JPH0634711B2 - Bacillus SP. KK-4645 - Google Patents
Bacillus SP. KK-4645Info
- Publication number
- JPH0634711B2 JPH0634711B2 JP61072455A JP7245586A JPH0634711B2 JP H0634711 B2 JPH0634711 B2 JP H0634711B2 JP 61072455 A JP61072455 A JP 61072455A JP 7245586 A JP7245586 A JP 7245586A JP H0634711 B2 JPH0634711 B2 JP H0634711B2
- Authority
- JP
- Japan
- Prior art keywords
- inulin
- enzyme
- bacillus
- reaction
- cells
- Prior art date
- Legal status (The legal status 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 status listed.)
- Expired - Fee Related
Links
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 title claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 15
- 108090000790 Enzymes Proteins 0.000 claims description 15
- 229920001202 Inulin Polymers 0.000 claims description 11
- 229940029339 inulin Drugs 0.000 claims description 11
- JYJIGFIDKWBXDU-MNNPPOADSA-N inulin Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@]1(OC[C@]2(OC[C@]3(OC[C@]4(OC[C@]5(OC[C@]6(OC[C@]7(OC[C@]8(OC[C@]9(OC[C@]%10(OC[C@]%11(OC[C@]%12(OC[C@]%13(OC[C@]%14(OC[C@]%15(OC[C@]%16(OC[C@]%17(OC[C@]%18(OC[C@]%19(OC[C@]%20(OC[C@]%21(OC[C@]%22(OC[C@]%23(OC[C@]%24(OC[C@]%25(OC[C@]%26(OC[C@]%27(OC[C@]%28(OC[C@]%29(OC[C@]%30(OC[C@]%31(OC[C@]%32(OC[C@]%33(OC[C@]%34(OC[C@]%35(OC[C@]%36(O[C@@H]%37[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O%37)O)[C@H]([C@H](O)[C@@H](CO)O%36)O)[C@H]([C@H](O)[C@@H](CO)O%35)O)[C@H]([C@H](O)[C@@H](CO)O%34)O)[C@H]([C@H](O)[C@@H](CO)O%33)O)[C@H]([C@H](O)[C@@H](CO)O%32)O)[C@H]([C@H](O)[C@@H](CO)O%31)O)[C@H]([C@H](O)[C@@H](CO)O%30)O)[C@H]([C@H](O)[C@@H](CO)O%29)O)[C@H]([C@H](O)[C@@H](CO)O%28)O)[C@H]([C@H](O)[C@@H](CO)O%27)O)[C@H]([C@H](O)[C@@H](CO)O%26)O)[C@H]([C@H](O)[C@@H](CO)O%25)O)[C@H]([C@H](O)[C@@H](CO)O%24)O)[C@H]([C@H](O)[C@@H](CO)O%23)O)[C@H]([C@H](O)[C@@H](CO)O%22)O)[C@H]([C@H](O)[C@@H](CO)O%21)O)[C@H]([C@H](O)[C@@H](CO)O%20)O)[C@H]([C@H](O)[C@@H](CO)O%19)O)[C@H]([C@H](O)[C@@H](CO)O%18)O)[C@H]([C@H](O)[C@@H](CO)O%17)O)[C@H]([C@H](O)[C@@H](CO)O%16)O)[C@H]([C@H](O)[C@@H](CO)O%15)O)[C@H]([C@H](O)[C@@H](CO)O%14)O)[C@H]([C@H](O)[C@@H](CO)O%13)O)[C@H]([C@H](O)[C@@H](CO)O%12)O)[C@H]([C@H](O)[C@@H](CO)O%11)O)[C@H]([C@H](O)[C@@H](CO)O%10)O)[C@H]([C@H](O)[C@@H](CO)O9)O)[C@H]([C@H](O)[C@@H](CO)O8)O)[C@H]([C@H](O)[C@@H](CO)O7)O)[C@H]([C@H](O)[C@@H](CO)O6)O)[C@H]([C@H](O)[C@@H](CO)O5)O)[C@H]([C@H](O)[C@@H](CO)O4)O)[C@H]([C@H](O)[C@@H](CO)O3)O)[C@H]([C@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@H](O)[C@@H](CO)O1 JYJIGFIDKWBXDU-MNNPPOADSA-N 0.000 claims description 11
- KVEFAMTVBXAETD-LJMGQZPQSA-N (3S,4R,5R)-1-[(2R,3S,4S,5R)-2-[[(2R,3S,4S,5R)-2-[[(2R,3S,4S,5R)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]oxymethyl]-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]oxymethyl]-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]oxy-3,4,5,6-tetrahydroxyhexan-2-one Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C(=O)CO[C@]1(CO[C@]2(CO[C@]3(CO)O[C@H](CO)[C@@H](O)[C@@H]3O)O[C@H](CO)[C@@H](O)[C@@H]2O)O[C@H](CO)[C@@H](O)[C@@H]1O KVEFAMTVBXAETD-LJMGQZPQSA-N 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 241000894006 Bacteria Species 0.000 claims description 6
- 210000004027 cell Anatomy 0.000 description 11
- 239000000203 mixture Substances 0.000 description 6
- 238000006116 polymerization reaction Methods 0.000 description 5
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical class OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 229920001542 oligosaccharide Polymers 0.000 description 4
- 150000002482 oligosaccharides Chemical class 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 210000002421 cell wall Anatomy 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 229930091371 Fructose Natural products 0.000 description 2
- 239000005715 Fructose Substances 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000000419 plant extract Substances 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- ODEHMIGXGLNAKK-OESPXIITSA-N 6-kestotriose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@@H]1[C@@H](O)[C@H](O)[C@](CO)(O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)O1 ODEHMIGXGLNAKK-OESPXIITSA-N 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000186000 Bifidobacterium Species 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
- 239000001828 Gelatine Substances 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- FLDFNEBHEXLZRX-DLQNOBSRSA-N Nystose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@]1(OC[C@]2(O[C@@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O)[C@H]([C@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@H](O)[C@@H](CO)O1 FLDFNEBHEXLZRX-DLQNOBSRSA-N 0.000 description 1
- 241000132152 Polymyxa Species 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 230000009603 aerobic growth Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000004042 decolorization Methods 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000013882 gravy Nutrition 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229910000037 hydrogen sulfide Inorganic materials 0.000 description 1
- UVEIHXHNEIMXTD-VORSWSGSSA-N inulotriose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C(=O)CO[C@]1(CO[C@]2(CO)O[C@H](CO)[C@@H](O)[C@@H]2O)O[C@H](CO)[C@@H](O)[C@@H]1O UVEIHXHNEIMXTD-VORSWSGSSA-N 0.000 description 1
- 229940046892 lead acetate Drugs 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- CEQFOVLGLXCDCX-WUKNDPDISA-N methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- FLDFNEBHEXLZRX-UHFFFAOYSA-N nystose Natural products OC1C(O)C(CO)OC1(CO)OCC1(OCC2(OC3C(C(O)C(O)C(CO)O3)O)C(C(O)C(CO)O2)O)C(O)C(O)C(CO)O1 FLDFNEBHEXLZRX-UHFFFAOYSA-N 0.000 description 1
- -1 nystose and kestose Chemical class 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 238000004816 paper chromatography Methods 0.000 description 1
- 238000005325 percolation Methods 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
【発明の詳細な説明】 ［産業上の利用分野］ 本発明は、イヌロオリゴ糖生成酵素を産生する新規な
菌、バチルスsp.ＫＫ−４６４５に関するものである。TECHNICAL FIELD The present invention relates to Bacillus sp. KK-4645, which is a novel bacterium that produces an inulooligosaccharide-forming enzyme.
［従来の技術］ 従来、イヌロオリゴ糖生成酵素としはアスペルギルス・
ニガー株の産生する酵素が知られている（中村豊彦等、
農化誌、第５２巻 第４号 Ｐ１５９〜１６６、１９７
８年）が、この酵素では一定重合度のイヌロオリゴ糖の
生産は困難であった。[Prior Art] Conventionally, as an inulooligosaccharide-forming enzyme, Aspergillus
The enzyme produced by the niger strain is known (Toyohiko Nakamura et al.,
Agriculture Journal, Vol. 52, No. 4, P 159-166, 197.
However, it was difficult to produce inulooligosaccharides with a certain degree of polymerization using this enzyme.
［発明が解決しようとする問題点］ 近年、ニストース、ケストース等のフラクトオリゴ糖に
ついては、ビフィダス菌増殖因子などの生理特性、物
性、甘味の質など、マルトオリゴ糖とは異なった特性を
持つことで注目され、イヌロオリゴ糖についても、フラ
クトオリゴ糖とほぼ同重合度で、一定重合度のイヌロオ
リゴ糖の生産が必要とされてきた。[Problems to be Solved by the Invention] Recently, attention has been paid to fructooligosaccharides such as nystose and kestose, which have characteristics different from maltooligosaccharides such as physiological characteristics of bifidobacteria growth factor, physical properties, and sweetness. As for inulooligosaccharides, it has been necessary to produce inulooligosaccharides having a degree of polymerization almost the same as that of fructooligosaccharides and a constant degree of polymerization.
［問題点を解決するための手段］ そこで本発明者等は、イヌリンからイヌロオリゴ糖の生
産を目的として、イヌリン分解酵素産生菌を検索し、イ
ヌリンから反応初期に、主としてイヌロテトラオースを
生成する酵素を産生する菌バチルスsp.ＫＫ−４６４５
を見い出した。[Means for Solving Problems] Therefore, the present inventors searched for inulin-degrading enzyme-producing bacteria for the purpose of producing inulooligosaccharide from inulin, and mainly produced inulotetraose at the early stage of the reaction. Enzyme-producing bacterium Bacillus sp. KK-4645
Found out.
本菌の産生する酵素をイヌリンに作用させることによ
り、ほぼ一定重合度のイヌロオリゴ糖を得ることができ
る。By reacting the enzyme produced by this bacterium with inulin, inulooligosaccharide having a substantially constant degree of polymerization can be obtained.
以下、本菌株の菌学的諸性質を述べる。The mycological properties of this strain will be described below.
１．細胞形態 形態：0.5〜0.6μ×４〜５μの桿菌 胞子：形成する 鞭毛：周鞭毛 グラム染色：弱陽性 ２．培養所見 寒天集落：円形、丘状、光沢あり、粘性あり、不透明、
鈍い黄色 寒天斜面：中程度の生育、光沢あり、粘性あり、鈍い黄
色 肉汁ゼラチン：液化する リトマスミルク：不変 ３．生理的性質 好気性 生育温度：30℃で良好な生育を示す、40℃まで生育する 生育pH：pH5.5〜8.0 硝酸塩の還元：あり メチルレッド試験：陽性 Ｖ−Ｐ反応：陽性 硫化水素の生成：あり（システイン添加培地、酢酸鉛試
験紙） クエン酸の利用：なし（クリステンセン培地） カタラーゼの生成：あり 澱粉分解性：あり カゼイン分解性：なし 糖より酸の生成：グルコース、シュクロース、マンニッ
ト、イヌリン、澱粉より酸を生成する、ガスは生成しな
い 上記の諸性質から、バージェイズ マニュアル オブ
デタミネイテイブ バクテリオロジー 第８版により検
索した結果、本菌株はバチルス属に属する。さらに種に
ついては同書によれば、本菌の類縁菌はサーキユラン
ス、ポリミキサ、マセランス、コアギュランス、フィル
ムスであるが、本菌株はこれらの菌の性質とは一致しな
いので、バチルスsp.ＫＫ−４６４５と命名した。本菌
株は工業技術院微生物工業技術研究所にＦＥＲＭ Ｐ−
８６８９として寄託されている。1. Cell morphology Morphology: 0.5-0.6 μ × 4-5 μ rod spores: Forming flagella: Periflagellates Gram stain: Weakly positive 2. Culture findings: Agar colonies: round, hill-like, glossy, viscous, opaque,
Dull yellow agar slope: medium growth, glossy, viscous, dull yellow gravy gelatine: liquefying Litmus milk: unchanged 3. Physiological properties Aerobic Growth temperature: Shows good growth at 30 ° C, grows up to 40 ° C Growth pH: pH 5.5-8.0 Nitrate reduction: Yes Methyl red test: Positive VP reaction: Positive Hydrogen sulfide formation : Yes (Cysteine added medium, lead acetate test paper) Utilization of citric acid: No (Kristensen medium) Catalase production: Yes Starch degradability: Yes Casein degradability: None Acid production from sugar: glucose, sucrose, mannite Acid from inulin and starch, no gas from the above properties
As a result of searching by Determinating Bacteriology 8th Edition, this strain belongs to the genus Bacillus. Regarding the species, according to the same book, the related fungi of this fungus are Sirkyurans, Polymyxa, Macerans, Coagulans, and Films, but since the present strain does not match the properties of these fungi, Bacillus sp.KK-4645 I named it. This strain was confirmed by the FERM P-
Deposited as 8689.
上記類縁菌は本菌と類似しているので、培養条件によっ
ては、イヌロオリゴ糖生成酵素を産生する可能性もあ
る。Since the above-mentioned related bacteria are similar to the present bacteria, they may produce inulooligosaccharide-forming enzyme depending on the culture conditions.
本菌株を、イヌリン及び／またはイヌリン含有植物の抽
出液を炭素源とし、ペプトンを窒素源とした培地に培養
すれば、高活性のイヌリン分解酵素が得られる。培養温
度は25〜30℃が適当である。また、培養時間は24〜72時
間が適当である。培養後の培養液をそのまま、あるいは
集菌して洗浄し、pH６〜７の緩衝液に菌体を懸濁したも
のをイヌリン溶液及び／またはイヌリン含有植物の抽出
液に加えて反応させると、反応初期に主としてイヌロテ
トラースが生成される。また反応時間の長短により、種
々の組成のイヌロオリゴ糖が生成される。例えば72時間
反応した後加熱して酵素を失活し、過、脱色、脱塩等
の方法を用いて精製すれば、各種重合度のイヌロオリゴ
糖を含む製品も得られる。すなわち、反応条件によりフ
ラクトースからイヌロテトラオースを主成分とする混合
物の生産も可能である。By culturing this strain in a medium containing an inulin and / or an inulin-containing plant extract as a carbon source and peptone as a nitrogen source, a highly active inulin-degrading enzyme can be obtained. A suitable culture temperature is 25 to 30 ° C. Further, the culture time is suitable for 24 to 72 hours. When the culture solution after culturing is used as it is, or the cells are collected and washed, and the cells are suspended in a buffer solution of pH 6 to 7 and added to an inulin solution and / or an inulin-containing plant extract to react, the reaction Inurotetrase is mainly produced in the early stage. Moreover, inulooligosaccharides of various compositions are produced depending on the length of the reaction time. For example, after reacting for 72 hours, the mixture is heated to inactivate the enzyme, and the product is purified by a method such as permeation, decolorization or desalting to obtain products containing inulooligosaccharides having various degrees of polymerization. That is, depending on the reaction conditions, it is possible to produce a mixture containing fructose as a main component of inulotetraose.
イヌロテトラオース生成酵素トフラクトース生成酵素の
存在部位については、前者が細胞壁、後者が細胞質内で
あり、細胞を破砕して細胞壁を集めた画分を用いれば、
イヌロテトラオース含量の多い製品が得られる。一方フ
ラクトース高含量製品を得たい場合は、菌体破砕物の全
体を用いればよい。したがって酵素材としては、目的に
応じて菌体そのまま又は従来法により固定化したもの、
菌体を破砕処理したもの又は細胞壁を取り出したものを
用いることもできる。Regarding the site where the inulotetraose producing enzyme tofructose producing enzyme is present, if the former is the cell wall and the latter is in the cytoplasm, and if the fraction obtained by crushing the cells and collecting the cell wall is used,
A product with a high content of inulotetraose is obtained. On the other hand, when it is desired to obtain a product having a high fructose content, the whole crushed cells may be used. Therefore, as the enzyme material, bacterial cells as they are or immobilized by a conventional method according to the purpose,
It is also possible to use a crushed bacterial cell or a cell wall taken out.
さらに高純度のイヌロオリゴ糖を得るには、精製した糖
液を活性炭カラムクロマトグラフィ、市販の充填剤を用
いたゲル過カラムクロマトグラフィ、種々の膜を用い
た膜分離濃縮などを行なえばよい。In order to obtain further highly pure inulooligosaccharide, the purified sugar solution may be subjected to activated carbon column chromatography, gel percolation column chromatography using a commercially available packing material, and membrane separation concentration using various membranes.
つぎに実施例を挙げて本発明をさらに詳しく説明する
が、これに限定されるものではない。Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
実施例 市販イヌリン１％、ＮＺアミン（シェフィールド社製ペ
プトン）３％、塩化アンモニウム0.2％、リン酸二カリ
ウム0.05％を含んだ培地50mlをpH７に調製し、120℃で1
5分間蒸気滅菌する。この滅菌した培地に、バチルスsp.
ＫＫ−４６４５（ＦＥＲＭ Ｐ−８６８９）を一白金耳
接種し、30℃で２日間振盪培養を行なう。培養終了後、
遠心分離機で集菌し、pH７の緩衝液で２〜３回洗浄す
る。ここで湿潤菌体約１ｇが得られる。この湿潤菌体の
酵素としての温度安定性は３０℃迄、反応至適温度は４
０℃、至適pHは７、pH安定性はpH5.5〜7.0の範囲で安定
である。この湿潤菌体を緩衝液５mlに懸濁させ、５％イ
ヌリン水溶液１０mlに本懸濁液１mlを加え、30℃で15時
間反応させる。反応液中のイヌロオリゴ糖の生成比率を
高速液体クロマトグラフィ及びペーパークロマトグラフ
ィを用いて求めると、イヌリンから全オリゴ糖の生成率
は５６％、全オリゴ糖中のそれぞれのオリゴ糖の組成
は、イヌロビオース７％、イヌロトリオース２７％、イ
ヌロテトラオース４７％、イヌロペンタオース１４％、
イヌロヘキサオース以上５％である。反応時間５時間で
のオリゴ糖組成は、イヌロテトラオース８０％にも達す
る。Example 50 ml of a medium containing 1% of commercially available inulin, 3% of NZ amine (Peptone manufactured by Sheffield), 0.2% of ammonium chloride and 0.05% of dipotassium phosphate was adjusted to pH 7 and the pH was adjusted to 1 at 120 ° C.
Sterilize by steam for 5 minutes. Bacillus sp.
One platinum loop of KK-4645 (FERM P-8689) is inoculated and shake culture is performed at 30 ° C. for 2 days. After culturing,
The cells are collected in a centrifuge and washed with a pH 7 buffer solution 2-3 times. Here, about 1 g of wet cells is obtained. The temperature stability of the wet cells as an enzyme is up to 30 ° C, and the optimum reaction temperature is 4
The temperature is 0 ° C, the optimum pH is 7, and the pH stability is stable in the range of pH 5.5 to 7.0. The wet cells are suspended in 5 ml of a buffer solution, 1 ml of this suspension is added to 10 ml of a 5% aqueous solution of inulin, and the mixture is reacted at 30 ° C. for 15 hours. When the production ratio of inulooligosaccharides in the reaction solution was determined using high performance liquid chromatography and paper chromatography, the production ratio of all oligosaccharides from inulin was 56%, and the composition of each oligosaccharide in all oligosaccharides was 7% inurobiose. , Inulotriose 27%, inulotetraose 47%, inulopentaose 14%,
It is 5% or more than inulohexaose. The oligosaccharide composition at the reaction time of 5 hours reaches 80% of inulotetraose.
フロントページの続き (72)発明者 本坊 慶吉 鹿児島県鹿児島市薬師１丁目７番33号 (72)発明者 門間 充 茨城県新治郡桜村吾妻２丁目1506番地809 −307 (72)発明者 貝沼 圭二 茨城県新治郡桜村並木３−619Front Page Continuation (72) Inventor Keikichi Honbo 1-373 Yakushi Yakushi, Kagoshima City, Kagoshima Prefecture (72) Inventor Mitsuru Monma 2-1506 Azuma Sakuramura, Shinji District, Ibaraki Prefecture 809-307 (72) Keiji Kainuma 3-619 Namiki, Sakura Village, Shinji District, Ibaraki Prefecture
Claims (1)
してイヌロテトラオースを生成する酵素を産生する新規
な菌、バチルスsp.ＫＫ−４６４５。1. A novel bacterium, Bacillus sp. KK-4645, which hydrolyzes inulin to produce an enzyme mainly producing inulotetraose in the early stage of the reaction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61072455A JPH0634711B2 (en) | 1986-04-01 | 1986-04-01 | Bacillus SP. KK-4645 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61072455A JPH0634711B2 (en) | 1986-04-01 | 1986-04-01 | Bacillus SP. KK-4645 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS62232380A JPS62232380A (en) | 1987-10-12 |
| JPH0634711B2 true JPH0634711B2 (en) | 1994-05-11 |
Family
ID=13489794
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61072455A Expired - Fee Related JPH0634711B2 (en) | 1986-04-01 | 1986-04-01 | Bacillus SP. KK-4645 |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0634711B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9829041B2 (en) | 2013-05-28 | 2017-11-28 | Ntn Corporation | Rolling bearing |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5089402A (en) * | 1989-02-28 | 1992-02-18 | Mitsubishi Kasei Corporation | Exo-type hydrolase capable of hydrolyzing a fructan only every 3 or 4 sugar units |
| CN108949857B (en) * | 2018-07-26 | 2020-09-04 | 江南大学 | Method for synthesizing inulin by using inulin as substrate |
-
1986
- 1986-04-01 JP JP61072455A patent/JPH0634711B2/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9829041B2 (en) | 2013-05-28 | 2017-11-28 | Ntn Corporation | Rolling bearing |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS62232380A (en) | 1987-10-12 |
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