JPH053280B2 - - Google Patents
Info
- Publication number
- JPH053280B2 JPH053280B2 JP13969485A JP13969485A JPH053280B2 JP H053280 B2 JPH053280 B2 JP H053280B2 JP 13969485 A JP13969485 A JP 13969485A JP 13969485 A JP13969485 A JP 13969485A JP H053280 B2 JPH053280 B2 JP H053280B2
- Authority
- JP
- Japan
- Prior art keywords
- rhamnose
- flavonoid
- highly purified
- enzyme
- solution
- 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 - Lifetime
Links
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims description 37
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 claims description 27
- PNNNRSAQSRJVSB-BXKVDMCESA-N aldehydo-L-rhamnose Chemical compound C[C@H](O)[C@H](O)[C@@H](O)[C@@H](O)C=O PNNNRSAQSRJVSB-BXKVDMCESA-N 0.000 claims description 26
- 108090000790 Enzymes Proteins 0.000 claims description 17
- 102000004190 Enzymes Human genes 0.000 claims description 17
- 239000000243 solution Substances 0.000 claims description 17
- 238000006243 chemical reaction Methods 0.000 claims description 13
- -1 flavonoid compound Chemical class 0.000 claims description 12
- 229930003935 flavonoid Natural products 0.000 claims description 11
- 229930182486 flavonoid glycoside Natural products 0.000 claims description 11
- 150000007955 flavonoid glycosides Chemical class 0.000 claims description 11
- 235000017173 flavonoids Nutrition 0.000 claims description 11
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 230000002378 acidificating effect Effects 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 239000003495 polar organic solvent Substances 0.000 claims description 3
- 238000001179 sorption measurement Methods 0.000 claims description 3
- 230000001376 precipitating effect Effects 0.000 claims description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 6
- ARGKVCXINMKCAZ-UHFFFAOYSA-N neohesperidine Natural products C1=C(O)C(OC)=CC=C1C1OC2=CC(OC3C(C(O)C(O)C(CO)O3)OC3C(C(O)C(O)C(C)O3)O)=CC(O)=C2C(=O)C1 ARGKVCXINMKCAZ-UHFFFAOYSA-N 0.000 description 6
- 239000001100 (2S)-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-one Substances 0.000 description 5
- QUQPHWDTPGMPEX-UHFFFAOYSA-N Hesperidine Natural products C1=C(O)C(OC)=CC=C1C1OC2=CC(OC3C(C(O)C(O)C(COC4C(C(O)C(O)C(C)O4)O)O3)O)=CC(O)=C2C(=O)C1 QUQPHWDTPGMPEX-UHFFFAOYSA-N 0.000 description 5
- QUQPHWDTPGMPEX-UTWYECKDSA-N aurantiamarin Natural products COc1ccc(cc1O)[C@H]1CC(=O)c2c(O)cc(O[C@@H]3O[C@H](CO[C@@H]4O[C@@H](C)[C@H](O)[C@@H](O)[C@H]4O)[C@@H](O)[C@H](O)[C@H]3O)cc2O1 QUQPHWDTPGMPEX-UTWYECKDSA-N 0.000 description 5
- APSNPMVGBGZYAJ-GLOOOPAXSA-N clematine Natural products COc1cc(ccc1O)[C@@H]2CC(=O)c3c(O)cc(O[C@@H]4O[C@H](CO[C@H]5O[C@@H](C)[C@H](O)[C@@H](O)[C@H]5O)[C@@H](O)[C@H](O)[C@H]4O)cc3O2 APSNPMVGBGZYAJ-GLOOOPAXSA-N 0.000 description 5
- ADSYMQORONDIDD-ZJHVPRRPSA-N hesperetin 7-O-beta-D-glucoside Chemical compound C1=C(O)C(OC)=CC=C1[C@H]1OC2=CC(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O)=CC(O)=C2C(=O)C1 ADSYMQORONDIDD-ZJHVPRRPSA-N 0.000 description 5
- VUYDGVRIQRPHFX-UHFFFAOYSA-N hesperidin Natural products COc1cc(ccc1O)C2CC(=O)c3c(O)cc(OC4OC(COC5OC(O)C(O)C(O)C5O)C(O)C(O)C4O)cc3O2 VUYDGVRIQRPHFX-UHFFFAOYSA-N 0.000 description 5
- QUQPHWDTPGMPEX-QJBIFVCTSA-N hesperidin Chemical compound C1=C(O)C(OC)=CC=C1[C@H]1OC2=CC(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO[C@H]4[C@@H]([C@H](O)[C@@H](O)[C@H](C)O4)O)O3)O)=CC(O)=C2C(=O)C1 QUQPHWDTPGMPEX-QJBIFVCTSA-N 0.000 description 5
- 229940025878 hesperidin Drugs 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 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 4
- 239000003463 adsorbent Substances 0.000 description 4
- 239000013078 crystal Substances 0.000 description 4
- JHIVVAPYMSGYDF-UHFFFAOYSA-N cyclohexanone Chemical compound O=C1CCCCC1 JHIVVAPYMSGYDF-UHFFFAOYSA-N 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 108010001078 naringinase Proteins 0.000 description 4
- BNRKZHXOBMEUGK-NRBMBCGPSA-N (2r,3r,4r,5r,6s)-6-methyloxane-2,3,4,5-tetrol;hydrate Chemical compound O.C[C@@H]1O[C@@H](O)[C@H](O)[C@H](O)[C@H]1O BNRKZHXOBMEUGK-NRBMBCGPSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 241001540751 Talaromyces ruber Species 0.000 description 3
- 239000003957 anion exchange resin Substances 0.000 description 3
- 108010030923 hesperidinase Proteins 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000001606 7-[(2S,3R,4S,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)-3-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxyoxan-2-yl]oxy-5-hydroxy-2-(4-hydroxyphenyl)chroman-4-one Substances 0.000 description 2
- 241000228212 Aspergillus Species 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 241000228143 Penicillium Species 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 229930182478 glucoside Natural products 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- ZXEKIIBDNHEJCQ-UHFFFAOYSA-N isobutanol Chemical compound CC(C)CO ZXEKIIBDNHEJCQ-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N isopropyl alcohol Natural products CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 229930019673 naringin Natural products 0.000 description 2
- DFPMSGMNTNDNHN-ZPHOTFPESA-N naringin Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](OC=2C=C3O[C@@H](CC(=O)C3=C(O)C=2)C=2C=CC(O)=CC=2)O[C@H](CO)[C@@H](O)[C@@H]1O DFPMSGMNTNDNHN-ZPHOTFPESA-N 0.000 description 2
- 229940052490 naringin Drugs 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- YFVGIJBUXMQFOF-PJOVQGMDSA-N 5-hydroxy-2-(4-methoxyphenyl)-7-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-[[(2r,3r,4r,5r,6s)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxymethyl]oxan-2-yl]oxychromen-4-one Chemical compound C1=CC(OC)=CC=C1C(OC1=C2)=CC(=O)C1=C(O)C=C2O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@H]2[C@@H]([C@H](O)[C@@H](O)[C@H](C)O2)O)O1 YFVGIJBUXMQFOF-PJOVQGMDSA-N 0.000 description 1
- TWCMVXMQHSVIOJ-UHFFFAOYSA-N Aglycone of yadanzioside D Natural products COC(=O)C12OCC34C(CC5C(=CC(O)C(O)C5(C)C3C(O)C1O)C)OC(=O)C(OC(=O)C)C24 TWCMVXMQHSVIOJ-UHFFFAOYSA-N 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
- PLMKQQMDOMTZGG-UHFFFAOYSA-N Astrantiagenin E-methylester Natural products CC12CCC(O)C(C)(CO)C1CCC1(C)C2CC=C2C3CC(C)(C)CCC3(C(=O)OC)CCC21C PLMKQQMDOMTZGG-UHFFFAOYSA-N 0.000 description 1
- YFVGIJBUXMQFOF-SAXLCNSLSA-N Linarin Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@H](O)[C@H](Oc2cc(O)c3C(=O)C=C(c4ccc(OC)cc4)Oc3c2)O1)[C@H]1[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O1 YFVGIJBUXMQFOF-SAXLCNSLSA-N 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 239000003929 acidic solution Substances 0.000 description 1
- 238000005377 adsorption chromatography Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- PFOARMALXZGCHY-UHFFFAOYSA-N homoegonol Natural products C1=C(OC)C(OC)=CC=C1C1=CC2=CC(CCCO)=CC(OC)=C2O1 PFOARMALXZGCHY-UHFFFAOYSA-N 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- ARGKVCXINMKCAZ-UZRWAPQLSA-N neohesperidin Chemical compound C1=C(O)C(OC)=CC=C1[C@H]1OC2=CC(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O[C@H]3[C@@H]([C@H](O)[C@@H](O)[C@H](C)O3)O)=CC(O)=C2C(=O)C1 ARGKVCXINMKCAZ-UZRWAPQLSA-N 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
Description
【発明の詳細な説明】
本発明は、Ｌ−ラムノースを有するフラボノイ
ド配糖体にラムノシド結合を選択的に開裂する酵
素を作用させ、Ｌ−ラムノースのみを該フラボノ
イド配糖体から遊離したのち、反応液からラムノ
シド結合の切断されたフラボノイド化合物を沈澱
させ除去して、残存する溶液から高純度のＬ−ラ
ムノースをうることを特徴とするＬ−ラムノース
の製造法に関する。
Ｌ−ラムノースは化学合成の困難な単糖である
が、近年種々の用途が開発され需要が拡大しつつ
ある化合物である。Ｌ−ラムノースは、天然には
微生物の細胞壁やフラボノイド配糖体の中に存在
している。特にわが国の特産品である温州ミカン
中に大量に含まれているヘスペリジン、グレープ
フルーツおよびザボンなどに含まれているナリン
ジン、ナツミカンおよび八朔中に含まれるネオヘ
スペリジンなどのフラボノイド配糖体において
は、Ｌ−ラムノースは、グルコースとアグリコン
とがグルコシド結合してできているフラボノイド
化合物（たとえば、ヘスペレチン−７−グルコシ
ド）のグルコースと、ラムノシド結合している。
これらのフラボノイド配糖体は用途が乏しいため
その利用が一般的に行なわれていなかつたが、本
発明者はこれらが有用なＬ−ラムノースを含むこ
とに注目し、Ｌ−ラムノースのすぐれた原料源と
して該フラボノイド配糖体から新規なプロセスに
より高純度のＬ−ラムノースを高収率で取得する
製法を開発し、本発明を完成するにいたつた。
本発明の方法はヘスペリジン、ナリンジン、ポ
ンシリン、リナリンおよびネオヘスペリジンなど
のラムノースを有するフラボノイド配糖体を、菌
体が産生するヘスペリジナーゼおよびナリンジナ
ーゼなどのアグリコンとグルコースとのグルコシ
ド結合部には作用せず、ラムノシド結合部のみを
選択的に開裂する酵素（ラムノシド結合を選択的
に開裂する酵素と称する）で処理し、ラムノース
を遊離させ、しかるのちに沈澱析出するフラボノ
イド化合物の大部分を濾別して除去したのち、溶
解している微量のフラボノイド化合物を吸着クロ
マトグラフまたは有機溶媒での処理によつて反応
液から除去し、高純度のＬ−ラムノースをうるこ
とを特徴とする。
本発明の方法によれば、Ｌ−ラムノースを有す
るフラボノイド配糖体からＬ−ラムノースを高収
量でうることができる。
Ｌ−ラムノースを有するフラボノイド配糖体の
基質からＬ−ラムノースのみを選択的に遊離させ
るために、基質のラムノシド結合を開裂する酵素
を使用する。酵素は、純品ばかりでなく種々の精
製段階の酵素を使用することができ、たとえば同
酵素を産生する菌体の培養液を使用してもよい。
使用する酵素としては、アスペルギルス属
（Aspergillus）に属する菌たとえばアスベルギル
ス・ニガー（Aspergillus niger）IAM2531が産
生する酵素（ナリンジナーゼとして市販されてい
る）、ペニシリウム属（Penicillium）に属する菌
たとえばペニシリウム・プルプロゲヌム
（Penicillium purpurogenum）IFO7756が産生す
る酵素（ヘスペリジナーゼとして市販されてい
る）、ハンゼニユラ属（Hansenula）が産生する
酵素など、ペニシリウム属、アスペルギルス属の
酵素またはその類似の反応を有するものが用いら
れる。
酵素または菌体培養液によるラムノシド結合の
開裂後、反応液を、たとえば氷冷することによ
り、Ｌ−ラムノースに切断されたフラボノイド化
合物の大部分が沈澱析出し濾別により反応液から
除去することができる。
微量のフラボノイド化合物が反応液中に溶解し
ているが、これを除去するために、酸性状態にし
水に難溶性の極性有機溶媒を用いてフラボノイド
化合物を完全に抽出除去する方法、またはフラボ
ノイド化合物を吸着する吸着剤たとえばスチレン
DVB系吸着剤カラム中に反応液を通過させる方
法が用いられる。つぎにアニオン交換樹脂カラム
中に水溶液を通し脱塩し濃縮したのち、メタノー
ルの添加により高純度のＬ−ラムノースを収率よ
く結晶として単離することができる。
反応条件としては、使用する酵素の至適PHにお
いて反応が行なわれ、酵素を種類によつて異なる
が、通常PH３〜７が適している。フラボノイド化
合物の除去は、酸性条件下、好ましくはPH３以下
で、イソ−ブタノール、シクロヘキサノン、イソ
−アミルアルコールまたはｎ−ブタノールのよう
な極性有機溶媒で抽出するか、または三菱化成工
業(株)製のSP−207、HP−20、Ｓ−861、Ｓ−862
またはXAD−４のようなスチレンPVB系吸着剤
（約３％）を用いて吸着除去することによつて行
なう。
つぎに実施例を用いて本発明をさらに詳しく説
明するが、本発明はもとよりこれらに限られるも
のではない。
実施例 １
ヘスペリジン25ｇを0.05N水酸化ナトリウム溶
液２に溶解し、塩酸でPHを3.5に調整し、これ
に市販のヘスペリジナーゼ100mgを添加し、50℃
にて４時間振とうして反応させた。ラムノシド結
合の開裂は、ほぼ理論値の100％であつた。反応
終了後、氷冷し、大部分のヘスペレチン−７−グ
ルコシドを沈澱させ除去した。つぎに硫酸の添加
によりPHを１としたのち、シクロヘキサノン50ml
によつて水溶液中に存在する微量のヘスペレチン
−７−グルコシドを２回抽出除去した。水相はア
ニオン交換樹脂IR−120で脱塩、濃縮し、つづい
てメタノールを加え冷却しＬ−ラムノース・１水
和物5.5ｇを白色結晶としてえた（収率：75％、
m.p.：93.5℃、［α］20 D＋9.1）。
実施例 ２
ナリンジン25ｇを0.05N水酸化ナトリウム溶液
２に溶解したのち、塩酸によりPHを4.5に調整
し、これに市販のナリンジナーゼ80mgを添加し、
40℃にて４時間振とうして反応させた。反応液を
実施例１と同じ操作にしたがつて処理してＬ−ラ
ムノース・１水和物6.0ｇを白色結晶としてえた
（収率：76％、m.p.：93.0℃、［α］20 D＋9.0）。
実施例 ３
実施例１にしたがつてヘスペリジン25ｇを加水
分解したのち、析出させたヘスペレチン−７−グ
ルコシドの大部分を冷却除去し、塩酸の添加によ
りPH３に調整した。一方、内径が２cmで長さが30
cmのカラムにスチレンDVB系吸着剤Sp−207を
充填し、イオン交換水およびPHが３の塩酸酸性液
により充分に洗浄したのち、上記の調整溶液をカ
ラム中に通し残存するヘスペレチン−７−グルコ
シドを吸着除去した。つづいてアニオン交換樹脂
IR−120カラムにより脱塩、濃縮し、メタノール
を加え冷却してＬ−ラムノース・１水和物5.0ｇ
を白色結晶としてえた（収率：67％、m.p.：93.0
℃、［α］20 D＋9.0）。
実施例 ４
ペブトン0.5部、イーストエキス0.3部およびグ
ルコース0.1部を有しPHを5.0に調整した培地にハ
ンゼニウラ属SP株を植菌して40℃にて48時間振
とう培養した培養液100mlを、0.2％のヘスペリジ
ンを有する水溶液100mlに加え酢酸バツフアーに
よりPH４に調整した。これを40℃にて２時間振と
うして反応させた。この反応によりヘスペリジン
に結合したＬ−ラムノースの約50％が選択的に切
断された。反応の進行状況については薄層クロマ
トグラム（展開液：酢酸エチル／イソプロピルア
ルコール／水の系）により確認を行なつた。
以下実施例５〜８を実施例１〜４と同様にして
行なつた結果を第１表に示す。
【表】Detailed Description of the Invention The present invention involves treating a flavonoid glycoside containing L-rhamnose with an enzyme that selectively cleaves rhamnoside bonds, releasing only L-rhamnose from the flavonoid glycoside, and then reacting the flavonoid glycoside. The present invention relates to a method for producing L-rhamnose, which comprises precipitating and removing a flavonoid compound having a cleaved rhamnoside bond from a solution, and obtaining highly purified L-rhamnose from the remaining solution. Although L-rhamnose is a monosaccharide that is difficult to chemically synthesize, it is a compound for which various uses have been developed in recent years and demand is increasing. L-rhamnose naturally exists in the cell walls of microorganisms and flavonoid glycosides. In particular, L- Rhamnose has a rhamnoside bond with glucose of a flavonoid compound (for example, hesperetin-7-glucoside), which is formed by a glucoside bond of glucose and an aglycone.
These flavonoid glycosides have not been generally utilized due to their lack of uses; however, the present inventor noticed that they contain useful L-rhamnose, and identified them as an excellent raw material source for L-rhamnose. As a result, we have developed a method for obtaining highly purified L-rhamnose in high yield from the flavonoid glycoside using a novel process, and have completed the present invention. The method of the present invention uses rhamnose-containing flavonoid glycosides such as hesperidin, naringin, poncillin, linarin, and neohesperidin without acting on the glucoside bond between glucose and aglycones such as hesperidinase and naringinase produced by bacterial cells. After treatment with an enzyme that selectively cleaves only the rhamnoside bond (referred to as an enzyme that selectively cleaves the rhamnoside bond) to liberate rhamnose, most of the flavonoid compounds that precipitate are then removed by filtration. , is characterized in that trace amounts of dissolved flavonoid compounds are removed from the reaction solution by adsorption chromatography or treatment with an organic solvent to obtain highly pure L-rhamnose. According to the method of the present invention, L-rhamnose can be obtained in high yield from a flavonoid glycoside containing L-rhamnose. In order to selectively release only L-rhamnose from a flavonoid glycoside substrate containing L-rhamnose, an enzyme that cleaves the rhamnoside bond of the substrate is used. Not only pure enzymes but also enzymes in various stages of purification can be used. For example, a culture solution of microbial cells that produce the enzyme may be used. The enzymes used include the enzyme produced by bacteria belonging to the genus Aspergillus, such as Aspergillus niger IAM2531 (commercially available as naringinase), and the enzyme produced by bacteria belonging to the genus Penicillium, such as Penicillium purpurogenum (commercially available as naringinase). Enzymes produced by Penicillium purpurogenum (Penicillium purpurogenum) IFO7756 (commercially available as hesperidinase), enzymes produced by Hansenula, Penicillium genus, Aspergillus genus enzymes, or those having similar reactions are used. After the rhamnoside bond is cleaved by an enzyme or a bacterial cell culture solution, most of the flavonoid compounds cleaved to L-rhamnose are precipitated by cooling the reaction solution, for example, on ice, and can be removed from the reaction solution by filtration. can. A trace amount of flavonoid compounds are dissolved in the reaction solution, but in order to remove this, the flavonoid compounds can be completely extracted and removed using a polar organic solvent that is poorly soluble in water in an acidic state, or Adsorbent such as styrene
A method is used in which the reaction solution is passed through a DVB-based adsorbent column. Next, the aqueous solution is passed through an anion exchange resin column to desalt and concentrate, and then, by adding methanol, highly purified L-rhamnose can be isolated as crystals in good yield. As for the reaction conditions, the reaction is carried out at the optimum pH of the enzyme used, and although it varies depending on the type of enzyme, a pH of 3 to 7 is usually suitable. Flavonoid compounds can be removed by extraction with polar organic solvents such as iso-butanol, cyclohexanone, iso-amyl alcohol or n-butanol under acidic conditions, preferably below pH 3, or with SP-207, HP-20, S-861, S-862
Alternatively, removal may be carried out by adsorption using a styrene PVB adsorbent (approximately 3%) such as XAD-4. Next, the present invention will be explained in more detail using Examples, but the present invention is not limited to these. Example 1 25g of hesperidin was dissolved in 0.05N sodium hydroxide solution 2, the pH was adjusted to 3.5 with hydrochloric acid, 100mg of commercially available hesperidinase was added, and the solution was heated at 50°C.
The mixture was shaken for 4 hours to react. The cleavage of the rhamnoside bond was approximately 100% of the theoretical value. After the reaction was completed, the mixture was cooled on ice to precipitate and remove most of hesperetin-7-glucoside. Next, after adjusting the pH to 1 by adding sulfuric acid, 50ml of cyclohexanone was added.
Trace amounts of hesperetin-7-glucoside present in the aqueous solution were extracted and removed twice. The aqueous phase was desalted and concentrated using anion exchange resin IR-120, and then methanol was added and cooled to obtain 5.5 g of L-rhamnose monohydrate as white crystals (yield: 75%,
mp: 93.5℃, [α] 20D +9.1 ). Example 2 After dissolving 25 g of naringin in 0.05N sodium hydroxide solution 2, the pH was adjusted to 4.5 with hydrochloric acid, and 80 mg of commercially available naringinase was added thereto.
The mixture was shaken and reacted at 40°C for 4 hours. The reaction solution was treated in the same manner as in Example 1 to obtain 6.0 g of L-rhamnose monohydrate as white crystals (yield: 76%, mp: 93.0°C, [α] 20 D +9. 0). Example 3 After 25 g of hesperidin was hydrolyzed according to Example 1, most of the precipitated hesperetin-7-glucoside was removed by cooling, and the pH was adjusted to 3 by adding hydrochloric acid. On the other hand, the inner diameter is 2cm and the length is 30cm.
A cm column was filled with styrene DVB adsorbent Sp-207, thoroughly washed with ion-exchanged water and an acidic solution of hydrochloric acid with a pH of 3, and the above prepared solution was passed through the column to remove residual hesperetin-7-glucoside. was removed by adsorption. Next, anion exchange resin
Desalt and concentrate using an IR-120 column, add methanol and cool to obtain 5.0 g of L-rhamnose monohydrate.
was obtained as white crystals (yield: 67%, mp: 93.0
°C, [α] 20 D +9.0). Example 4 A medium containing 0.5 part of pebtone, 0.3 part of yeast extract, and 0.1 part of glucose and adjusted to PH 5.0 was inoculated with SP strain of Hanseniura and cultured with shaking at 40°C for 48 hours. The pH was adjusted to 4 with an acetic acid buffer in addition to 100 ml of an aqueous solution containing 0.2% hesperidin. This was shaken and reacted at 40°C for 2 hours. Approximately 50% of L-rhamnose bound to hesperidin was selectively cleaved by this reaction. The progress of the reaction was confirmed by thin layer chromatography (developing solution: ethyl acetate/isopropyl alcohol/water system). Examples 5 to 8 were carried out in the same manner as Examples 1 to 4, and the results are shown in Table 1. 【table】
Claims (1)
のラムノシド結合を選択的に開裂する酵素を作用
させ、Ｌ−ラムノースのみを該フラボノイド配糖
体から遊離したのち、反応液からラムノシド結合
の切断されたフラボノイド化合物を沈澱させ除去
して、残存する溶液から高純度のＬ−ラムノース
をうることを特徴とするＬ−ラムノースの製造
法。 ２ 前記高純度のＬ−ラムノースを含む水溶液中
に残存する微量のフラボノイド化合物を酸性状態
において水と混和しがたい極性有機溶媒で抽出除
去することによつてさらに高純度のＬ−ラムノー
スをうる特許請求の範囲第１項記載の製造法。 ３ 前記高純度のＬ−ラムノースを含む水溶液中
に残存する微量のフラボノイド化合物を酸性状態
において吸着クロマトグラフにより吸着除去する
ことによつてさらに高純度のＬ−ラムノースをう
る特許請求の範囲第１項記載の製造法。[Scope of Claims] 1. After releasing only L-rhamnose from the flavonoid glycoside by acting with an enzyme that selectively cleaves the rhamnoside bond of the flavonoid glycoside containing L-rhamnose, the rhamnoside bond is removed from the reaction solution. 1. A method for producing L-rhamnose, which comprises precipitating and removing the cleaved flavonoid compound and obtaining highly purified L-rhamnose from the remaining solution. 2 A patent for obtaining even higher purity L-rhamnose by extracting and removing trace amounts of flavonoid compounds remaining in the aqueous solution containing the high-purity L-rhamnose in an acidic state with a polar organic solvent that is immiscible with water. The manufacturing method according to claim 1. 3. Claim 1: Further highly purified L-rhamnose is obtained by adsorbing and removing trace amounts of flavonoid compounds remaining in the aqueous solution containing the highly purified L-rhamnose using an adsorption chromatograph in an acidic state. Manufacturing method described.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP13969485A JPS62293A (en) | 1985-06-26 | 1985-06-26 | Production of l-rhamnose |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP13969485A JPS62293A (en) | 1985-06-26 | 1985-06-26 | Production of l-rhamnose |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS62293A JPS62293A (en) | 1987-01-06 |
| JPH053280B2 true JPH053280B2 (en) | 1993-01-14 |
Family
ID=15251239
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP13969485A Granted JPS62293A (en) | 1985-06-26 | 1985-06-26 | Production of l-rhamnose |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS62293A (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ATE190665T1 (en) * | 1992-06-25 | 2000-04-15 | Aventis Res & Tech Gmbh & Co | PSEUDOMONAS AERUGINOSA AND ITS USE FOR PRODUCING L-RHAMNOSE |
| SG54274A1 (en) | 1992-11-27 | 1998-11-16 | Hoechst Ag | Alpha-l-rhamnosidase for obtaining rhamnose a process for its preparation and its use |
| DE19850029A1 (en) | 1998-10-30 | 2000-05-04 | Merck Patent Gmbh | Process for the enzymatic cleavage of rutinosides |
-
1985
- 1985-06-26 JP JP13969485A patent/JPS62293A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS62293A (en) | 1987-01-06 |
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