WO1994010295A1 - Procede de production d'un inulo-oligosaccharide cyclique et d'une enzyme le produisant - Google Patents

Procede de production d'un inulo-oligosaccharide cyclique et d'une enzyme le produisant Download PDF

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Publication number
WO1994010295A1
WO1994010295A1 PCT/JP1993/001607 JP9301607W WO9410295A1 WO 1994010295 A1 WO1994010295 A1 WO 1994010295A1 JP 9301607 W JP9301607 W JP 9301607W WO 9410295 A1 WO9410295 A1 WO 9410295A1
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WO
WIPO (PCT)
Prior art keywords
cyclic
oligosaccharide
producing
inulo
inulin
Prior art date
Application number
PCT/JP1993/001607
Other languages
English (en)
Japanese (ja)
Inventor
Sachiko Kushibe
Yuuki Morimoto
Original Assignee
Mitsubishi Kasei Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from JP4296136A external-priority patent/JPH06141856A/ja
Priority claimed from JP29613792A external-priority patent/JPH06141879A/ja
Application filed by Mitsubishi Kasei Corporation filed Critical Mitsubishi Kasei Corporation
Publication of WO1994010295A1 publication Critical patent/WO1994010295A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

Definitions

  • the present invention relates to a method for producing a cyclic inulo-oligosaccharide and a method for producing a cyclic inulo-oligosaccharide-forming enzyme.
  • cyclodextrin in which glucose is bound to ⁇ - (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.
  • cyclodextrin has low solubility in water, and its application to food and pharmaceuticals is currently limited.
  • 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).
  • 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.
  • 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.
  • cyclic canine ligose sugar 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.
  • 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.
  • 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.
  • the amount of inulin is 0.1% or more, preferably 0.5 to 2%, in the culture solution.
  • the present invention 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%.
  • the content is 0.05 to 0.5%
  • the ratio (C / N ratio) between the organic carbon source and the organic nitrogen source is 1 to 15.
  • the CZN ratio has been about 20.
  • 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.
  • a culture solution having the following composition can be suitably used. Per liter of culture
  • 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.
  • 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.
  • cyclic inulooligosaccharides can be obtained in the culture supernatant.
  • 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
  • a DEAE-Tyopear 1 M, QAE-Toyopear 1 650M column manufactured by Tosoichi
  • 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.
  • 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.
  • cyclic canine ligose sugar examples include cyclic inulooligosaccharides such as cycloine mouth hexose, cycloine mouth heptaose, and cycloine mouthful kutaose.
  • 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.
  • 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.
  • 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.
  • these silica carriers are not particularly limited, but silica gel (ODS) in which octadecylsilyl groups are chemically bonded is particularly preferably used.
  • ODS silica gel
  • 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.
  • 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.
  • the fraction 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.
  • 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 ⁇ 7.5, 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.
  • CFTase activity in the crude enzyme solution was measured, it was 1.9 U / m1.
  • 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) ( ⁇ / ml)
  • 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.
  • 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.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Inulo-oligosaccharide cyclique produit par la culture d'un microbe apte à produire une enzyme produisant l'oligosacharide, dans un milieu contenant de l'inuline et de 0,01 à 1,0 % d'une source d'azote organique, et par la mise en réaction de l'inuline et des cellules ainsi produites, lesquelles sont optiquement traitées, ou du surnageant du milieu. On purifie l'inulo-oligosaccharide cyclique par la mise en contact de la solution de sucre contenant l'oligosaccharide ainsi obtenu, et d'un véhicule en silice de type siloxane, afin de provoquer l'adsorption sélective de l'oligosaccharide seul, et par l'élution de l'oligosaccharide adsorbé à l'aide d'une solution aqueuse d'alcool.
PCT/JP1993/001607 1992-11-05 1993-11-05 Procede de production d'un inulo-oligosaccharide cyclique et d'une enzyme le produisant WO1994010295A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP4/296137 1992-11-05
JP4/296136 1992-11-05
JP4296136A JPH06141856A (ja) 1992-11-05 1992-11-05 環状イヌロオリゴ糖及び環状イヌロオリゴ糖生成酵素の製造方法
JP29613792A JPH06141879A (ja) 1992-11-05 1992-11-05 環状イヌロオリゴ糖の精製方法

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WO1994010295A1 true WO1994010295A1 (fr) 1994-05-11

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0682118A2 (fr) * 1994-04-15 1995-11-15 Mitsubishi Chemical Corporation Procédé pour purifier des inulooligosaccharides cycliques

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02252701A (ja) * 1989-03-28 1990-10-11 Maruzen Kasei Co Ltd 新規環状イヌロオリゴ糖およびその製造法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02252701A (ja) * 1989-03-28 1990-10-11 Maruzen Kasei Co Ltd 新規環状イヌロオリゴ糖およびその製造法

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0682118A2 (fr) * 1994-04-15 1995-11-15 Mitsubishi Chemical Corporation Procédé pour purifier des inulooligosaccharides cycliques
EP0682118A3 (fr) * 1994-04-15 1997-05-02 Mitsubishi Chem Corp Procédé pour purifier des inulooligosaccharides cycliques.
US5672482A (en) * 1994-04-15 1997-09-30 Mitsubishi Chemical Corporation Method for purifying cyclic inulooligosaccharide

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