JPH04234983A - Dextrin dextranase and its production - Google Patents
Dextrin dextranase and its productionInfo
- Publication number
- JPH04234983A JPH04234983A JP6959091A JP6959091A JPH04234983A JP H04234983 A JPH04234983 A JP H04234983A JP 6959091 A JP6959091 A JP 6959091A JP 6959091 A JP6959091 A JP 6959091A JP H04234983 A JPH04234983 A JP H04234983A
- Authority
- JP
- Japan
- Prior art keywords
- ddase
- dextrin dextranase
- enzyme
- organic solvent
- dextranase
- 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
- 108010012023 Dextrin dextranase Proteins 0.000 title claims abstract description 14
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 4
- 239000003960 organic solvent Substances 0.000 claims abstract description 16
- 241000589236 Gluconobacter Species 0.000 claims abstract description 6
- 238000012258 culturing Methods 0.000 claims abstract description 6
- 241000894006 Bacteria Species 0.000 claims description 5
- 229920002307 Dextran Polymers 0.000 abstract description 7
- 230000000813 microbial effect Effects 0.000 abstract 1
- 244000005700 microbiome Species 0.000 abstract 1
- 239000002994 raw material Substances 0.000 abstract 1
- 108090000790 Enzymes Proteins 0.000 description 24
- 102000004190 Enzymes Human genes 0.000 description 24
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 18
- 210000004027 cell Anatomy 0.000 description 17
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 15
- 230000000694 effects Effects 0.000 description 11
- 230000001580 bacterial effect Effects 0.000 description 10
- 238000004587 chromatography analysis Methods 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 241000589232 Gluconobacter oxydans Species 0.000 description 5
- 238000005119 centrifugation Methods 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
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 239000008351 acetate buffer Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 3
- 238000001502 gel electrophoresis Methods 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical group CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- FYGDTMLNYKFZSV-DZOUCCHMSA-N alpha-D-Glcp-(1->4)-alpha-D-Glcp-(1->4)-D-Glcp Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)O[C@H](O[C@@H]2[C@H](OC(O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-DZOUCCHMSA-N 0.000 description 2
- 239000012223 aqueous fraction Substances 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 229920000856 Amylose Polymers 0.000 description 1
- 229920001503 Glucan Polymers 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000005984 hydrogenation reaction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- -1 maltose Chemical compound 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000003495 polar organic solvent Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
Description
【０００１】0001
【産業上の利用分野】本発明は、医療あるいは生化学な
どの分野で利用されているデキストラン（α１，６グル
カン）を、マルトオリゴ糖，澱粉及びその分解物より生
成する能力を有するデキストリンデキストラナーゼ（以
下、ＤＤａｓｅという。）及びその生産方法に関するも
のである。[Industrial Application Field] The present invention is directed to a dextrin dextranase that has the ability to produce dextran (α1,6 glucan) from maltooligosaccharides, starch, and their decomposition products, which is used in fields such as medicine and biochemistry. (hereinafter referred to as DDase) and its production method.
【０００２】0002
【従来の技術】ＤＤａｓｅについては約４０年前に報告
（Ｈｅｈｒｅ，Ｅ．Ｊ．＆ Ｈａｍｉｌｔｏｎ，Ｄ．
Ｍ．（１９５１）：Ｊｏｕｒｎａｌ ｏｆ Ｂｉｏ
ｌｏｇｉｃａｌ Ｃｈｅｍｉｓｔｒｙ，５５，１６１
１７４．）が行われているが、それ以降全く研究が
されていない。同報告による精製手法とは、菌体を除去
しないままの状態の菌体培養液に適量の硫酸アンモニウ
ムを加えることによって生じる沈澱を集めた後、これを
適量の蒸留水に溶かし、溶けない細胞などを除去するこ
とによって酵素を取得する方法である。また、現在にお
ける菌体内酵素を精製する通常の方法は、細胞破壊によ
り無細胞抽出液を調製した後、有機溶媒を含まない緩衝
液を用いる各種クロマトグラフィーによる。[Prior Art] DDase was reported about 40 years ago (Hehre, E.J. & Hamilton, D.
M. (1951): Journal of Bio
Logical Chemistry, 55, 161
174. ), but no research has been conducted since then. The purification method described in the same report involves adding an appropriate amount of ammonium sulfate to the bacterial culture solution without removing the bacterial cells, collecting the precipitate, and then dissolving this in an appropriate amount of distilled water to remove undissolved cells. This is a method of obtaining enzymes by removing them. Furthermore, the current common method for purifying intracellular enzymes is to prepare a cell-free extract by disrupting the cells, followed by various types of chromatography using a buffer solution that does not contain an organic solvent.
【０００３】0003
【発明が解決しようとする課題】発明者は、前述の報告
に基ずき実験によりＤＤａｓｅの生産と精製を行ったと
ころ、同精製方法では菌体中に存在する酵素量のわずか
数％の粗製酵素が得られただけで、従来の技術を使用し
てはこれ以上の精製はできなかった。また、細胞破壊に
よる精製では、超音波を用いた通常の菌体破壊処理によ
ってもなおＤＤａｓｅは菌体内にとどまったままであっ
た。この時得られるＤＤａｓｅを含む液を常法通り有機
溶媒を含まない緩衝液を用いた各種クロマトグラフィー
によって精製することを試みたが、ほとんど吸着せず精
製はできなかった。[Problems to be Solved by the Invention] The inventor conducted experiments to produce and purify DDase based on the above-mentioned report, and found that the purification method produced only a few percent of the amount of crude enzyme present in the bacterial cells. Only the enzyme was obtained and no further purification was possible using conventional techniques. Furthermore, in purification by cell destruction, DDase remained within the cells even after normal cell destruction treatment using ultrasound. Attempts were made to purify the DDase-containing solution obtained at this time by various chromatography methods using a buffer solution that does not contain an organic solvent, but almost no adsorption occurred and purification was not possible.
【０００４】0004
【課題を解決するための手段】本グルコノバクター属に
属するデキストリンデキストラナーゼ生産菌とは、グル
コノバクターオキシダンスＡＴＣＣ１１８９４株（Ｇｌ
ｕｃｏｎｏｂａｃｔｅｒ ｏｘｙｄａｎｓ Ａｍｅ
ｒｉｃａｎ ＴｙｐｅＣｕｌｔｕｒｅ Ｃｏｌｌｅ
ｃｔｉｏｎ ｓｔｒａｉｎ Ｎｏ．１１８９４），
グルコノバクターオキシダンスＡＴＣＣ１１８９５株（
Ｇｌｕｃｏｎｏｂａｃｔｅｒ ｏｘｙｄａｎｓ Ａ
ｍｅｒｉｃａｎ Ｔｙｐｅ Ｃｕｌｔｕｒｅ Ｃ
ｏｌｌｅｃｔｉｏｎ ｓｔｒａｉｎ Ｎｏ．１１８
９５）又はグルコノバクターに属するその他のＤＤａｓ
ｅ生産菌をいう。その培養は、常法によればよい。[Means for solving the problem] The dextrin dextranase-producing bacteria belonging to the genus Gluconobacter is Gluconobacter oxidans ATCC 11894 strain (Gl
uconobacter oxydans Ame
rican TypeCulture Colle
ction strain No. 11894),
Gluconobacter oxydans ATCC11895 strain (
Gluconobacter oxydans A
merican Type Culture C
Collection strain No. 118
95) or other DDas belonging to Gluconobacter
e-producing bacteria. The culture may be carried out by a conventional method.
【０００５】本発明において有機溶媒とは、ｎ−ブタノ
ール，エチレングリコール，エタノール，プロパノール
，ジエチレングリコール等のアルコール類やアセトン等
をいう。特に菌体から酵素を抽出する際にはｎ−ブタノ
ール，クロロホルム等の水に溶けにくいあるいは水にほ
とんど溶けない、つまり極性の低い有機溶媒が好適に用
いられる。この極性の低い有機溶媒とはヘキサンを０，
水を９とした分類（以下、有機溶媒の極性という。）に
より表すと５より低いものをいう。またクロマトグラフ
ィーに吸着した酵素を溶出する際には、たとえばエチレ
ングリコールのように有機溶媒の極性が５以上のものが
好適に用いられる。有機溶媒を用いて採取するには次の
とおりに行う。培養後採取した菌体をそのままあるいは
細胞破壊した後、緩衝液に懸濁する。緩衝液には酢酸緩
衝液などＤＤａｓｅが安定に存在できるＰＨの水溶液を
用いることが望ましい。この懸濁液にたとえばｎ−ブタ
ノールなどの極性の低い有機溶媒を同容量程度加え分散
させる。分散は手で軽く振とうする程度で容易に行うこ
とができる。この分散液を適時放置した後、遠心分離す
ると有機溶媒を主とする層，水を主とする層及び菌体あ
るいは菌体破砕沈澱物に分かれる。このうち水を主とす
る層を集め、透析した後、疎水クロマトグラフィーに供
し、吸着したタンパク質を、たとえばエチレングリコー
ル等の水で容易に溶かすことのできるアルコール類など
を含む緩衝液で溶出した後、これを濃縮し、同じく緩衝
液で平衡化したゲルろ過カラムに供し回収する。[0005] In the present invention, the organic solvent refers to alcohols such as n-butanol, ethylene glycol, ethanol, propanol, diethylene glycol, and acetone. In particular, when extracting enzymes from bacterial cells, organic solvents such as n-butanol and chloroform that are poorly or almost insoluble in water, that is, have low polarity, are preferably used. This organic solvent with low polarity is hexane,
When expressed in a classification with water being 9 (hereinafter referred to as organic solvent polarity), it refers to something lower than 5. Furthermore, when eluting the adsorbed enzyme during chromatography, an organic solvent having a polarity of 5 or more, such as ethylene glycol, is preferably used. To collect using an organic solvent, proceed as follows. The bacterial cells collected after culturing are suspended in a buffer solution either as is or after the cells have been disrupted. As the buffer, it is desirable to use an aqueous solution such as acetic acid buffer with a pH that allows DDase to exist stably. About the same volume of a less polar organic solvent such as n-butanol is added to this suspension and dispersed. Dispersion can be easily accomplished by shaking lightly by hand. After this dispersion is left to stand for a suitable period of time, it is centrifuged and separated into a layer mainly containing an organic solvent, a layer mainly containing water, and bacterial cells or a precipitate of crushed bacterial cells. After collecting and dialyzing the water-based layer, it is subjected to hydrophobic chromatography, and the adsorbed proteins are eluted with a buffer containing alcohols that can be easily dissolved in water, such as ethylene glycol. , this is concentrated, subjected to a gel filtration column equilibrated with the same buffer solution, and recovered.
【０００６】本発明のＤＤａｓｅは以下の特徴を有する
。
１）作用
本酵素はアミロースなどを含めて重合度３以上のマルト
オリゴ糖に作用し、それら基質の非還元末端側のグルコ
ース残基を転移することによってデキストランを生産す
る。この作用は、反応に用いた物質がマルトースなどの
重合度が２つとなるマルトオリゴ糖となるまで進行する
。このＤＤａｓｅの作用はマルトオリゴ糖の還元末端に
位置するグルコースが水素添加などの修飾を受けていて
も同様である。
２）最適ＰＨ及び安定ＰＨ
ＤＤａｓｅの最適作用ＰＨは４．０〜４．５であり、各
種ＰＨ下で３０分置いた時にＰＨは２．５〜６．０で安
定であった。
３）最適温度及び安定温度
ＤＤａｓｅの最適作用温度は３７〜４５℃であり各種温
度下で３０分置いた時に温度は４５℃以下で安定であっ
た。また、本酵素は５５℃まで活性を示した。
４）分子量
電気泳動によるＤＤａｓｅの分子量は約３０万であった
。５）力価測定法
酵素反応基質には還元低加水分解澱粉を用い、常法によ
り測定した。力価は１分間に１μｍｏｌのグルコース単
位がデキストランとなる時の酵素量を１Ｕとした。The DDase of the present invention has the following characteristics. 1) Action This enzyme acts on maltooligosaccharides with a degree of polymerization of 3 or higher, including amylose, and produces dextran by transferring glucose residues on the non-reducing terminal side of these substrates. This action progresses until the substance used in the reaction becomes a maltooligosaccharide such as maltose, which has a degree of polymerization of two. This action of DDase is the same even if the glucose located at the reducing end of the maltooligosaccharide has been modified such as hydrogenation. 2) Optimum pH and Stable PH The optimal pH for DDase's action was 4.0 to 4.5, and the pH was stable at 2.5 to 6.0 when left for 30 minutes under various pH conditions. 3) Optimal temperature and stable temperature The optimal operating temperature of DDase was 37 to 45°C, and the temperature was stable at 45°C or lower when left at various temperatures for 30 minutes. Moreover, this enzyme showed activity up to 55°C. 4) Molecular weight The molecular weight of DDase by electrophoresis was approximately 300,000. 5) Titer measurement method The titer was measured by a conventional method using reduced low hydrolyzed starch as the enzyme reaction substrate. The titer was defined as 1 U, which is the amount of enzyme required to convert 1 μmol of glucose units into dextran in 1 minute.
【０００７】[0007]
【作用】ＤＤａｓｅには有機溶媒に対する親和性が強く
且つ菌の細胞膜の近辺に存在するという特異性がある。
また有機溶媒は細胞膜の成分である脂質を溶かすことが
できる。したがって細胞を破壊しなくても有機溶媒によ
り直接作用させることにより、ＤＤａｓｅが抽出できる
。一般的に、酵素は有機触媒の存在下ですみやかに活性
を失うが、ＤＤａｓｅは有機溶媒下で失活しない。[Operation] DDase has the specificity of having a strong affinity for organic solvents and being present near the cell membrane of bacteria. Organic solvents can also dissolve lipids, which are components of cell membranes. Therefore, DDase can be extracted by directly acting on an organic solvent without destroying cells. Generally, enzymes quickly lose their activity in the presence of an organic catalyst, but DDase does not become inactive in an organic solvent.
【０００８】[0008]
【実施例】（実施例１）
０．５％酵母エキス，０．３％ポリペプトン，０．５％
グルコース，１％エタノールを含む培地４リットルを殺
菌後、グルコノバクターオキシダンスＡＴＣＣ１１８９
４株を接種し、３０℃で１６時間振とう培養した。得ら
れた菌体を１０ｍＭ酢酸緩衝液に懸濁し、等容積のｎ−
ブタノールを加えてよく混和した後、遠心分離すること
によって水層画分にＤＤａｓｅ活性を得た。この時の酵
素活性は０．５４Ｕ／ｍリットルであった。次にこの時
得られたＤＤａｓｅ画分を透析してｎ−ブタノールを除
去した後、４０％エチレングリコールを用いてフェニル
トヨパール，トヨパールＨＷ−６５Ｓクロマトグラフィ
ーを行い、ＤＤａｓｅを精製した。得られた精製酵素は
ゲル電気泳動によって単一のバンドを示し、この時の酵
素回収率は４９．７％であった。[Example] (Example 1) 0.5% yeast extract, 0.3% polypeptone, 0.5%
After sterilizing 4 liters of medium containing glucose and 1% ethanol, Gluconobacter oxydans ATCC1189
Four strains were inoculated and cultured with shaking at 30°C for 16 hours. The obtained bacterial cells were suspended in 10mM acetate buffer, and an equal volume of n-
After adding butanol and mixing well, DDase activity was obtained in the aqueous layer fraction by centrifugation. The enzyme activity at this time was 0.54 U/ml. Next, the DDase fraction obtained at this time was dialyzed to remove n-butanol, and then phenylToyopearl, Toyopearl HW-65S chromatography was performed using 40% ethylene glycol to purify DDase. The obtained purified enzyme showed a single band by gel electrophoresis, and the enzyme recovery rate at this time was 49.7%.
【０００９】（実施例２）
０．５％酵母エキス，０．３％ポリペプトン，０．５％
グルコース，２％グリセリンを含む培地１リッ卜ルを殺
菌後、グルコノバクターオキシダンスＡＴＣＣ１１８９
４株を接種し、３０℃で１６時間振とう培養した。培養
後、遠心分離により得た菌体２．０ｇを２５ｍリットル
，１０ｍＭ酢酸緩衝液に懸濁し、等容積のｎ−ブタノー
ルを加えてよく混和した後、遠心分離することによって
水層画分にＤＤａｓｅ活性を得た。この時の酵素活性は
０．３９Ｕ／ｍリットルであった。次にこの時得られた
ＤＤａｓｅ画分を透析してｎ−ブタノールを除去した後
、４０％エチレングリコールを用いてフェニルトヨパー
ル，トヨパールＨＷ−６５Ｓクロマトグラフィーを行い
、ＤＤａｓｅを精製した。得られた精製酵素はゲル電気
泳動によって単一のバンドを示した。その酵素活性から
算出された酵素回収率は２１．８％であった。(Example 2) 0.5% yeast extract, 0.3% polypeptone, 0.5%
After sterilizing 1 liter of medium containing glucose and 2% glycerin, Gluconobacter oxydans ATCC1189
Four strains were inoculated and cultured with shaking at 30°C for 16 hours. After culturing, 2.0 g of bacterial cells obtained by centrifugation were suspended in 25 ml of 10 mM acetate buffer, an equal volume of n-butanol was added, and the mixture was thoroughly mixed. DDase was added to the aqueous fraction by centrifugation. Obtained activity. The enzyme activity at this time was 0.39 U/ml. Next, the DDase fraction obtained at this time was dialyzed to remove n-butanol, and then phenylToyopearl, Toyopearl HW-65S chromatography was performed using 40% ethylene glycol to purify DDase. The obtained purified enzyme showed a single band by gel electrophoresis. The enzyme recovery rate calculated from the enzyme activity was 21.8%.
【００１０】（実施例３）
０．５％酵母エキス，０．３％ポリペプトン，０．２％
グルコース，１％グリセリンを含む培地２リッ卜ルを殺
菌後、グルコノバクターオキシダンスＡＴＣＣ１１８９
５株を接種し、３０℃で４０時間振とう培養した。培養
後、遠心分離により得た菌体３．９ｇを４５ｍリットル
，１０ｍＭ酢酸緩衝液に懸濁し、等容積のｎ−ブタノー
ルを加えてよく混和した後、遠心分離することによって
水層画分にＤＤａｓｅ活性を得た。この時の酵素活性は
０．２４Ｕ／ｍリットルであった。次にこの時得られた
ＤＤａｓｅ画分を透析してｎ−ブタノールを除去した後
、４０％ｎ−プロパノールを用いてフェニルトヨパール
，トヨパールＨＷ−６５Ｓクロマトグラフィーを行い、
ＤＤａｓｅを精製した。得られた精製酵素はゲル電気泳
動によって単一のバンドを示た。その酵素活性から算出
された酵素回収率は２３．４％であった。(Example 3) 0.5% yeast extract, 0.3% polypeptone, 0.2%
After sterilizing 2 liters of medium containing glucose and 1% glycerin, Gluconobacter oxydans ATCC1189
Five strains were inoculated and cultured with shaking at 30°C for 40 hours. After culturing, 3.9 g of bacterial cells obtained by centrifugation were suspended in 45 ml of 10 mM acetate buffer, an equal volume of n-butanol was added, and the mixture was thoroughly mixed. DDase was added to the aqueous fraction by centrifugation. Obtained activity. The enzyme activity at this time was 0.24 U/ml. Next, the DDase fraction obtained at this time was dialyzed to remove n-butanol, and then subjected to phenylToyopearl and Toyopearl HW-65S chromatography using 40% n-propanol.
DDase was purified. The obtained purified enzyme showed a single band by gel electrophoresis. The enzyme recovery rate calculated from the enzyme activity was 23.4%.
【００１１】[0011]
【効果】本発明によりＤＤａｓｅが採取できた。また細
胞破壊を行わない容易な方法で且つ高い回収率でＤＤａ
ｓｅが精製できた。ＤＤａｓｅを精製することによって
、例えばマルトオリゴ糖等からデキストランを生産する
際に従来法によって得られている粗製酵素中に含まれて
いるＤＤａｓｅ以外の酵素やその他の成分による影響等
を受けることがなくなる。また目的のデキストラン中に
不純物の混入が少なく、その精製は極めて容易になる。
特に生成物であるデキストランは高分子物質であるので
、混入している異物の除去は困難であることから、精製
酵素を使用した方が良い。[Effect] DDase could be collected according to the present invention. In addition, DDa can be easily extracted without cell destruction and with a high recovery rate.
se was purified. By purifying DDase, it is no longer affected by enzymes other than DDase or other components contained in crude enzymes obtained by conventional methods when producing dextran from maltooligosaccharides, etc., for example. Moreover, there are few impurities mixed into the target dextran, and its purification becomes extremely easy. In particular, since the product dextran is a high-molecular substance, it is difficult to remove contaminated foreign substances, so it is better to use purified enzymes.
Claims (2)
リンデキストラナーゼ生産菌を培養し、これから有機溶
媒を用いて採取したものであることを特徴とするデキス
トリンデキストラナーゼ。1. Dextrin dextranase, which is obtained by culturing a dextrin dextranase-producing bacterium belonging to the genus Gluconobacter and collecting the same using an organic solvent.
リンデキストラナーゼ生産菌を培養し、これから有機溶
媒を用いて採取することを特徴とするデキストリンデキ
ストラナーゼの生産方法。2. A method for producing dextrin dextranase, which comprises culturing a dextrin dextranase producing bacterium belonging to the genus Gluconobacter and collecting the dextrin dextranase using an organic solvent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6959091A JPH066057B2 (en) | 1991-01-10 | 1991-01-10 | Dextrin dextranase and method for producing the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6959091A JPH066057B2 (en) | 1991-01-10 | 1991-01-10 | Dextrin dextranase and method for producing the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH04234983A true JPH04234983A (en) | 1992-08-24 |
| JPH066057B2 JPH066057B2 (en) | 1994-01-26 |
Family
ID=13407195
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP6959091A Expired - Fee Related JPH066057B2 (en) | 1991-01-10 | 1991-01-10 | Dextrin dextranase and method for producing the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH066057B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007181452A (en) * | 2005-12-06 | 2007-07-19 | Hokkaido Univ | Dextran-producing enzyme gene, dextran-producing enzyme and method for producing the same, and method for producing dextran |
-
1991
- 1991-01-10 JP JP6959091A patent/JPH066057B2/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007181452A (en) * | 2005-12-06 | 2007-07-19 | Hokkaido Univ | Dextran-producing enzyme gene, dextran-producing enzyme and method for producing the same, and method for producing dextran |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH066057B2 (en) | 1994-01-26 |
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