JP3870264B2 - Method for producing fructose disaccharide and fructose disaccharide synthase - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing fructose disaccharides and fructose disaccharide synthase, and more specifically, a method for producing linear disaccharide levambiose comprising fructose from a plant substrate such as ryegrass, and production of the synthase It is about the law.
[0002]
[Prior art]
Fructose linear disaccharide levanbiose and cyclic disaccharide difructose dianhydride IV are produced by a microbial enzyme using as a raw material a polysaccharide in which a number of fructose polymers called levan are polymerized. These fructose disaccharides have a sweetness that is about half that of sucrose, and are promising saccharides as functional food materials having a calcium absorption promoting effect, an improvement of the intestinal mycelia layer, and an intestinal regulating action (for example, Patent Document 1). And Non-Patent Document 1). Therefore, the development of an enzyme that synthesizes these fructose disaccharides from levan is underway.
[0003]
Examples of microorganisms that produce levanbiose synthase include Bacillus subtilis (for example, see Non-Patent Document 2), Pseudomonas putida (for example, see Non-Patent Document 3), Streptomyces griseus (Streptomyces) griseus) (for example, see Patent Document 2), Streptomyces sp (Streptomyces sp) (for example, see Non-Patent Document 3), and Streptomyces exfoliatus (Streptomyces exfoliatus) ( For example, see Patent Document 3).
[0004]
Examples of microorganisms that produce difructose dianhydride IV synthase include Arthrobacter ureafaciens (see, for example, non-patent documents 4 to 6), Arthrobacter sp. (For example, non-patent documents). 7), and Arthrobacter nicotinovorans (see, for example, Patent Document 4).
[0005]
In addition, a gene for fructose disaccharide synthase is isolated from these microorganisms and is produced in large quantities by genetic recombination techniques using Escherichia coli or yeast (see, for example, Patent Documents 5 and 6) .
[0006]
On the other hand, levan as a raw material is fermentatively produced from sucrose by the action of Serratia bacteria and the like, and a technique for increasing the yield of levan from sucrose is disclosed (for example, see Patent Document 7 ). However, (1) sucrose production by a conventional method, (2) levan production by microbial fermentation, (3) production of fructose disaccharide synthase, (4) production of fructose disaccharide, In this case, since the yield of sucrose for levan is about 20%, the manufacturing technology for these fructose disaccharides has not been established from the viewpoint of cost and efficiency.
[0007]
[Patent Document 1]
JP 2000-204042 A [Patent Document 2]
JP-A-48-99385 [Patent Document 3]
JP-A-5-244974 [Patent Document 4]
Japanese Patent Laid-Open No. 9-154594 [Patent Document 5]
JP-A-11-69978 [Patent Document 6]
JP 2002-17366 A [Patent Document 7]
JP 2002-17390 A [Non-Patent Document 1]
K. Saito et al., Biosci. Biotechnol. Biochem. 64, 1321-1327 (2000)
[Non-Patent Document 2]
Yamamoto S. Et al., Agric. Biol. Chem. 49 (2), 343-350, 1985
[Non-Patent Document 3]
MurakamiH. Et al., Agric. Biol. Chem. 54 (9), 2247-2256, 1990
[Non-Patent Document 4]
K. Tanaka et al. Biochem. 90, 1545-1548 (1981))
[Non-Patent Document 5]
K. Tanaka et al. Biochem. 94, 1569-1578 (1983)
[Non-Patent Document 6]
K. Tanaka et al. Biochem. 97, 1679-1688 (1985)
[Non-Patent Document 7]
Murakami Hiroshi et al., Science and Industry, 67 (9), 365-370 (1993)
[0008]
On the other hand, it is known that fractans in which several molecules of fructose are connected to one molecule of glucose are present in the leaves and stems of grasses or in the straws dried from them. Fructan has a much lower number of fructose polymerization than levan, and although it has a slightly different chemical structure from levan in terms of glucose addition, if it can be used for fructose disaccharide production, the cost of fructose disaccharides Reduction and process simplification are possible.
[0009]
[Problems to be solved by the invention]
An object of the present invention is to provide a method for producing levanbiose, which is a linear fructose disaccharide, by using an inexpensive and simple raw material instead of levan synthesis by microbial fermentation, and a method for producing the synthase thereof. .
[0010]
[Means for Solving the Problems]
As a result of intensive studies by the present inventors to achieve the above-mentioned object, a fructan in which several molecules of fructose are linked to one molecule of glucose exists in the leaves, stems or dried straws of the grass family plants. Although it is known that this fructan has a much smaller number of fructose polymerization than levan, and although it has a slightly different chemical structure from levan in terms of addition of glucose, it is linear by allowing levanbiose synthase to act as a substrate. Synthesize | combined levambiose which is a fructose disaccharide of the shape, and completed this invention in which the said objective is achieved. The present invention was invented and filed on the basis of the results of “Functional analysis and utilization of alkalophilic fermenting microorganisms” conducted by the National Institute of Agricultural Technology as a commissioned work by the Ministry of Education, Culture, Sports, Science and Technology's Science and Technology Promotion Adjustment Cost. Has been reached.
[0011]
That is, the present invention is characterized in that a leaf, stem or cocoon is used as a raw material in the production of fructose disaccharides using a microbial enzyme. Lebanbiose is released in the aqueous solution by reacting by direct contact with levambiose synthase directly on the stem or pod , or by adding water to the raw material and boiling, and then adding and reacting with microbial levambiose synthase. This is a method for producing levanbiose that facilitates the process. The present invention, instead of the addition of Rebanbiosu synthase, by adding a producing bacteria Rebanbiosu synthase culture are readily method for producing Rebanbiosu synthase Rebanbiosu synthase can be produced.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
The leaves and stems of the gramineous plant of the present invention may be any as long as they are derived from gramineous plants such as ryegrass, rye, rye wheat, wheat, oat, pearl barley, orchard grass, and ryegrass is particularly preferred. Moreover, the thing using the bran which dried the leaf, stem, grain, etc. of these Gramineae plants, or the bran which was further processed as a single or multiple mixture is also included. Liquids or suspensions in which any aqueous solution such as water, buffer solution, enzyme other than fructose disaccharide synthase derived from microorganisms is added to the leaves, stems, or pods of this Gramineae plant, or solids containing some water All of the above conditions are included, and the leaves, stems or straws of the grass family may be used as raw materials regardless of the presence or absence of sterilization, sterilization or heating.
[0013]
The levanbiose synthase can be used regardless of whether it is a cloned enzyme or an uncloned enzyme, as long as it is an enzyme produced by a microorganism that produces even the desired target levanbiose . More specifically, the microorganism is brought into contact with levan and reacted with the culture medium or the culture substrate components and the environmental conditions such as content, volume, temperature, humidity, pH, and air permeability. This refers to the liquid that produces levanbiose . At this time, the culture solution may contain microbial cells, and has been produced under at least one of the combinations of environmental conditions such as content, volume, temperature, humidity, pH, and air permeability after contact with levan. If you say this. Any state in which concentration, freeze-drying, immobilization, purification, or the like is performed based on this culture solution is included. In addition, any microorganisms such as bacteria, yeasts, and molds can be used for the production of the enzyme.
[0014]
The production of levanbiose is carried out by bringing the above grass family material and a microorganism-derived levanbiose synthase into contact with each other and carrying out an enzymatic reaction. The enzyme reaction may take any form such as a method in which levambiose synthase is brought into direct contact with the raw material, a method in which an aqueous solution such as water or a buffer solution is added to the raw material and boiled, and then a microorganism-derived levanbiose synthase is added. The inclusion may be in contact with levanbiose synthase . The ratio of the raw material to the amount of enzyme added may be an optimum amount in consideration of the type of raw material and the like, and it is appropriate to add 20 volumes of enzyme solution to about 1 weight of solid raw material. After starting the reaction, the desired levanbiose is produced by keeping the temperature constant for a certain period of time, but the conditions such as shape, temperature, pH, and time may be any conditions as long as they produce fructose disaccharides. In the state, it is desirable to carry out at temperatures of 20-60 ° C., pH 5-8, and 24-72 hours, respectively.
[0015]
Production of Rebanbiosu synthase is performed by a microorganism that produces Rebanbiosu synthase and culture substrate the Gramineae plant material culture. Culturing is carried out by inoculating the microorganism in a mixed medium containing plant materials alone or other additives. The shape of the medium at this time is not limited to liquid or solid, and the conditions such as volume, temperature, pH, aeration, and culture time during culture can be obtained as long as a culture solution in which levanbiose synthase is produced can be obtained. Any of them may be used, but it is preferably performed in a liquid state at a temperature of 30-50 ° C., pH 5-8, and 24-72 hours.
[0016]
A feature of the present invention is that levanbiose , which is a linear fructose disaccharide that is promising as a functional food material, is produced from leaves, stems, or straws produced in large quantities as by-products in agricultural production. There is a feature. In addition, levanbiose synthase is derived from microorganisms that are relatively easy to handle, and the production method thereof is characterized in that it can be carried out using the above plant raw materials.
[0017]
Recovery and purification of levanbiose from the reaction solution or culture solution after the constant temperature can be performed according to a commonly used conventional method, for example, silica gel column chromatography, activated carbon column chromatography, lyophilization or the like .
[0018]
Confirmation that the product is levanbiose can be performed by thin layer chromatography (thin layer plate: silica gel, developing solvent: n-butanol / 2-propanol / water / acetic acid = 7/5/4/2). it can. First, it is confirmed from the fact that the Rf value of the product coincides with levanbiose and that when the hydrochloric acid decomposition product of the product is developed, it coincides with fructose. Furthermore, it can also confirm from a liquid high-speed chromatography, a mass spectrum, or a NMR spectrum.
[0019]
Although the details of the components in the grass family material that are direct substrates for levanbiose synthase are unknown, it is presumed that the substrate is a fructan containing at least 3 molecules of fructose.
[0020]
【Example】
EXAMPLES Next, although this invention is demonstrated in detail based on an Example, this invention is not limited to these Examples at all.
[Example 1] For ryegrass, stalks and leaves, rye, rye wheat, and for wheat, potatoes with dried stem portions were collected and cut into approximately 1-2 cm. To 100 g of the cut raw material, 1 L of 10 mM sodium phosphate buffer solution (pH 6) was added and boiled at 100 ° C. for 10 minutes. This suspension was used as a substrate solution for enzyme reaction.
[0021]
On the other hand, for the production of levanbiose synthase, Arthrobacter sp. B25 (FERM P-18691) was applied to slant medium (5 g polypeptone, 5 g meat extract, 10 g glucose, 3 g sodium chloride, 15 g agar 15 g). , PH 7.2), and 1 platinum loop of the culture was added to 100 mL of main culture medium (Levan 10 g, NH ₄ NO ₃ 2.0 g, MgSO ₄ .7H ₂ O 0.5 g, KCl per liter). 0.5 g, 0.5 g of KH ₂ PO ₄ , 0.01 g of FeSO ₄ .7H ₂ O, 0.2 g of yeast extract, 20 g of CaCO ₃ , pH 7.0), and inoculated at 30 ° C. And cultured for 48 hours with shaking. This culture solution was centrifuged to remove the cells, and the supernatant was used as an enzyme solution.
[0022]
1 L of enzyme solution was added to the above substrate solution, and the temperature was kept constant at 40 ° C. After 24 hours, each enzyme reaction analysis of high performance liquid chromatography (column: YMC-Pack ODS-AQ, eluent: water, column temperature: room temperature Detection: RI) to subjecting result of analysis of Rebanbiosu, FIG As can be seen from FIG. 1, the production of levambiose was confirmed particularly when ryegrass was used as a substrate, and the yield was about 0.5-1% based on the initial dry matter weight. The production of other substrates was confirmed although it was a trace amount.
[0023]
[Example 2] 100 mL of 10 mM sodium phosphate buffer (pH 6) was added to each 10 g of ryegrass, rye, rye wheat, wheat straw cut into approximately 1-2 cm as in Example 1, Sterilized for 10 minutes.
[0024]
Microorganisms similar to those in Example 1 were grown on a slant culture medium, and then sterilized samples were inoculated with 1 platinum loop and cultured with shaking at 30 ° C. for 48 hours. After centrifuging the culture solution and removing the cells, the supernatant was used as an enzyme solution, and the enzyme activity during the culture was measured. Enzyme activity was determined by mixing an equal amount of the above enzyme solution with a substrate solution in which levan (manufactured by Serratia) was dissolved in 0.1 M sodium phosphate buffer to 2% (w / v), and incubated at 37 ° C. for 30 minutes. The reaction was stopped in boiling water for 5 minutes, and the amount of levanbiose in the liquid was quantified by high performance liquid chromatography described in Example 1. Enzyme activity was defined as 1 unit of enzyme amount when 1 μmol of levanbiose was produced per minute. As can be seen from FIG. 2, it was confirmed that enzyme production was possible with each culture substrate , and in particular, when ryegrass was used, the enzyme could be produced without much inferior to when Levan was used.
[0025]
【The invention's effect】
As described above, according to the present invention, instead of levan, which is a conventional raw material for fructose disaccharides, it is a linear fructose disaccharide that is promising as a functional food material, using leaves, stems or straws as a raw material. It became possible to manufacture a certain levanbiose . This makes it possible to produce levanbiose cheaply and efficiently, since koji etc. are produced as a by-product in agricultural production, compared to levan, which was conventionally produced by microbial fermentation from sucrose and its yield was low. It is possible to provide a new treatment method for these by-products.
[Brief description of the drawings]
FIG. 1 is a graph showing the amount of levanbiose produced when a grass sample is used as an enzyme substrate.
FIG. 2 is a graph showing the enzyme activity of levanbiose synthase when a gramineous plant sample is used as a culture substrate.

Claims (4)

A method for producing fructose disaccharides by causing microorganism-derived enzymes to act on leaves, stems, and pods of gramineous plants,
Using Arthrobacter sp B25 as above Symbol microorganism, to produce a Reban'osu synthase, fructose disaccharides manufacturing method characterized by the production of Reban'osu by the Reban'osu synthase.   2. The method for producing fructose disaccharide according to claim 1, wherein the gramineous plant is ryegrass, rye, rye wheat or wheat. A method for producing fructose disaccharide synthase by growing microorganisms on leaves, stems, and pods of gramineous plants ,
A method for producing fructose disaccharide synthase, characterized in that Arthrobacter sp. B25 is used as the microorganism and levanose synthase is produced .   4. The method for producing a fructose disaccharide synthase according to claim 3, wherein the gramineous plant is ryegrass, rye, rye wheat or wheat.

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