained, but also a high fructose-containing product can be easily produced from sucrose by using the fructosyltransferase and glucose isomerase in combination to act on sucrose. is known (US Pat. No. 4,317,880). In addition, fructooligosaccharide, in which several 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 has no caries properties 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.
issue). Conventionally, these fructooligosaccharides have been obtained mainly by aerobically culturing Aureobasidium pullulans and allowing fructosyltransferase, which is produced and accumulated within the bacterial cells, to act on a high concentration of sucrose. However, (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 this may cause secondary reactions by other enzymes present within the bacterial body, resulting in the production of the desired fructooligosaccharide. Not only may the yield be reduced, but the added bacterial cells (enzymes)
This causes clogging in the filtration and purification process of the rearrangement reaction product, making purification of the sugar solution substantially difficult. 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 several types of alkaliphilic 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. Heading: This completes the present invention. The novel extracellular fructosyltransferase-producing strains used in the present invention were searched and isolated from the natural world by the present inventors, and these strains were published in Bergey's Manual of Determinative Bacteriology.
of Determinative Bacteriology (8th edition) and The Genus Bacillus
When identified according to (USDept, Agriculture),
Both are aerobic sporobacilli and are motile.
It was clear that it belonged to the genus Bacillus because of its positive Gram stain and catalase test, but it was thought to be a new strain taxonomically different from known neutral bacteria because it grows well in alkaline conditions around pH 10. . Table 1 shows the mycological properties of the three isolated extracellular fructosyltransferase producing bacteria.

[Table] +; Grows -; Does not grow
It has been deposited as 8256. Next, a novel method for producing extracellular fructosyltransferase will be described. First, the enzyme-producing bacteria are mixed with, for example, 5% sucrose and 0.5% polypeptone.
%, yeast extract 0.5%, K ₂ HPO ₄ 0.1%, MgSO ₄ .
By culturing aerobically at 35 to 55°C for 20 to 50 hours using a liquid medium containing 0.02% 7H ₂ O and 1% sodium carbonate, fructosyltransferase is present in the culture supernatant (outside the cells). is generated and stored. 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. Next, an example of the method for purifying fructosyltransferase of the present invention is shown below. Alkaliphilic Bacillus No.S-442 (FERM P-8255) was inoculated into the above alkaline medium and cultured aerobically at 37°C for 48 hours. The resulting culture was centrifuged at 10,000 rpm for 10 minutes at 0°C. The cells were separated and the bacterial cells were removed to obtain 10 liters of supernatant. Then, the supernatant liquid was concentrated about 10 times using an ultrafiltration membrane with an average molecular weight cut off of 10,000, ammonium sulfate (75% saturation) was added, and the mixture was left in a cold room overnight. The resulting enzyme precipitate was collected by centrifugation, dissolved in 10 mM acetate buffer (PH6.0), and then dialyzed against the same buffer overnight. The precipitate generated after dialysis was removed by centrifugation, and the supernatant was diluted with 10mM acetate buffer (PH
The enzyme was adsorbed onto a DEAE-Toyopearl-650M column equilibrated with 6.0) and eluted using the concentration gradient method using the same buffer containing 0 to 0.8 mol NaCl.
The eluted active fractions were collected and concentrated using an ultrafiltration membrane, and then dialyzed using 10mM acetate buffer (PH6.0). The dialyzed enzyme was then loaded into a Sephacryl S-200 gel filtration column equilibrated with the above buffer containing 0.1 mol NaCl, and eluted with the above buffer.
After concentrating the active fraction, it was rechromatographed on a Sephacryl S-200 column and subjected to polyacrylamide gel disc electrophoresis (gel concentration 7.5%, PH4.0).
185 mg of a single enzyme preparation was obtained. The activity yield was 24%. In addition, No-S- according to the present invention
Bacterium 432 (FERMP-8254) and Bacterium No-S-452 (FERM P-8256) can be purified in the same manner except for changing the gel filtration resin as appropriate. 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 boiling water for 10 minutes to inactivate the enzyme. After that, the reducing sugars produced in the transglycosylation reaction 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. Next, the general characteristics of fructosyltransferase obtained by the present invention will be described. (b) Action: The β-D-fructofuranoside bond of sugars having β-D-fructofuranoside bonds such as seuculose, 1-kestose, nystose, and raffinose is cleaved, and the generated fructose is converted into seuucrose, 1-kestose, and raffinose. Transfers to sugars such as nystose and raffinose. (b) Substrate specificity: acts well on seuucrose, 1-kestose, nystose, ruffinose, but not on turanose,
It has no effect on multiulose and trehalose. (c) Optimal PH and stable PH range: - Optimal 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 due to PH, temperature, etc. Under treatment conditions of 55°C and 30 minutes, it is completely inactivated at pH 3.5 and 10.5. 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 present invention will be explained in more detail with reference to Examples below. Example 1 Alkaliphilic bacterium Bacillus No-S-432 strain (FERM P-8254) was grown in a 1 L Erlenmeyer flask with 5% sucrose, 0.5% yeast extract, 0.5% polypeptone, 0.1% K ₂ HPO ₄ , MgSO ₄ . 7H ₂ O0.02
% and 300 ml of medium containing 1% soda (PH10.3)
The cells were inoculated and cultured at 37°C for 48 hours with rotational shaking at 250 rpm, and the culture solution was centrifuged to obtain a crude enzyme solution containing 27.5 units/ml. Example 2 The alkaliphilic bacterium Bacillus No-S-442 strain (FERM P-8255) was inoculated into 300 ml of a medium (PH8.6) containing 0.25% sodium bicarbonate in place of the sodium carbonate in the medium composition described in Example 1. A shaking culture was performed under the same conditions as in Example 1 to obtain a crude enzyme solution containing 20.3 units/ml. Example 3 The alkaliphilic bacterium Bacillus No-S-452 strain (FERM P-8256) was inoculated into 300 ml of the medium composition described in Example 1, adjusted to pH 7.5 with caustic soda instead of soda 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.

Claims (1)

[Scope of Claims] 1. A novel extracellular fructosyltransferer obtained by culturing an alkaliphilic microorganism belonging to the genus Bacillus and having an optimum pH for growth from neutral to alkaline, and having the following physicochemical properties: Ze. (a) Action: It cleaves the β-D-fructofuranoside bond of sugars having a β-D-fructofuranoside bond, such as seuucrose, 1-kestose, ruffinose, etc., and transfers the produced fructose to the sugar. (b) Substrate specificity It acts well on seuucrose, 1-kestose, nystose, and ruffinose, but not on turanose, maltulose, and trehalose. (c) Optimal PH and stable PH range The optimal PH is 5.0 to 7.0, and is stable at PH 5.0 to 8.5 under heating conditions at 55° C. for 30 minutes. (d) Range of optimal temperature for action The optimal temperature for action is around 55°C. (e) Conditions for inactivation due to PH, temperature, etc. Under treatment conditions of 55°C and 30 minutes, it is completely inactivated at pH 3.5 and 10.5. At pH 5.5 and 15 minutes, it is completely inactivated at 70℃. (f) Inhibitors and stabilizers Inactivated by mercury, copper, and EDTA. Stabilized by calcium. (G) Molecular weight: 70,000 to 160,000 (gel filtration method) 2. A novel fructosyltransferase production ability that belongs to the genus Bacillus, has an optimal pH for growth from neutral to alkaline, and has the following physical and chemical properties. 1. A method for producing a novel extracellular fructosyltransferase, which comprises culturing a microorganism having the above-mentioned fructosyltransferase, producing and accumulating the fructosyltransferase in a culture solution, and collecting the same. (a) Action: It cleaves the β-D-fructofuranoside bond of sugars having a β-D-fructofuranoside bond, such as seuucrose, 1-kestose, ruffinose, etc., and transfers the produced fructose to the sugar. (b) Substrate specificity It acts well on seuucrose, 1-kestose, nystose, and ruffinose, but not on turanose, maltulose, and trehalose. (c) Optimal PH and stable PH range The optimal PH is 5.0 to 7.0, and is stable at PH 5.0 to 8.5 under heating conditions at 55° C. for 30 minutes. (d) Range of optimal temperature for action The optimal temperature for action is around 55°C. (e) Conditions for inactivation due to PH, temperature, etc. Under treatment conditions of 55°C and 30 minutes, it is completely inactivated at pH 3.5 and 10.5. At pH 5.5 and 15 minutes, it is completely inactivated at 70℃. (f) Inhibitors and stabilizers Inactivated by mercury, copper, and EDTA. Stabilized by calcium. (g) Molecular weight 70,000 to 160,000 (gel filtration method).