JPH0191777A - Production of levan fructotransferase - Google Patents
Production of levan fructotransferaseInfo
- Publication number
- JPH0191777A JPH0191777A JP24588687A JP24588687A JPH0191777A JP H0191777 A JPH0191777 A JP H0191777A JP 24588687 A JP24588687 A JP 24588687A JP 24588687 A JP24588687 A JP 24588687A JP H0191777 A JPH0191777 A JP H0191777A
- Authority
- JP
- Japan
- Prior art keywords
- levan
- fructotransferase
- pseudomonas
- medium containing
- production
- 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.)
- Granted
Links
- 108010022991 levan fructotransferase Proteins 0.000 title claims abstract description 14
- 238000004519 manufacturing process Methods 0.000 title claims description 13
- 241000589516 Pseudomonas Species 0.000 claims abstract description 10
- AIHDCSAXVMAMJH-GFBKWZILSA-N levan 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)(CO[C@@H]2[C@H]([C@H](O)[C@@](O)(CO)O2)O)O1 AIHDCSAXVMAMJH-GFBKWZILSA-N 0.000 claims abstract description 10
- 229930091371 Fructose Natural products 0.000 claims abstract description 7
- 239000005715 Fructose Substances 0.000 claims abstract description 7
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims abstract description 7
- 229920000642 polymer Polymers 0.000 claims abstract description 7
- 238000012258 culturing Methods 0.000 claims abstract description 6
- 241000589540 Pseudomonas fluorescens Species 0.000 claims abstract description 5
- 241000894006 Bacteria Species 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 abstract description 20
- 102000004190 Enzymes Human genes 0.000 abstract description 20
- 235000003599 food sweetener Nutrition 0.000 abstract description 2
- 239000003765 sweetening agent Substances 0.000 abstract description 2
- 108090000992 Transferases Proteins 0.000 abstract 2
- 102000004357 Transferases Human genes 0.000 abstract 2
- 230000000813 microbial effect Effects 0.000 abstract 2
- 239000000243 solution Substances 0.000 description 10
- 239000002609 medium Substances 0.000 description 9
- 230000000694 effects Effects 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 5
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 230000001580 bacterial effect Effects 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
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 2
- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical compound CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 230000005526 G1 to G0 transition Effects 0.000 description 2
- GLZPCOQZEFWAFX-UHFFFAOYSA-N Geraniol Chemical compound CC(C)=CCCC(C)=CCO GLZPCOQZEFWAFX-UHFFFAOYSA-N 0.000 description 2
- 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 2
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 241001524178 Paenarthrobacter ureafaciens Species 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- 229940090248 4-hydroxybenzoic acid Drugs 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 239000005792 Geraniol Substances 0.000 description 1
- GLZPCOQZEFWAFX-YFHOEESVSA-N Geraniol Natural products CC(C)=CCC\C(C)=C/CO GLZPCOQZEFWAFX-YFHOEESVSA-N 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 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
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 229920002472 Starch Polymers 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
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- GTDPSWPPOUPBNX-UHFFFAOYSA-N ac1mqpva Chemical compound CC12C(=O)OC(=O)C1(C)C1(C)C2(C)C(=O)OC1=O GTDPSWPPOUPBNX-UHFFFAOYSA-N 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 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
- 108010051210 beta-Fructofuranosidase Proteins 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000006114 decarboxylation reaction Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000003028 enzyme activity measurement method Methods 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 229940113087 geraniol Drugs 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229910000037 hydrogen sulfide Inorganic materials 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- -1 inositone Chemical compound 0.000 description 1
- 235000011073 invertase Nutrition 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 235000002867 manganese chloride Nutrition 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- 229940099607 manganese chloride Drugs 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000019086 sulfide ion homeostasis Effects 0.000 description 1
- 235000019605 sweet taste sensations Nutrition 0.000 description 1
- 229960004295 valine Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
産業上の利用分野
本発明は、レバンまたはフレインよりジ−ローフラクト
シルクラノース ２．６’：６．２’ジアンハイドライ
ドを生成させる酵素・レバンフラクトトランスフェラー
ゼの製造法に関するものである。DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for producing levan fructotransferase, an enzyme that produces g-low fructosyl curanose 2.6':6.2' dianhydride from levan or Frein. It is.
従来の技術
レバンフラクトトランスフェラーゼは、多糖類・レバン
またはフレインを分解して下記構造式のジーＤ−７ラク
トシルフラノース ２，６’：６，２’アンハイドライ
ド（略称ＤＦＡＩ！／）を生成させる酵素である。Conventional technology Levan fructotransferase is an enzyme that decomposes polysaccharides such as levan or frein to produce G-D-7 lactosylfuranose 2,6':6,2' anhydride (abbreviated as DFAI!/) having the following structural formula. It is.
ＬＪ
−ＩＱ
ＤＦＡＩＶはフラクトースに似たされやかな甘味を有す
る物質である。したがって、レバンまたはフレインから
ＤＦＡＩＶを安価に製造することができれば、この物質
を甘味料として利用することが可能になるが、それに必
要なレバンフラクトトランスフェラーゼが従来は入手困
難であった。LJ-IQ DFAIV is a substance with a mild sweet taste similar to fructose. Therefore, if DFAIV could be produced inexpensively from levan or Frein, it would be possible to use this substance as a sweetener, but the levan fructotransferase required for this has hitherto been difficult to obtain.
従来、レバン７ラクトトランス７エラーゼはアースロバ
フタ−・ウレアファシェンスにより生産されることが知
られている（Ｃａｒｂｏｈｙｄｒａｔｅ Ｉｔｅｓｅｓ
ｒｃｈ、第９９巻。It has been known that levan-7 lactotrans-7 erase is produced by Arthrobacter ureafaciens (Carbohydrate Iteses).
rch, Volume 99.
１９７〜２０４頁、１００年）。しかしながら、アース
ロバクターーウレアファシエンスのレバンフラクトトラ
ンスフェラーゼ産生力は弱く、この菌を用いる方法によ
っテ高品質のレバン７ラクトトランスフエラーゼヲ経済
的に得ることは困難である。pp. 197-204, 100). However, the levan 7 lactotransferase production ability of Arthrobacter ureafaciens is weak, and it is difficult to economically obtain high quality levan 7 lactotransferase by a method using this bacterium.
発明が解決しようとする問題点
したがって本発明の目的は、レバンフラクトトランスフ
ェラーゼを安価に且つ豊富に生産し得る方法を提供する
ことにある。Problems to be Solved by the Invention Therefore, an object of the present invention is to provide a method for producing levanfructotransferase at low cost and in abundance.
問題点を解決するための手段
上記目的を達成するため、本発明者らは多くの微生物に
ついてレバンフラクトトランスフェラーゼ産生能の観点
からスクリーニングを行い、シュードモナス属に属する
一細菌すなわちシュードモナス・フルオレッセンスＭＺ
、 Ｎｏ、９４９ （微工研菌寄第９２３５号）がすぐ
れた性質を有することを知った。Means for Solving the Problems In order to achieve the above object, the present inventors screened many microorganisms from the viewpoint of their ability to produce levanfructotransferase, and found one bacterium belonging to the genus Pseudomonas, namely Pseudomonas fluorescens MZ.
, No. 949 (Feikoken Bibori No. 9235) was found to have excellent properties.
本発明は上記知見に基づくものであって、フラクトース
のポリマーを含有する培地を用いてシュードモナス属に
属するレバン７ラクトトランスフエラーゼ生産菌を培養
し、培養物からレバンフラクトトランスフェラーゼを採
取することを特徴とするレバンフラクトトランスフェラ
ーゼの製造法を提供するものである。The present invention is based on the above findings, and is characterized by culturing levan-7 lactotransferase-producing bacteria belonging to the genus Pseudomonas using a medium containing a fructose polymer, and collecting levan-fructotransferase from the culture. The present invention provides a method for producing levan fructotransferase.
上記ＭＺ Ｎｏ、９４９株は、広島県尾道市の土壌より
分離されたものであって、その菌学的性質は次のとおり
である。The above MZ No. 949 strain was isolated from the soil of Onomichi City, Hiroshima Prefecture, and its mycological properties are as follows.
（ａ）形態
■細胞の形および大きさ
０．５〜０．７Ｘ２．０〜３．５μ重の桿菌■運動性：
あり。１本以上の極鞭毛を有する。(a) Morphology ■ Cell shape and size 0.5-0.7 x 2.0-3.5μ rods ■ Motility:
can be. It has one or more polar flagella.
■胞子：なし ■グラム染色性：陰性 ■抗酸性：なし くｂ）生育 ■肉汁寒天斜面培養 中程度の発育；透明；やや赤味を帯びる。■Spores: None ■Gram staining: negative ■Anti-acidity: None b) Growth ■Meat juice agar slant culture Medium growth; transparent; slightly reddish.
■肉汁液体培養：中程度の発育；表面に膜形成。■ Broth liquid culture: Moderate growth; film formation on the surface.
■肉汁ゼラチン穿刺培養：液化しない。■Meat juice gelatin puncture culture: Does not liquefy.
■リドマスミルク ：変化しない。■ Lidmus milk: No change.
■ＢＣＰミルク ：変化しない。■BCP milk: No change.
（ｃ）生理的性質 ■硝酸塩の還元ニする。(c) Physiological properties ■Reduces nitrates.
■脱窒：しない。■Denitrification: No.
■ＭＲテスト ：陰性
■ｖｐテスト ：陰性
■インドールの生成：なし
■硫化水素の生成：なしくＴＳＩ寒天培地）■デン粉の
加水分解：なし
■クエン酸の利用：あり （クリステンセン培地）■無
機窒素源の利用
硝酸塩およびアンモニウム塩を利用する。■MR test: Negative ■VP test: Negative ■Indole production: None ■Hydrogen sulfide production: None (TSI agar medium) ■Starch hydrolysis: None ■Use of citric acid: Yes (Christensen medium) ■Inorganic nitrogen Source Utilization Utilizes nitrate and ammonium salts.
［相］色素の生成：水溶性、蛍光性の色素を生成する。[Phase] Pigment generation: Produces water-soluble, fluorescent dyes.
■オキシダーゼ：陽性 ＠カタラーゼ：陽性 ■生育の範囲：ｐＨ４〜１０で生育する。■Oxidase: positive @catalase: positive ■Growth range: Grows at pH 4-10.
４℃、２７℃で生育する。３７℃で生育しない。Grows at 4℃ and 27℃. Does not grow at 37°C.
■酸素に対する態度：好気性 ［相］０−Ｆテスト ニゲルコースを酸化する。■Attitude towards oxygen: aerobic [Phase] 0-F test oxidizes nigercose.
（ＨＨｈ Ｌ ｅｉｆｓｏｎ法）
■炭水化物の利用＝Ｄ−グルコース、Ｌ−アラビノース
、トレハロース、イノシトーノ呟 Ｌ−バリン、ｐ−ヒ
ドロキシ安息香酸を利用する。シュクロース、ゲラニオ
ール、アセトアミド、エタノールを利用しない。(HHh Leifson method) ■ Utilization of carbohydrates = D-glucose, L-arabinose, trehalose, inositone, L-valine, p-hydroxybenzoic acid. Do not use sucrose, geraniol, acetamide, or ethanol.
Ｏアルギニンの脱炭酸：陽性
［相］グロトカテキン酸の分解：オルト型の開裂以上の
諸性状をバージエイのマニュアル・オブ・デターミネイ
ティブ・バクテリオロジー、第３版（１９７４）の記載
と照合することにより、本菌がシュードモナス・フルオ
レッセンス［ミグラ １８９５］であることを確認した
。Decarboxylation of O-arginine: positive [phase] Decomposition of glotocatechuic acid: cleavage of ortho-type and other properties by comparing with the description in Bergey's Manual of Determinative Bacteriology, 3rd edition (1974) It was confirmed that this bacterium was Pseudomonas fluorescens [Migra 1895].
本発明を実施する場合に用いる培地としては、７うクト
ースのポリマーを酵素誘導基質として含有させることを
除けば、特殊なものを必要としない。シュードモナス属
細菌の培養に通常使用される培地、すなわちグルコース
等の炭素源および酵母エキス、ペプトン等の有機窒素源
、その他硝酸ナトリウム、硫酸マグネシウム、塩化マン
ガンなどの無機塩類等を基本成分として適宜組合せたｐ
Ｈ５〜８程度の培地を用いることができる。培地に含有
させるフラクトースのポリマーとしては、レバン、フレ
イン等を用いることができ、その好適濃度は０．０１〜
３０％、特に好ましくは１〜５％である。The medium used in carrying out the present invention does not require any special medium, except that it contains a polymer of 7 ctose as an enzyme-inducing substrate. A medium normally used for culturing Pseudomonas bacteria, that is, a carbon source such as glucose, yeast extract, an organic nitrogen source such as peptone, and other inorganic salts such as sodium nitrate, magnesium sulfate, and manganese chloride, etc., were appropriately combined as basic ingredients. p
A medium of about H5 to H8 can be used. Levan, Frein, etc. can be used as the fructose polymer to be contained in the medium, and the preferred concentration thereof is 0.01 to 0.01.
30%, particularly preferably 1-5%.
培養は、シュードモナス属細菌培養の常法に従って行う
ことができる。目的とする酵素は菌体外に生産される。The culture can be performed according to a conventional method for culturing Pseudomonas bacteria. The target enzyme is produced outside the bacterial cell.
生産は菌の対数増殖期に始まり、定常期にほぼ最大生産
量に達するので、通常は定常期に達したあと適当な時期
に培養を打切る。Production begins during the logarithmic growth phase of the bacterium and reaches almost maximum production during the stationary phase, so culture is usually discontinued at an appropriate time after the stationary phase is reached.
培養終了後は直ちに遠心分離して菌体を除去する。Immediately after culturing, centrifuge to remove bacterial cells.
シュードモナス菌はβ−フラクトシダーゼを生産しない
ので、菌体を除いただけの培養液でも粗酵素液として利
用できるが、これを酵素精製の常法に従って精製すれば
、より活性が強く、利用し易いレバンフラクトトランス
フェラーゼを得ることができる。Since Pseudomonas does not produce β-fructosidase, the culture solution from which the bacterial cells have been removed can be used as a crude enzyme solution, but if this is purified according to the conventional enzyme purification method, it is possible to obtain a highly active and easily used enzyme solution. fructotransferase can be obtained.
得られるレバンフラクトトランスフェラーゼは安定で、
冷蔵庫で数カ月間保存可能であり、冷凍庫中ならばさら
に長期間保存することができる。The resulting levan fructotransferase is stable and
It can be stored in the refrigerator for several months, and even longer in the freezer.
発明の効果
後記実施例が実証するように、本発明の製法によればア
ースロバ、フタ−属細菌を用いる従来の製法よりもはる
かに高い収率でレバン７ラクトトランス７エラーゼが得
られ、したがってこの酵素を安価に且つ豊富に提供する
ことが可能になる。Effects of the Invention As demonstrated by the Examples described below, the production method of the present invention allows levan-7 lactotrans-7 erase to be obtained in a much higher yield than the conventional production method using bacteria belonging to the genus Arthrobacteria and Futa. Enzymes can be provided inexpensively and in abundance.
実施例
以下、実施例を示して本発明を説明する。なお、各側に
おいて示した酵素活性は、次の方法により測定したもの
である。EXAMPLES The present invention will be explained below with reference to Examples. The enzyme activity shown on each side was measured by the following method.
酵素活性測定法：適当に希釈した酵素液１鳳１と２％レ
バン溶液（ｐＨ６，０の０．１Ｍ酢酸緩衝液使用）１ｍ
ｌとを混合し、４０℃で１時間反応させたのち、生成し
たＤＦＡＩＶを高速液体クロマトグラフィーで定量する
。Enzyme activity measurement method: Appropriately diluted enzyme solution 1 Otori and 2% levan solution (using 0.1M acetate buffer with pH 6.0) 1m
After reacting at 40° C. for 1 hour, the produced DFAIV is quantified by high performance liquid chromatography.
１分間に１μモルのＤＦＡＩＶを生成させる酵素量を、
１単位とする。なお、高速液体クロマトグラフィーの条
件は次のとおりである。The amount of enzyme that produces 1 μmol of DFAIV per minute is
It is considered as 1 unit. Note that the conditions for high performance liquid chromatography are as follows.
カラム： ５ｈｏｄｅｘ Ｎｉｌ ｐｘｋ Ｊ−４１１
キャリヤー：アセトニトリル／水（８５／１５）流速：
１ 、Ｏｎｌ／ ｍｉｎ
カラム温度：４０℃
検出：ＲＩ
実施例 ル
レバン １％、ペプトン １％、Ｋ、ＨＰｏ、０．１％
、ＮａＮ０，０．５％、Ｍ［ＳＯ４”７ＨｚＯｏ、ｏｓ
％、およびＭｎＣＩｓ ・４ Ｈｚ８ ０ 、０２％を
含むｐＨ７，０の培地１００ｍ１を５００ｍ１容の坂ロ
コルベンに分注し、１２０°Ｃで１５分間加熱滅菌した
。冷却後、シュードモナス・フルオレッセンスＭＺ Ｎ
ｏ、９４９を１白金耳植菌し、２７℃で４日間振とう培
養した。培養終了後、遠心分離して培養液から菌体を除
き、粗酵素液を得た。Column: 5hodex Nil pxk J-411
Carrier: Acetonitrile/Water (85/15) Flow rate:
1, Onl/min Column temperature: 40°C Detection: RI Example Relevan 1%, Peptone 1%, K, HPo, 0.1%
, NaN0,0.5%, M[SO4”7HzOo, os
% and MnCIs 4 Hz80, 02% at pH 7.0 was dispensed into a 500 ml volume of Sakalokolben and heat sterilized at 120°C for 15 minutes. After cooling, Pseudomonas fluorescens MZ N
949 was inoculated into one platinum loop and cultured with shaking at 27°C for 4 days. After the culture was completed, the cells were removed from the culture solution by centrifugation to obtain a crude enzyme solution.
その酵素活性は１１．６単位／１００ｍ１であった。Its enzyme activity was 11.6 units/100ml.
比較のためシュードモナス菌にかえてアースロバフタ−
・ウレアファシェンスを用い、他は同様にして得られた
粗酵素液の酵素活性は、１．０単位／１００ｍ１であっ
た。For comparison, Arthrobafter was used instead of Pseudomonas.
- The enzyme activity of the crude enzyme solution obtained using Urea Fascens in the same manner as above was 1.0 unit/100ml.
実施例 ２
レバンにかえて同量のフレインを含有させた培地を用い
たほかは実施例１と同様にして、粗酵素液を得た。Example 2 A crude enzyme solution was obtained in the same manner as in Example 1, except that a medium containing the same amount of Frein instead of levan was used.
その酵素活性は１２．０単位／１００ｍ１であった。Its enzyme activity was 12.0 units/100ml.
比較のためシュードモナス菌にかえてアースロバフタ−
・ウレアファシェンスを用い、他は同様にして得られた
粗酵素液の酵素活性は、１．０単位／１００ｍ１であっ
た。For comparison, Arthrobafter was used instead of Pseudomonas.
- The enzyme activity of the crude enzyme solution obtained using Urea Fascens in the same manner as above was 1.0 unit/100ml.
Claims (3)
シュードモナス属に属するレバンフラクトトランスフェ
ラーゼ生産菌を培養し、培養物からレバンフラクトトラ
ンスフェラーゼを採取することを特徴とするレバンフラ
クトトランスフェラーゼの製造法。(1) A method for producing levan fructotransferase, which comprises culturing a levan fructotransferase-producing bacterium belonging to the genus Pseudomonas using a medium containing a fructose polymer, and collecting levan fructotransferase from the culture.
シュードモナス・フルオレッセンスＭＺＮｏ．９４９（
微工研菌寄第９２３５号）を用いる特許請求の範囲第１
項記載の製造法。(2) Pseudomonas fluorescens MZNo. as a levanfructotransferase producing bacterium. 949(
Claim 1 using the method
Manufacturing method described in section.
インを含有する培地を用いる特許請求の範囲第１項記載
の製造法。(3) The production method according to claim 1, which uses a medium containing levan or phlein as a fructose polymer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP24588687A JP2561100B2 (en) | 1987-10-01 | 1987-10-01 | Method for producing levanfructotransferase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP24588687A JP2561100B2 (en) | 1987-10-01 | 1987-10-01 | Method for producing levanfructotransferase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0191777A true JPH0191777A (en) | 1989-04-11 |
| JP2561100B2 JP2561100B2 (en) | 1996-12-04 |
Family
ID=17140269
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP24588687A Expired - Fee Related JP2561100B2 (en) | 1987-10-01 | 1987-10-01 | Method for producing levanfructotransferase |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2561100B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001029185A1 (en) * | 1999-10-19 | 2001-04-26 | Korea Research Institute Of Bioscience And Biotechnology | Enzymatic production of difructose dianhydride iv from sucrose and relevant enzymes and genes coding for them |
-
1987
- 1987-10-01 JP JP24588687A patent/JP2561100B2/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001029185A1 (en) * | 1999-10-19 | 2001-04-26 | Korea Research Institute Of Bioscience And Biotechnology | Enzymatic production of difructose dianhydride iv from sucrose and relevant enzymes and genes coding for them |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2561100B2 (en) | 1996-12-04 |
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