JP3819094B2 - 1,5-Anhydroglucitol dehydrogenase, method for producing the same, and method for quantifying 1,5-anhydroglucitol using the same - Google Patents

Description

【００２０】
本発明の酵素の生産に用いられる微生物としては、アグロバクテリウム属に属し、1,5-AG脱水素酵素を生産するものであればいずれの菌株でもよいが、好適な例としては、財団法人発酵研究所に寄託され、分譲可能な公知菌株であるアグロバクテリウム・ツメファシエンスIFO13532、アグロバクテリウム・ツメファシエンスIFO13533等が挙げられる。
【００２１】
上記菌体を培養する培地としては、炭素源、無機窒素源及び無機塩を含む培地が用いられる。炭素源としては基本的に糖類を利用し、L-ソルボース、グルコース等を酵素誘導するものであればいずれも使用できる。無機窒素源としては硫酸アンモニウム、塩化アンモニウム、リン酸アンモニウム等が、無機塩としてはナトリウム、カリウム、マグネシウム、鉄、マンガン等の塩類が使用できる。
【００２２】
培養は、振とう、通気撹拌等の方法による好気的条件下で行うのががよく、pH６〜８、25〜35℃で行うのが好ましい。培養期間は１〜７日間、通常は２〜４日間で完了する。上記方法で培養することにより、主に菌体の膜画分中に1,5-AG脱水素酵素が生成蓄積される。
【００２３】
培養後、微生物中から本発明酵素を取得するには、適当な緩衝液中で菌体をダイノミル、超音波処理等により破壊し、その処理液から膜画分を超遠心等により分離する。次いでトリトンX-100等の非イオン界面活性剤により膜画分から酵素を遊離させ、不溶分を遠心分離により除去すると、1,5-AG脱水素酵素含有抽出液を得ることができる。精製酵素は、この抽出液を、疎水クロマトグラフィー、イオン交換クロマトグラフィー、ヒドロキシアパタイト、ゲル濾過等の酵素の精製に一般的に使われている技術を適宜組み合せて精製することにより得られる。
【００２４】
次に、微生物としてアグロバクテリウム・ツメファシエンスIFO13533を用いて得られた本発明の1,5-AG脱水素酵素の性質を示す。なお、以下における1,5-AG脱水素酵素の活性は、次の方法で測定した。
【００２５】
〈酵素活性測定法〉
0.2Mリン酸カリウム緩衝液（pH8.0）1.5ml、0.25重量％ニトロテトラゾリウムブルー 0.3ml、２重量％トリトンX-100 0.3ml、50mM 1,5-AG水溶液0.3ml、水0.45ml、及び酵素溶液0.15mlより成る全量3.0mlの酵素含有液を37℃で反応させ、540nmにおける吸光度変化（ΔmOD/min）を測定する。この条件下で生成するホルマザン色素の分子吸光係数を16.7×10³とし、１分間に１μmolのホルマザンを生成する酵素量を１単位（unit）として、1,5-AG脱水素酵素の活性を次式に従って求めた。
【００２６】
【数２】

【００２７】
〈理化学的性質〉
(1) 作用：
１mM 2,6-ジクロロフェノールインドフェノール及び2.5mM 1,5-AGを含有する100mMトリス・塩酸緩衝液（pH9.0）に本発明酵素を添加し、37℃で反応させ経時的に反応液中の生成物と反応のモルバランスを表１に示す条件でHPLCにて分析した。
【００２８】
【表１】

【００２９】
その結果、反応の進行とともにリテンションタイム10.0分の1,5-AGのピークが減少し、新たにリテンションタイム9.6分の位置に反応物のピークが現れた。この反応のモルバランスは一致していた。また、1,5-AGの３位の水酸基を酸化する作用を持つグルコシド-3-デヒドロゲナーゼ（EC 1.1.99.13）を同様の条件で反応させ、HPLCで分析した結果、1,5-AGのピーク減少とともに、新たにリテンションタイム10.8分の位置に反応物のピークが現れた。一方、1,5-AGの２位の水酸基を酸化する作用を持つピラノースオキシダーゼ（EC 1.1.3.10）を、2.5mM 1,5-AGを含有する40mMホウ酸緩衝液（pH7.5）に添加し、37℃で反応させた結果、1,5-AGのピーク減少とともに、新たにリテンションタイム9.6分の位置に反応物のピークが現れた。これらの結果において、1,5-AGに本発明酵素を作用させた場合の反応生成物とピラノースオキシダーゼを作用させた場合の反応生成物のリテンションタイムが一致していることから、本発明酵素による1,5-AGの酸化部位は２位の水酸基であることが分かる。
【００３０】
(2) 基質特異性：
前述の酵素活性測定法において、1,5-AGの代わりに各種糖溶液（いずれも終濃度５mM）を用いた場合の相対反応性（基質特異性）を表２に示す。本発明酵素は1,5-AGに対して強く、L-ソルボースに対しては弱く作用し、D-グルコースに若干作用した。
【００３１】
【表２】

【００３２】
(3) 至適pH：
本発明酵素を用いて、前述の酵素活性測定法中における緩衝液の代わりに、種々のpHを有するクエン酸緩衝液、リン酸カリウム緩衝液、トリス−塩酸緩衝液及びグリシン緩衝液を用いた場合の酵素活性を測定した。この結果を図１に示す。図１より、本発明酵素の至適pHは8.0〜9.0付近にあることが分かる。
【００３３】
(4) pH安定性：
種々のpHを有するクエン酸緩衝液、リン酸カリウム緩衝液、トリス−塩酸緩衝液及びグリシン緩衝液（各緩衝液とも200mM）に本発明酵素を添加し、37℃で60分間処理後、残存酵素活性を測定した。この結果を図２に示す。図２より、本発明酵素の安定pH範囲は7.0〜9.0であることが分かる。
【００３４】
(5) 至適温度：
本発明酵素を用いて、前述の酵素活性測定法における組成で、種々の温度で10分間反応させた。この結果を図３に示す。図３より、本発明酵素の至適温度は35〜40℃付近であることが分かる。
【００３５】
(6) 温度安定性：
本発明酵素を20mMリン酸カリウム緩衝液に添加し、種々の温度で10分間処理後、残存酵素活性を測定した。この結果を図４に示す。図４より、本発明酵素は40℃付近まで90％以上安定であることが分かる。
【００３６】
(7) 電子受容体：
100mMリン酸カリウム緩衝液（pH8.0）中に、本発明酵素とともに種々の電子受容体を共存させた場合の、各電子受容体での活性を相対活性で表した。表３より、2,6-ジクロロフェノールインドフェノール及びフェリシアン化合物が電子受容体としての活性が高いが、ニトロテトラゾリウムブルーについては電子キャリアー非存在下においても電子受容体となり得ることが分かる。
【００３７】
【表３】

【００３８】
(8) 分子量：
ドデシル硫酸−ポリアクリルアミド電気泳動法により求めた本発明酵素の分子量は、約55,000であった。
【００３９】
(9) 等電点：
クロマトフォーカシングカラム（ファルマシア社製）を用いた本発明酵素の等電点は、約4.0であった。
【００４０】
(10) Km値：
ラインウイバーバーク（Lineweaver-Burk）プロットにより、本発明酵素の1,5-AGに対するKm値を求めた結果、約1.0mMであった。
【００４１】
本発明の1,5-AGの定量法は、上記本発明の1,5-AG脱水素酵素を用い、次の如くして行われる。すなわち、被検体を還元性発色剤の存在下、1,5-AG脱水素酵素又はこれを含有する酵素製剤とともにインキュベートし、2-ケト-1,5-AG及び還元された発色物質を生成せしめ、生成した発色物質量を測定することにより、被検体中の1,5-AG量を定量することができる。
【００４２】
本発明の定量法に使用できる緩衝液としてはpH６〜10のリン酸緩衝液、トリス塩酸緩衝液、グッド緩衝液、ホウ酸緩衝液等、通常使用されるものであればいずれも使用可能である。
【００４３】
還元性発色剤としては、テトラゾリウム又はその塩が挙げられるが、具体的には、ニトロテトラゾリウムブルー（以下NTBと略す）、2-(4-ヨードフェニル)-3-(4-ニトロフェニル)-5-フェニル-2Hテトラゾリウムクロライド、3-(4,5-ジメチル-2-チアゾリル)-2,5-ジフェニル-2Hテトラゾリウムブロマイド、2-(4-ヨードフェニル)-3-(4-ニトロフェニル)-5-(2,4-ジスルホフェニル)-2Hテトラゾリウム塩（以下WST-1と略す）等が利用でき、その使用濃度は、50〜2000μmol/l、特に100〜1000μmol/lの範囲が好ましい。
【００４４】
本発明の定量法においては、1,5-AG脱水素酵素は20〜10,000単位／リットル、特に200〜2,000単位／リットルの範囲で使用するのが好ましい。
【００４５】
本発明の定量法が適用できる試料としては、1,5-アンヒドログルシトールを含む血清、血漿、尿等の生体由来の試料が挙げられる。
【００４６】
反応液中の還元された発色性物質の測定は、上記の如くして反応せしめた後、発色物質特有の吸収波長において、一定時間後の吸光度あるいは一定時間内の吸光度変化を測定することにより行うことができる。
【００４７】
【実施例】
以下、実施例を挙げて本発明を更に詳細に説明するが、本発明はこれらに限定されるものではない。
【００４８】
実施例１ 〔アグロバクテリウム・ツメファシエンスIFO13533由来1,5-AG脱水素酵素の取得〕
リン酸二カリウム２％、リン酸一カリウム0.5％、塩化カリウム0.75％、塩化ナトリウム2.5％、塩化アンモニウム1.25％、硫酸ナトリウム0.025％、硫酸マグネシウム0.005％及びL-ソルボース0.1％より成る培地（pH7.0）50mlを200ml容の培養フラスコに添加し、120℃で15分間殺菌後冷却し、アグロバクテリウム・ツメファシエンスIFO13533を１白金耳接種し、30℃で３日間振とう培養して種培養液とした。得られた種培養液10mlを、２リットル培養フラスコ中、上記と同じ組成の液体培地1000mlに接種し、30℃で３日間振とう培養した。
培養終了後、遠心分離して菌体を回収した。得られた菌体量は培養液１リットル当たり約２ｇであった。得られた菌体を１ｇ当たり2.5mlの20mMリン酸カリウム緩衝液（pH7.2）に懸濁させ、超音波ホモジナイザーにより、菌体を細断した。得られた破砕液を10,000ｇで遠心分離し、その上清を更に100,000ｇで遠心分離して膜画分を得た。膜画分１ｇ当たり10mlの１％トリトンX-100含有20mMリン酸カリウム緩衝液（pH7.2）で懸濁させ、撹拌しながら一晩膜画分から酵素の可溶化を行った。得られた可溶化液を再度100,000ｇで遠心分離して膜残渣を除き、粗酵素液を得た。この粗酵素液の酵素活性を前述の測定法に基づいて測定したところ、菌体１ｇ当たり0.5単位の活性が得られた。
【００４９】
実施例２ 〔1,5-AG脱水素酵素の精製〕
実施例１で得られた可溶化液を、予め１％トリトンX-100含有20mMリン酸カリウム緩衝液（pH7.2）で平衡化したハイドロキシアパタイトカラムに通して酵素を吸着させ、同組成の緩衝液で洗浄後、１％トリトンX-100含有20mMリン酸カリウム緩衝液（pH7.2）の直線グラジエントで溶出し、酵素の活性画分を集めた。更に比活性を上げるため、再度ハイドロキシアパタイトカラムに通して上記同様の操作を行い、活性画分を集め限外濾過により濃縮し、比活性が12単位/mg蛋白質である精製酵素を得た。該精製標品は、ポリアクリルアミド電気泳動において単一バンドを示した。
【００５０】
実施例３ 〔NTBを利用した1,5-AGの定量〕
0.35mM NTB及び１％トリトンX-100を含む350mMリン酸カリウム緩衝液（pH8.0）2.55mlに、各種濃度の1,5-AG溶液0.15ml及び実施例２により得られた1,5-AG脱水素酵素溶液0.3ml（0.54単位）を加えた全量3.0mlの反応液を、37℃で10分間反応させ、540nmの吸光度を測定して検量線を作成した。図５にこの結果を示した。検量線は、０〜50μg/mlまで直線となり、還元性発色剤としてNTBを用いた1,5-AGの定量が可能であることが分かる。
【００５１】
実施例４ 〔WST-1を利用した1,5-AGの定量〕
実施例３の還元性発色剤に代えてWST-1を用い、吸光度の測定波長を450nmとした以外は、上記と同様の操作にて測定を行い検量線を作成した。図６にこの結果を示した。検量線は、０〜50μｇ／mlまで直線となり、還元性発色剤としてWST-1を用いた1,5-AGの定量が可能であることが分かる。
【００５２】
【発明の効果】
以上のように、本発明の1,5-AG脱水素酵素は、電子キャリアー非存在下において1,5-AGに特異的に作用し直接還元性発色剤を還元する作用を有する。従って、これを用いた本発明の1,5-AGの定量法は、特異酵素の作用により直接生成される還元された発色物質を測定するものであるため、試料中の1,5-AGを簡便かつ精度良く測定することができ、特に糖尿病等の診断に極めて有用である。
【図面の簡単な説明】
【図１】アグロバクテリウム・ツメファシエンスIFO13533を培養して得られた本発明の1,5-AG脱水素酵素の最適pHを示すグラフである。
【図２】アグロバクテリウム・ツメファシエンスIFO13533を培養して得られた本発明の1,5-AG脱水素酵素のpH安定性を示すグラフである。
【図３】アグロバクテリウム・ツメファシエンスIFO13533を培養して得られた本発明の1,5-AG脱水素酵素の最適温度を示すグラフである。
【図４】アグロバクテリウム・ツメファシエンスIFO13533を培養して得られた本発明の1,5-AG脱水素酵素の温度安定性を示すグラフである。
【図５】アグロバクテリウム・ツメファシエンスIFO13533を培養して得られた本発明の1,5-AG脱水素酵素及び還元性発色剤としてNTBを用いた1,5-AG測定の検量線である。
【図６】アグロバクテリウム・ツメファシエンスIFO13533を培養して得られた本発明の1,5-AG脱水素酵素及び還元性発色剤としてWST-1を用いた1,5-AG測定の検量線である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel 1,5-anhydroglucitol dehydrogenase and a method for producing the same, and a 1,5-anhydroglucose that is useful for diagnosis of diabetes and the like simply and with high accuracy using the enzyme. The present invention relates to a method for quantifying cytosole.
[0002]
[Prior art]
Since 1,5-anhydroglucitol (hereinafter referred to as “1,5-AG”) varies greatly in body fluids such as serum, plasma and urine in certain diseases, particularly diabetes, In recent years, it has become an important test item in the clinic as a useful diagnostic index. As a method for quantifying 1,5-AG, a method in which pyranose oxidase is allowed to act and the generated hydrogen peroxide is colorimetrically determined with a peroxidase coloring system (Japanese Patent Publication No. 5-41238) has become the mainstream. It is applied to general-purpose automatic analyzers. As another method, there is a method of measuring an oxygen consumption, a reduced form of an electron acceptor or an oxidized 1,5-AG reaction product by allowing an oxidase having 1,5-AG oxidizing ability to act. Can be mentioned.
[0003]
[Problems to be solved by the invention]
However, the concentration level of 1,5-AG in human-derived specimens, such as serum, is low and is further lowered in diabetic patients. Therefore, peroxidase coloring systems using pyranose oxidase are sensitive in terms of sensitivity. It is hard to say that it is enough. Also, in the method of measuring the reductant using an oxidoreductase that uses an electron acceptor, the reductant itself such as 2,6-dichlorophenolindophenol or ferricyan compound as an electron acceptor is unstable. However, it is not sufficient in terms of sensitivity, and it is difficult to apply to a general-purpose automatic analyzer for processing a large number of samples.
[0004]
Accordingly, there has been a demand for the development of a method for quantifying 1,5-AG that can measure 1,5-AG easily and accurately and can be easily applied to a general-purpose automatic analyzer.
[0005]
[Means for Solving the Problems]
Under such circumstances, the present inventors examined a reaction system that can be used for the analysis of 1,5-AG, and searched for enzymes that can be used for them. As a result, a novel enzyme that specifically oxidizes 1,5-AG (hereinafter referred to as “1,5-AG dehydrogenase”) is present in the membrane fraction of microorganisms belonging to the genus Agrobacterium. I found out. When this 1,5-AG dehydrogenase acts on 1,5-AG, it oxidizes the hydroxyl group at the 2-position of 1,5-AG and converts an electron acceptor such as 2,6-dichlorophenolindophenol. Catalyzing the reduction reaction, and when the reaction is carried out using a reducing color former, oxidizes the 2-position hydroxyl group of 1,5-AG and directly reduces the reducing color former even in the absence of an electron carrier. The inventors have found that a coloring material can be produced and completed the present invention.
[0006]
That is, the present invention provides a 1,5-AG dehydrogenase having the following physicochemical properties.
[0007]
(1) Action:
In the absence of an electron carrier, it acts specifically on 1,5-anhydroglucitol to oxidize the hydroxyl group at the 2-position and catalyze the reaction of directly reducing the reducing color former.
[0008]
(2) Substrate specificity:
It acts strongly on 1,5-anhydroglucitol and weakly acts on L-sorbose. It also acts on D-glucose slightly.
[0009]
(3) Optimum pH:
The optimum pH is around 8.0-9.0.
[0010]
(4) pH stability:
Stable at pH 7.0-9.0.
[0011]
(5) Optimal temperature:
The optimum temperature is around 35-40 ° C.
[0012]
(6) Temperature stability:
It is more than 90% stable up to around 40 ℃.
[0013]
(7) Electron acceptor:
In addition to 2,6-dichlorophenolindophenol and ferricyan compounds, reducing color formers can also be used. Also, coenzymes such as NAD and NADP and dissolved enzymes cannot be used.
[0014]
(8) Molecular weight:
The molecular weight by dodecyl sulfate-polyacrylamide electrophoresis is about 55,000.
[0015]
The present invention belongs to the genus Agrobacterium, a microorganism having a 1,5-AG dehydrogenase producing ability cultured nutrient medium, producing and accumulating the 1,5-AG dehydrogenase in the membrane fraction The present invention provides a method for producing 1,5-AG dehydrogenase, characterized by collecting the same.
[0016]
Furthermore, the present invention comprises measuring the reduced color-developing substance produced by allowing the 1,5-AG dehydrogenase to act on a sample containing 1,5-anhydroglucitol in the presence of a reducing color former. A quantitative method for 1,5-AG characterized by
[0017]
DETAILED DESCRIPTION OF THE INVENTION
Conventionally, from microorganisms belonging to the genus Agrobacterium, Hayano et al. (Journal of Biological Chemistry, 242, 3665-3672, 1967) confirmed the presence of glucoside-3-dehydrogenase in the cytoplasmic fraction. Separately, Takeuchi et al. (Journal of Biochemistry, 100, 1049-1055, 1986) have reported that 1,5-AG can be a substrate for glucoside-3-dehydrogenase. Also, from strains belonging to the genus Pseudomonas, NAKAMURA et al. (Journal of Biochemistry, 99, 607-613, 1986), 1,5-AG only in the presence of an electron acceptor in the membrane fraction. Has been reported to have an oxidative activity that converts oxygen to 1,5-anhydro-D-fructose and consumes oxygen.
[0018]
However, the existence of a metabolic enzyme that is specific to 1,5-AG and oxidizes 1,5-AG in the absence of an electron carrier and directly reduces the reducing color former has not yet been reported. That is, the action of the enzyme of the present invention is shown in the following formula, but the enzyme showing such action is not yet known and is novel.
[0019]
[Expression 1]

[0020]
The microorganism used for the production of the enzyme of the present invention may be any strain as long as it belongs to the genus Agrobacterium and produces 1,5-AG dehydrogenase. Examples include Agrobacterium tumefaciens IFO13532 and Agrobacterium tumefaciens IFO13533, which are known strains deposited at the Fermentation Research Institute and can be distributed.
[0021]
A medium containing a carbon source, an inorganic nitrogen source and an inorganic salt is used as the medium for culturing the cells. Any carbon source can be used as long as it basically utilizes sugars and can induce L-sorbose, glucose and the like. As the inorganic nitrogen source, ammonium sulfate, ammonium chloride, ammonium phosphate and the like can be used, and as the inorganic salt, salts such as sodium, potassium, magnesium, iron and manganese can be used.
[0022]
The culture is preferably performed under aerobic conditions such as shaking and aeration stirring, and is preferably performed at pH 6 to 8 and 25 to 35 ° C. The culture period is 1 to 7 days, usually 2 to 4 days. By culturing by the above method, 1,5-AG dehydrogenase is produced and accumulated mainly in the cell membrane fraction.
[0023]
In order to obtain the enzyme of the present invention from microorganisms after culturing, the cells are disrupted in a suitable buffer solution by dynomill, ultrasonic treatment, etc., and the membrane fraction is separated from the treated solution by ultracentrifugation or the like. Next, the enzyme is released from the membrane fraction with a nonionic surfactant such as Triton X-100, and the insoluble matter is removed by centrifugation, whereby an extract containing 1,5-AG dehydrogenase can be obtained. The purified enzyme can be obtained by purifying the extract by appropriately combining techniques generally used for enzyme purification such as hydrophobic chromatography, ion exchange chromatography, hydroxyapatite, and gel filtration.
[0024]
Next, the properties of the 1,5-AG dehydrogenase of the present invention obtained using Agrobacterium tumefaciens IFO13533 as a microorganism will be shown. The activity of 1,5-AG dehydrogenase in the following was measured by the following method.
[0025]
<Enzyme activity measurement method>
0.2M potassium phosphate buffer (pH 8.0) 1.5ml, 0.25wt% nitrotetrazolium blue 0.3ml, 2wt% Triton X-100 0.3ml, 50mM 1,5-AG aqueous solution 0.3ml, water 0.45ml, and enzyme A total of 3.0 ml of the enzyme-containing solution consisting of 0.15 ml of the solution is reacted at 37 ° C., and the change in absorbance at 540 nm (ΔmOD / min) is measured. The activity of 1,5-AG dehydrogenase is as follows, assuming that the molecular extinction coefficient of the formazan dye produced under these conditions is 16.7 × 10 ³ and the amount of enzyme that produces 1 μmol of formazan per unit is 1 unit. Obtained according to the formula.
[0026]
[Expression 2]

[0027]
<Physical and chemical properties>
(1) Action:
The enzyme of the present invention is added to 100 mM Tris / hydrochloric acid buffer (pH 9.0) containing 1 mM 2,6-dichlorophenolindophenol and 2.5 mM 1,5-AG, and reacted at 37 ° C. over time. The product and the molar balance of the reaction were analyzed by HPLC under the conditions shown in Table 1.
[0028]
[Table 1]

[0029]
As a result, the peak of 1,5-AG with a retention time of 10.0 / 0.0 decreased with the progress of the reaction, and a new reactant peak appeared at a position with a retention time of 9.6 minutes. The molar balance of this reaction was consistent. In addition, glucoside-3-dehydrogenase (EC 1.1.99.13), which has the action of oxidizing the hydroxyl group at the 3-position of 1,5-AG, was reacted under the same conditions and analyzed by HPLC. Along with the decrease, a peak of the reactant appeared at a new retention time of 10.8 minutes. On the other hand, pyranose oxidase (EC 1.1.3.10), which acts to oxidize the hydroxyl group at the 2-position of 1,5-AG, is added to 40 mM borate buffer (pH 7.5) containing 2.5 mM 1,5-AG. As a result of the reaction at 37 ° C., a peak of the reaction product appeared at a retention time of 9.6 minutes as well as a decrease in the peak of 1,5-AG. In these results, since the retention time of the reaction product when the enzyme of the present invention is allowed to act on 1,5-AG and the reaction product of when the pyranose oxidase is allowed to act are the same, It can be seen that the oxidation site of 1,5-AG is a hydroxyl group at the 2-position.
[0030]
(2) Substrate specificity:
Table 2 shows the relative reactivity (substrate specificity) when various sugar solutions (each having a final concentration of 5 mM) were used in place of 1,5-AG in the enzyme activity measurement method described above. The enzyme of the present invention acted strongly against 1,5-AG, weakly against L-sorbose and slightly acted on D-glucose.
[0031]
[Table 2]

[0032]
(3) Optimum pH:
When using the enzyme of the present invention, citrate buffer, potassium phosphate buffer, Tris-HCl buffer and glycine buffer having various pHs are used instead of the buffer in the enzyme activity measurement method described above. The enzyme activity of was measured. The result is shown in FIG. FIG. 1 shows that the optimum pH of the enzyme of the present invention is in the vicinity of 8.0 to 9.0.
[0033]
(4) pH stability:
The enzyme of the present invention is added to citrate buffer, potassium phosphate buffer, Tris-HCl buffer and glycine buffer (200 mM for each buffer) having various pHs, treated at 37 ° C. for 60 minutes, and then the remaining enzyme Activity was measured. The result is shown in FIG. FIG. 2 shows that the stable pH range of the enzyme of the present invention is 7.0 to 9.0.
[0034]
(5) Optimal temperature:
Using the enzyme of the present invention, the reaction was carried out at various temperatures for 10 minutes with the composition in the enzyme activity measurement method described above. The result is shown in FIG. FIG. 3 shows that the optimum temperature of the enzyme of the present invention is around 35 to 40 ° C.
[0035]
(6) Temperature stability:
The enzyme of the present invention was added to 20 mM potassium phosphate buffer, treated for 10 minutes at various temperatures, and the residual enzyme activity was measured. The result is shown in FIG. FIG. 4 shows that the enzyme of the present invention is more than 90% stable up to around 40 ° C.
[0036]
(7) Electron acceptor:
The activity of each electron acceptor in the presence of various electron acceptors together with the enzyme of the present invention in a 100 mM potassium phosphate buffer (pH 8.0) was expressed as relative activity. From Table 3, it can be seen that 2,6-dichlorophenolindophenol and ferricyan compound have high activity as an electron acceptor, but nitrotetrazolium blue can be an electron acceptor even in the absence of an electron carrier.
[0037]
[Table 3]

[0038]
(8) Molecular weight:
The molecular weight of the enzyme of the present invention determined by dodecyl sulfate-polyacrylamide electrophoresis was about 55,000.
[0039]
(9) Isoelectric point:
The isoelectric point of the enzyme of the present invention using a chromatofocusing column (Pharmacia) was about 4.0.
[0040]
(10) Km value:
The Km value for 1,5-AG of the enzyme of the present invention was determined by a Lineweaver-Burk plot and found to be about 1.0 mM.
[0041]
The quantification method of 1,5-AG of the present invention is performed as follows using the above-described 1,5-AG dehydrogenase of the present invention. That is, the specimen is incubated with 1,5-AG dehydrogenase or an enzyme preparation containing this in the presence of a reducing chromogenic agent to produce 2-keto-1,5-AG and a reduced chromogenic substance. The amount of 1,5-AG in the specimen can be quantified by measuring the amount of the color developing substance produced.
[0042]
As the buffer that can be used in the quantification method of the present invention, any phosphate buffer solution, pH 6-10 phosphate buffer solution, Tris-HCl buffer solution, Good buffer solution, borate buffer solution, and the like can be used. .
[0043]
Examples of the reducing color former include tetrazolium or a salt thereof. Specifically, nitrotetrazolium blue (hereinafter abbreviated as NTB), 2- (4-iodophenyl) -3- (4-nitrophenyl) -5 -Phenyl-2H tetrazolium chloride, 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H tetrazolium bromide, 2- (4-iodophenyl) -3- (4-nitrophenyl) -5 -(2,4-Disulfophenyl) -2H tetrazolium salt (hereinafter abbreviated as WST-1) can be used, and the concentration used is preferably in the range of 50 to 2000 μmol / l, particularly 100 to 1000 μmol / l.
[0044]
In the quantification method of the present invention, 1,5-AG dehydrogenase is preferably used in the range of 20 to 10,000 units / liter, particularly 200 to 2,000 units / liter.
[0045]
Samples to which the quantification method of the present invention can be applied include samples derived from living bodies such as serum, plasma and urine containing 1,5-anhydroglucitol.
[0046]
Measurement of the reduced chromogenic substance in the reaction solution is carried out by measuring the absorbance after a certain time or the change in absorbance within a certain time at the absorption wavelength peculiar to the chromogenic substance after reacting as described above. be able to.
[0047]
【Example】
EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these.
[0048]
Example 1 [Acquisition of 1,5-AG dehydrogenase derived from Agrobacterium tumefaciens IFO13533]
Medium consisting of 2% dipotassium phosphate, 0.5% monopotassium phosphate, 0.75% potassium chloride, 2.5% sodium chloride, 1.25% ammonium chloride, 0.025% sodium sulfate, 0.005% magnesium sulfate and 0.1% L-sorbose (pH 7. 0) Add 50 ml to a 200 ml culture flask, sterilize at 120 ° C for 15 minutes, cool, inoculate 1 platinum ear of Agrobacterium tumefaciens IFO13533, and incubate with shaking at 30 ° C for 3 days did. 10 ml of the obtained seed culture solution was inoculated into 1000 ml of a liquid medium having the same composition as described above in a 2 liter culture flask and cultured with shaking at 30 ° C. for 3 days.
After completion of the culture, the cells were collected by centrifugation. The amount of the obtained microbial cells was about 2 g per liter of the culture solution. The obtained bacterial cells were suspended in 2.5 ml of 20 mM potassium phosphate buffer (pH 7.2) per gram, and the bacterial cells were shredded by an ultrasonic homogenizer. The obtained crushed liquid was centrifuged at 10,000 g, and the supernatant was further centrifuged at 100,000 g to obtain a membrane fraction. The enzyme was solubilized from the membrane fraction overnight while being suspended in 10 ml of 20% potassium phosphate buffer (pH 7.2) containing 1% Triton X-100 per gram of membrane fraction. The obtained solubilized solution was centrifuged again at 100,000 g to remove the membrane residue to obtain a crude enzyme solution. When the enzyme activity of this crude enzyme solution was measured based on the above-described measurement method, 0.5 units of activity per 1 g of cells were obtained.
[0049]
Example 2 [Purification of 1,5-AG dehydrogenase]
The solubilized solution obtained in Example 1 was passed through a hydroxyapatite column equilibrated in advance with 20 mM potassium phosphate buffer (pH 7.2) containing 1% Triton X-100 to adsorb the enzyme, and the buffer having the same composition. After washing with a liquid, the enzyme active fraction was collected by eluting with a linear gradient of 20 mM potassium phosphate buffer (pH 7.2) containing 1% Triton X-100. In order to further increase the specific activity, the same operation as described above was performed again through a hydroxyapatite column, and the active fractions were collected and concentrated by ultrafiltration to obtain a purified enzyme having a specific activity of 12 units / mg protein. The purified sample showed a single band in polyacrylamide electrophoresis.
[0050]
Example 3 [Quantification of 1,5-AG using NTB]
To 2.55 ml of 350 mM potassium phosphate buffer (pH 8.0) containing 0.35 mM NTB and 1% Triton X-100, 0.15 ml of 1,5-AG solution of various concentrations and 1,5- A total of 3.0 ml of the reaction solution added with 0.3 ml of AG dehydrogenase solution (0.54 units) was reacted at 37 ° C. for 10 minutes, and the absorbance at 540 nm was measured to prepare a calibration curve. FIG. 5 shows the result. The calibration curve is linear from 0 to 50 μg / ml, indicating that 1,5-AG can be quantified using NTB as the reducing color former.
[0051]
Example 4 [Quantification of 1,5-AG using WST-1]
A calibration curve was prepared by carrying out the measurement in the same manner as described above except that WST-1 was used instead of the reducing color former of Example 3 and the measurement wavelength of absorbance was 450 nm. FIG. 6 shows the result. The calibration curve is a straight line from 0 to 50 μg / ml, indicating that 1,5-AG can be quantified using WST-1 as the reducing color former.
[0052]
【The invention's effect】
As described above, the 1,5-AG dehydrogenase of the present invention acts specifically on 1,5-AG and directly reduces the reducing color former in the absence of an electron carrier. Therefore, since the method for quantifying 1,5-AG of the present invention using this method measures a reduced chromogenic substance directly produced by the action of a specific enzyme, 1,5-AG in a sample is measured. It can be measured easily and accurately, and is particularly useful for diagnosis of diabetes and the like.
[Brief description of the drawings]
FIG. 1 is a graph showing the optimum pH of 1,5-AG dehydrogenase of the present invention obtained by culturing Agrobacterium tumefaciens IFO13533.
FIG. 2 is a graph showing the pH stability of 1,5-AG dehydrogenase of the present invention obtained by culturing Agrobacterium tumefaciens IFO13533.
FIG. 3 is a graph showing the optimum temperature of the 1,5-AG dehydrogenase of the present invention obtained by culturing Agrobacterium tumefaciens IFO13533.
FIG. 4 is a graph showing the temperature stability of 1,5-AG dehydrogenase of the present invention obtained by culturing Agrobacterium tumefaciens IFO13533.
FIG. 5 is a calibration curve of 1,5-AG measurement using NTB as a 1,5-AG dehydrogenase of the present invention and a reducing color former obtained by culturing Agrobacterium tumefaciens IFO13533.
FIG. 6 is a calibration curve of 1,5-AG measurement using 1,5-AG dehydrogenase of the present invention obtained by culturing Agrobacterium tumefaciens IFO13533 and WST-1 as a reducing chromogenic agent. is there.

Claims (6)

1,5-anhydroglucitol dehydrogenase having the following physicochemical properties:
(1) Action:
In the absence of an electron carrier, it acts specifically on 1,5-anhydroglucitol to oxidize the hydroxyl group at the 2-position and catalyze the reaction of directly reducing the reducing color former.
(2) Substrate specificity:
It acts strongly on 1,5-anhydroglucitol and weakly acts on L-sorbose. It also acts on D-glucose slightly.
(3) Optimum pH:
The optimum pH is around 8.0-9.0.
(4) pH stability:
Stable at pH 7.0-9.0.
(5) Optimal temperature:
The optimum temperature is around 35-40 ° C.
(6) Temperature stability:
It is more than 90% stable up to around 40 ℃.
(7) Electron acceptor:
In addition to 2,6-dichlorophenolindophenol and ferricyan compounds, reducing color formers can also be used. Also, coenzymes such as NAD and NADP and dissolved enzymes cannot be used.
(8) Molecular weight:
The molecular weight by dodecyl sulfate-polyacrylamide electrophoresis is about 55,000.   The 1,5-anhydroglucitol dehydrogenase according to claim 1, which belongs to the genus Agrobacterium and is produced by a microorganism having the ability to produce 1,5-anhydroglucitol dehydrogenase. A microorganism belonging to the genus Agrobacterium and capable of producing 1,5-anhydroglucitol dehydrogenase is cultured in a nutrient medium, and 1,5-anhydroglucitol according to claim 1 is contained in the membrane fraction. A method for producing 1,5-anhydroglucitol dehydrogenase, which comprises collecting and collecting dehydrogenase.   The method according to claim 3, wherein the microorganism belonging to the genus Agrobacterium and having the ability to produce 1,5-anhydroglucitol dehydrogenase is Agrobacterium tumefaciens.   The reduced color produced by reacting the sample containing 1,5-anhydroglucitol with the 1,5-anhydroglucitol dehydrogenase according to claim 1 or 2 in the presence of a reducing color former. A method for quantifying 1,5-anhydroglucitol, comprising measuring a substance.   6. The method according to claim 5, wherein the reducing color former is tetrazolium or a salt thereof.

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