WO1994010295A1 - Processes for producing cyclic inulo-oligosaccharide and for producing enzyme that produces the same - Google Patents

Abstract

A cyclic inulo-oligosaccharide is produced by culturing a microbe capable of producing an enzyme that produces the oligosaccharide in a medium containing inulin and 0.01-1.0 % of an organic nitrogen source and reacting inulin with the produced cells, which have been optionally treated, or the supernatant of the medium. The cyclic inulo-oligosaccharide is purified by contacting the sugar liquor containing the oligosaccharide thus produced with a siloxane-type silica carrier to selectively adsorb only the oligosaccharide and eluting the adsorbed oligosaccharide with an aqueous alcohol solution.

Description

 Description Method for producing cyclic canine liposaccharide and its producing enzyme

 The present invention relates to a method for producing a cyclic inulo-oligosaccharide and a method for producing a cyclic inulo-oligosaccharide-forming enzyme.

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 Conventionally, cyclodextrin in which glucose is bound to α- (1-4) darcoside has been known as a cyclic inurioligosaccharide. Cyclodextrins are attracting attention as food clathrates, and contribute to increasing the value of foods for the purpose of retaining flavor components, masking specific odors, removing bitterness, and preventing oxidation. It is also used in various fields other than food. However, cyclodextrin has low solubility in water, and its application to food and pharmaceuticals is currently limited.

 On the other hand, cyclic inulooligosaccharides are cyclic oligosaccharides in which six or more fructose molecules are linked in a cyclic manner by a; 3- (2-1) bond, and were isolated and identified in 1989 by Kawamura et al. search, 192, 83—90, 1989). As the cyclic inulooligosaccharides, there are known cyclonuclear hexoses each having 6 fructose residues, cyclonuclear heptaose composed of 7 fructose residues, and cyclonucleated kutaose composed of 8 fructose residues. This cyclic inulooligosaccharide has attracted attention as a new functional sugar different from cyclodextrin due to its extremely high solubility in water.

In recent years, as a method for producing cyclic inulo-oligosaccharides, Takayama et al. Have proposed that Bacillus circulans MZ No. 31 (Fujin-Kyo-Ken No. 9943) be added to inulin, a polysaccharide composed mainly of fructose. A method has been proposed in which a cyclic inulooligosaccharide is obtained by reacting an enzyme produced by Japanese Patent Application Laid-Open No. 2-252701 (C arb). ohydrate Re search, 192, 83-90, 1989). In addition, a method using a cyclic inulo-oligosaccharide-forming enzyme produced by Bacillus saccurans MC 1-2554 (Japanese Patent Application Laid-Open No. 11-237496) has also been proposed. However, the productivity of cyclic inulo-oligosaccharides by these enzymes is not yet sufficient for industrial use, and it is still difficult to use cyclic inulo-oligosaccharides economically as a method for producing cyclic inulo-oligosaccharides. It has been desired to provide a method for producing cyclic canine ligose sugar by specifically producing enzymes.

 By the way, when a cyclic canine oligosaccharide is produced by allowing CFTase to act on inulin or the like as in the above method, other contaminants present in the reaction solution, such as unreacted By-products such as fructan and monosaccharides such as fructose, disaccharides, and linear oligosaccharides during the reaction are unavoidable, and a process for separation and purification of cyclic canine sugar cane is required. Conventionally, as a method for separating and purifying only cyclic inulo-oligosaccharide from a sugar solution containing cyclic inulo-oligosaccharide, only a method of fractionating and collecting using activated carbon is known (see Japanese Patent Application Laid-Open No. — 252701 Publication). Moreover, the method for purifying cyclic inulo-oligosaccharides using activated carbon requires that the alcohol be eluted with the alcohol concentration being changed stepwise, and cannot be said to be an efficient method in terms of yield. Studies on purification methods are not yet sufficient, and it is currently difficult to produce them industrially. Therefore, development of an efficient method for purifying cyclic inulo-oligosaccharides is desired.

Disclosure of the invention

The present inventors have conducted intensive studies on a method for efficiently producing cyclic inulooligosaccharides from inulin, focusing on the culture conditions, and as a result, have been able to culture microorganisms having CFTase-producing ability with a specific ratio of nitrogen source. It has been found that CFTase can be satisfactorily produced by culturing in a liquid, and that the cyclic sugar cane ligose can be produced more efficiently by using the CFTase. Furthermore, in the step of producing such a cyclic canine ligose sugar, other contaminants present in the reaction solution, for example, unreacted fructan, monosaccharides such as fructose by-produced during the reaction, disaccharides, Siloxane is used to remove linear oligosaccharides and obtain cyclic inulooligosaccharides. The present inventors have found that the sily gel has very efficiently and selectively adsorbed cyclic canine ligose sugar and is easily eluted by alcohol, and thus completed the present invention.

 That is, the gist of the present invention is to provide a cell of a microorganism or a cell thereof capable of producing a cyclic canine liposaccharide-forming enzyme, which is cultured in a culture solution containing inulin and an organic nitrogen source of 0.01 to 1.0%. A process for producing a cyclic inulo-oligosaccharide, which comprises reacting the treated product or the culture supernatant with inulin, and further comprising reacting the cells of a microorganism having an ability to produce a cyclic inulo-oligosaccharide-forming enzyme or a treated product thereof with inulin. The sugar solution containing the cyclic canine ligose sugar obtained is contacted with a siloxane-based silicic acid carrier to selectively adsorb only the cyclic canine ligose sugar, and then the cyclic canine mouth adsorbed on the carrier is contacted. A method for producing a cyclic inulo-oligosaccharide, characterized in that the oligosaccharide is purified by elution with an aqueous alcohol solution, and a microorganism capable of producing a cyclic inulo-oligosaccharide-producing enzyme, which is capable of producing a cyclic inulo-oligosaccharide-forming enzyme. Resides in emissions and 0.01 to 1.0% of the production method of the cyclic I j clever speech oligosaccharides forming enzyme, which comprises culturing in a culture medium containing an organic nitrogen source. Hereinafter, the present invention will be described in detail.

 The CFTase used in the method of the present invention for producing a cyclic canine rigorous sugar is an enzyme derived from a microorganism having CFTase-producing ability, but a purified enzyme may be used, or a microorganism producing this enzyme may be used. May be used. The term “processed product” as used herein refers to a product obtained by physically or enzymatically disrupting cells, and a product obtained by separating and purifying CFTase therefrom. These CFT aces, bacterial cells, or their crushed products may be used by immobilizing them on an insoluble carrier or the like. Further, a culture supernatant obtained by culturing the microorganism may be used as it is.

 Microorganisms having the ability to produce a cyclic canine liposaccharide-forming enzyme include, for example, Bacillus circulans MZ-No. No. 11940), but other than the above-mentioned bacteria may be used as long as they have the ability to produce a cyclic canine sugar-forming enzyme.

The culture solution used in the present invention contains an extract of a plant having a high inulin content such as Jerusalem artichoke, burdock, and chicory, and / or inulin as a sole carbon source. Usually, the amount of inulin is 0.1% or more, preferably 0.5 to 2%, in the culture solution. The present invention The culture solution contains soybean flour, wheat germ, corn steep liquor, cottonseed liquor, meat extract, peptone, yeast extract, preferably corn steep liquor as an organic nitrogen source, preferably in a range of 0.01% to 1.0%. Preferably, the content is 0.05 to 0.5%, and the ratio (C / N ratio) between the organic carbon source and the organic nitrogen source is 1 to 15. Conventionally, when culturing a microorganism belonging to Bacillus circulans having the ability to produce a cyclic canine oligosaccharide-forming enzyme, the CZN ratio has been about 20. Can be efficiently produced. In addition, the culture solution contains inorganic nitrogen sources such as ammonium sulfate, sodium nitrate, urea, potassium nitrate, etc. It is effective to add an inorganic salt which produces the following ions. Specifically, for example, a culture solution having the following composition can be suitably used. Per liter of culture

 Inulin 10 g

 5g corn steep liquor

NaNOs 2 g

 KC 1 0.5 g

F e S 0 ₄ 7H ₂ 0 0.01

 (pH 7.5) For example, when cultivating the bacterium belonging to the above-mentioned Bacillus sucurans in such a culture solution, perform shaking cultivation at a culturing temperature of 20 to 50 for about 12 to 120 hours. Thus, CFTase can be produced efficiently.

Inulooligosaccharides can be obtained by reacting inulin with the cells of the microorganism producing CFTase, the processed cells thereof, the culture supernatant, or the enzyme purified therefrom, or the enzyme purified therefrom. At this time, Inurin used as substrate may be used a solution in a buffer or the like, the medium used as such which may _c further used for culture, Inurin or Jerusalem artichoke, high Inurin content of burdock etc. Culture CFTase-producing bacteria under the above conditions in a solution containing plant extracts as the sole carbon source. Thus, cyclic inulooligosaccharides can be obtained in the culture supernatant.

 When CFTase was allowed to act on inulin, the culture broth cultured by the above method was first sterilized by centrifugation, and the resulting filtrate was salted out with ammonium sulfate (65% saturation). The precipitate is collected, suspended in a small amount of water, and dialyzed to obtain a crude enzyme solution. By reacting this crude enzyme solution with inulin in a 0.01-0.1 M phosphate buffer adjusted to, for example, pH 7.0, a desired cyclic canine sugar ligose can be obtained. This enzyme solution is purified by ion exchange chromatography using, for example, a DEAE-Tyopear 1 M, QAE-Toyopear 1 650M column (manufactured by Tosoichi) and, if necessary, a Toyopear 1 HW55F By carrying out gel filtration gel chromatography using a gel, an enzyme preparation showing a single band electrophoretically can be obtained.

 The reaction solution containing the cyclic inulo-oligosaccharide obtained by reacting the cells of the microorganism producing the CFTase, the treated cells thereof, the culture supernatant, or the enzyme purified therefrom with inulin as described above is contained in the cells. In addition to the cyclic inulooligosaccharides, saccharides such as monosaccharides, disaccharides, and linear oligosaccharides are contained (hereinafter, this solution is also referred to as a “cyclic canine liposaccharide-containing solution”). By removing these, a purified cyclic inulooligosaccharide can be produced.

 Examples of the cyclic canine ligose sugar that can be purified include cyclic inulooligosaccharides such as cycloine mouth hexose, cycloine mouth heptaose, and cycloine mouthful kutaose.

 On the other hand, monosaccharides to be removed include fructose, glucose, etc., disaccharides, sucrose, inulobiose, etc., and linear oligosaccharides, such as fructo-oligosaccharide, have a reducing end such as glucose and several to several tens of fructose. Sugars bound to a certain extent can be mentioned.

Hereinafter, a method for purifying the cyclic inulo-oligosaccharide by removing saccharides such as monosaccharides, disaccharides, and linear oligosaccharides from the solution containing the cyclic inulo-oligosaccharide will be specifically described. First, the siloxane-based silica carrier is washed with acetone, methanol, or the like by a predetermined method. The siloxane-based silica carrier used in the present invention means that some hydrogen atoms of silanol groups on the surface of a silica carrier such as silica gel are converted to alkylsilyl groups or the like, preferably C ₈ to It means those having a structure substituted with a C18 straight-chain alkylsilyl group or those having a structure in which the hydrogen atom of the remaining unreacted silanol group is further substituted to various extents with a trimethylsilyl group. These siloxane-based silica carriers can be produced by reacting an alkylchlorosilane with the silica carrier, or by further reacting the reaction product with trimethylchlorosilane. Specifically, a silica gel surface in which an octadecylsilyl group is introduced into a silanol group so that the carbon content is 7 to 20%, and a trimethylsilyl group in various proportions to the remaining silanol group of the substance. The commercially available products, or those in which octylsilyl groups have been introduced into some of the silanol groups on the silica gel surface, are commercially available. These commercially available products can be used.

 In the present invention, these silica carriers are not particularly limited, but silica gel (ODS) in which octadecylsilyl groups are chemically bonded is particularly preferably used. Specifically, Silica Gel ODS'G-5, Silica Ge1ODS Q-3 (Fuji Gel Sales Co., Ltd.), Cosmoseal 75C18-OPN (Nakarai Tesque Co., Ltd.) And the like.

 The washed silica carrier is then gradually replaced with water. These are brought into contact with a cyclic inulo-oligosaccharide-containing solution and washed with water. Thereafter, extraction is performed with 5 to 80% alcohol, and the extract is decolorized and desalted, and then concentrated to dryness to obtain a desired cyclic inulooligosaccharide. It can also be carried out by packing a gel into a column. After loading the resin containing the cyclic inulooligosaccharide in an amount of 1 to 100 to 1/10 of the resin packed in the column and eluting monosaccharides, disaccharides and linear oligosaccharides with water, elute with a 5 to 80% aqueous alcohol solution I do. The eluate is not particularly limited as long as it can dissociate the cyclic inulo-oligosaccharide from the resin. For example, a 25% aqueous methanol solution is used. Since the cyclic inulo-oligosaccharide is obtained in the eluted fraction, the fraction is decolorized, desalted, and concentrated to dryness, whereby a purified cyclic inulo-oligosaccharide can be produced.

The above-mentioned purification method is not limited to the method for producing a cyclic inulooligosaccharide of the present invention, and may be used for purifying cyclic canine oligosaccharide from a sugar solution containing cyclic inulooligosaccharide produced by a conventional production method. Needless to say, this can also be applied. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the method of the present invention will be described more specifically with reference to examples, but the present invention is not limited to these without departing from the gist thereof.

<Example 1>

 Inulin was added to a basal medium containing 0.2% sodium nitrate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.05% potassium phosphate, 0.05% ferric chloride, and Table 1 1 100 ml of a medium to which the amount shown in Table 1 and the amount of corn steep liquor were added as shown in Table 11 was adjusted to ρΗ7.5, and the mixture was placed in a 500 ml Sakaguchi flask and steam-sterilized for 120 to 120 minutes. A loopful of Bacillus circulans MC I-2554 was inoculated into the sterilized medium and cultured at 160 rpm T27 ° C for 30 hours. After completion of the culture, the cells were removed by centrifugation to obtain a culture filtrate. The obtained culture filtrate is used as a crude enzyme solution. When the CFTase activity in the crude enzyme solution was measured, it was 1.9 U / m1. Using this crude enzyme solution, cyclic inulooligosaccharides were produced. The reaction solution contained 5 UmM phosphate buffer pH 7.0, 25% inulin and a crude enzyme solution of 0.8 UZm1 as final activities, and the total amount was 5 Om1. The reaction was carried out at 37 for 10 hours, and the amount of cyclic inulo-oligosaccharide accumulated in the reaction solution was quantified. As a result, 12.5 g of inulin produced 8.12 g of cyclic inulo-oligosaccharide.

<Examples 2 to 11> As shown in Table 11, the same procedure as in Example 1 was performed except that the composition of the organic carbon source and the organic nitrogen source was changed. Table 1 shows the results. Inulin corn steep liquor C / N CFTase activity Example (g / L) (g / L) (ϋ / ml)

1 10 5 2 1.9

2 10 1 10 1.8

3 10 10 1 1.4

4 5 1 5 1.3

5 5 5 1 1.4

6 20 5 4 1.8

7 20 10 2 1.3

8 5 20 0.25 0.6

9 1 5 0.2 0.9

10 1 10 0. 1 0.5

11 1 20 0.05 0.2

<Example 12>

Contains inulin 4%, yeast extract 0.2%, sodium nitrate 0.5%, magnesium sulfate, potassium chloride 0.055%, monopotassium phosphate 0.055%, ferric chloride 0.001% The fermentation medium (150 ml) was adjusted to pH 7.0 and steam sterilized at 120 ° C for 20 minutes. A loopful of Bacillus' circulans MCI-2554 was inoculated into the sterilized medium, and cultured with shaking at 160 rpm for 30 to 30 hours. The OD value of the culture at 660 nm was about 5. After completion of the culture, the cells were removed by centrifugation to obtain a culture filtrate. The obtained culture filtrate was used as a crude enzyme solution. 3 g of inulin was finally added to 10% of the crude enzyme solution to 3%, and the mixture was reacted at 37 ° C for 8 hours. After the reaction, the product was analyzed by high performance liquid chromatography (HPLC). As a result, cyclohexahexaose was obtained at a concentration of 10.37 gZL, and cyclohexaheptaose was obtained at a concentration of 5.19 g / L. The reaction solution contains cyclohexahexaose, cyclohinoheptaose, cyclonautose, kutaose, unreacted inulin, linear oligosaccharides such as fructooligosaccharides, and fructose. included.

 Using the above reaction solution, a resin having the physical properties shown in Table 2 was examined for adsorption of cyclic canine sugar. The resin was gradually replaced with water after washing with methanol. The column was packed with 2 Om1 of resin thoroughly washed with water. After 100 ml of water was passed through the column, 2 Om1 of the reaction solution was passed at s V = 1 to adsorb. 0.207 g of cyclohexahexaose was contained in the reaction solution 2 Om1. After that, unadsorbed substances were eluted with water, and then eluted with 25% ethanol. Cyclonus hexaose in the ethanol eluate was quantified by HPLC. As a result, 0.180 g of cyclohexahexaose was obtained at a recovery rate of 87%.

<Comparative Examples 1 to 4>

 As shown in Table 2, purification was carried out by adsorption in the same manner as in Example 12 except that the type of resin was changed. Table 2 shows the obtained recovery rates. Table 2 Eluate obtained cycle π

 (ml) (g) Recovery (! ¾) Remark Example 12 35 0.180 87.0 ODS

 Siloxane Cosmoseal 75C18-OPN silica carrier (Nakarai Tesque, Ltd.) Comparative Example 1 46 0.080 38.6 Sepabeads

 (Synthetic adsorption resin) SP 800 (Mitsubishi Kasei Co., Ltd.) Comparative Example 2 45 0.090 43.5 Diaion

 (Synthetic adsorption resin) HP 20 (Mitsubishi Kasei Co., Ltd.) Comparative Example 3 35 0.110 53.1 Diaion

 (Synthetic adsorption resin) HP 21 (Mitsubishi Kasei Corporation) Comparative Example 4 40 0.085 41.0 For chromatography

(Activated carbon) Activated carbon (Wako Pure Chemicals) Industrial applicability

 According to the production method of the present invention, a cyclic inulo-oligosaccharide-forming enzyme can be efficiently produced, and by reacting the enzyme with inulin, a cyclic inulo-oligosaccharide can be efficiently and economically obtained. . Further, a purified cyclic inulooligosaccharide can be produced by using a siloxane-based silica carrier. The obtained cyclic canine sugar can be expected to be used not only in the field of food but also in other fields.

Claims

The scope of the claims 1. Cells of a microorganism capable of producing a cyclic dog canine sucrose-forming enzyme, or a treated product thereof or a culture thereof, which is cultured in a culture solution containing 1.0 inulin and 0.01 to 1.0% of an organic nitrogen source. A method for producing cyclic dog ligose sugar, comprising reacting Qing with inulin.  2. The method according to claim 1, wherein the mixing ratio of the carbon source and the nitrogen source is 1 to 15 (carbon source and nitrogen source).  3. Cyclic inulooligosaccharide-containing sugar solution obtained by reacting microbial cells capable of producing a cyclic inulooligosaccharide-forming enzyme or a processed product thereof with inulin is contacted with a siloxane-based carrier to form a cyclic inulooligosaccharide. A method for producing cyclic canine rigorous saccharides, comprising selectively adsorbing only canine ligneous sugar and then purifying the cyclic inulo-oligosaccharide adsorbed on the carrier by dissolving it with an aqueous alcohol solution. 4. The method for producing a cyclic inulo-oligosaccharide according to claim 3, wherein the sugar solution containing the cyclic inulo-oligosaccharide is a reaction solution containing the cyclic inulo-oligosaccharide obtained in claim 1 or 2.  5. The method according to claim 3 or 4, wherein the step of contacting the sugar solution containing the cyclic inulooligosaccharide with a siloxane-based silica carrier is performed by passing the sugar solution through a column filled with the siloxane-based silica carrier. A method for purifying cyclic inulo-oligosaccharides.  6. A cyclic canine oligosaccharide-forming enzyme, which is characterized by culturing a microorganism capable of producing a cyclic canine-oligosaccharide-forming enzyme in a culture medium containing inulin and an organic nitrogen source in an amount of 0.01 to 1.0%. Production method.