JP3035685B2 - Steroid β-sulfate sulfatase, method for producing the same, and method for determination of bile acid 3β-sulfate - Google Patents
Steroid β-sulfate sulfatase, method for producing the same, and method for determination of bile acid 3β-sulfateInfo
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- JP3035685B2 JP3035685B2 JP3674893A JP3674893A JP3035685B2 JP 3035685 B2 JP3035685 B2 JP 3035685B2 JP 3674893 A JP3674893 A JP 3674893A JP 3674893 A JP3674893 A JP 3674893A JP 3035685 B2 JP3035685 B2 JP 3035685B2
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- sulfate
- steroid
- sulfatase
- bile acid
- acid
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Description
【0001】[0001]
【産業上の利用分野】本発明は、ステロイド核の3β−
硫酸エステルを加水分解するサルファターゼ、その製造
法及び胆汁酸の3β−硫酸エステルの定量法に関する。BACKGROUND OF THE INVENTION The present invention relates to a steroid nucleus containing 3β-
The present invention relates to a sulfatase that hydrolyzes a sulfate, a method for producing the same, and a method for determining 3β-sulfate of bile acid.
【0002】[0002]
【従来の技術および問題点】肝胆道系疾患によって、血
清中の胆汁酸濃度が増加し、同時に尿中で3位の水酸基
が硫酸エステル化された胆汁酸(硫酸抱合型胆汁酸)が
増加することはよく知られている。血清中の胆汁酸の定
量については、酵素を用いる簡便な直接測定法が開発さ
れていて、臨床検査において肝機能を調べる上で重要な
検査項目となっている。胆汁酸の硫酸エステル化は、胆
汁酸の水溶性を高め、有害な胆汁酸を速やかに血中から
体外、即ち尿中への排泄促進するための一種の解毒機構
と考えられていて、実際、尿中には血清中の胆汁酸と同
程度、或いはそれ以上の濃度で硫酸抱合型胆汁酸が存在
することが知られている。しかしながら、硫酸抱合型胆
汁酸は、その硫酸エステルを効率良く加水分解するサル
ファターゼがなかったことから容易には測定できず、検
査の一般化が遅れていた。本発明者は、先に胆汁酸の3
α−硫酸エステルに特異的なサルファターゼを見出し、
簡便な酵素法による胆汁酸の3α硫酸エステル体の直接
比色定量法を開発した(特開平2−145183号)。2. Description of the Related Art Serum bile acid concentration increases due to hepatobiliary disease, and at the same time, bile acid (sulfate-conjugated bile acid) in which the hydroxyl group at position 3 is sulfated in urine increases. It is well known. For the determination of bile acids in serum, a simple direct measurement method using an enzyme has been developed and is an important test item for examining liver function in clinical tests. Sulfate esterification of bile acids is considered to be a kind of detoxification mechanism for increasing the water solubility of bile acids and promptly excreting harmful bile acids from the blood to the outside of the body, that is, to urine. It is known that sulfate-conjugated bile acids are present in urine at a concentration similar to or higher than that of bile acids in serum. However, sulfate-conjugated bile acids could not be easily measured because there was no sulfatase that efficiently hydrolyzes the sulfate ester, and the generalization of the test was delayed. The inventor of the present invention has previously identified 3 bile acids.
Finding a specific sulfatase for α-sulfate,
A method for direct colorimetric determination of 3α-sulfate of bile acid by a simple enzymatic method was developed (JP-A-2-145183).
【0003】3α−ヒドロキシ胆汁酸は、主要な胆汁酸
であるが、胆汁酸の研究が進むにつれて、異常胆汁酸と
称されている3β−ヒドロキシ胆汁酸量の変動が特定の
病態と深く関与することが分かってきた。例えば、肝ガ
ンの患者の血清中には健常者よりも有意に高い濃度の3
β−ヒドロキシ−5−コレン酸が含まれていることが明
らかになった(第6回腫瘍マーカー研究会記録、254
−256頁、1986年)。[0003] Although 3α-hydroxy bile acid is a major bile acid, fluctuations in the amount of 3β-hydroxy bile acid, which is referred to as abnormal bile acid, is deeply involved in a specific disease state as research on bile acid progresses. I understand that. For example, the serum of a liver cancer patient has a significantly higher concentration of 3 than that of a healthy person.
β-hydroxy-5-cholenic acid was found to be contained (Record of the 6th meeting of the tumor marker workshop, 254
-256, 1986).
【0004】また、新生児胆道閉鎖症の患者の尿中の3
β−ヒドロキシ−5−コレン酸の定量結果は、尿では高
率で硫酸エステル化されていることが示されており、尿
での3β−ヒドロキシ胆汁酸の測定は、3α−ヒドロキ
シ胆汁酸と同様に、硫酸エステル型(硫酸抱合型)の定
量の重要性が示唆されている。しかしながら、3β−ヒ
ドロキシ胆汁酸の硫酸抱合型の簡便な定量法の確立に必
要な該3β−硫酸エステルを効率良く加水分解する酵素
の存在は、知られていなかった。[0004] In addition, 3 in urine of patients with neonatal biliary atresia.
The results of quantification of β-hydroxy-5-cholenic acid show that the urine is highly sulfated in urine, and the measurement of 3β-hydroxy bile acid in urine is similar to that of 3α-hydroxy bile acid. The importance of quantification of the sulfate ester type (sulfate conjugate type) is suggested. However, the existence of an enzyme that efficiently hydrolyzes the 3β-sulfate ester required for establishing a simple method for quantitative determination of the sulfate-conjugated form of 3β-hydroxybile acid was not known.
【0005】[0005]
【発明が解決しようとする課題】本発明は、3β−ヒド
ロキシ胆汁酸の3β−硫酸エステルの総量を特異的に検
出するための試薬および該試薬を用いた3β−ヒドロキ
シ胆汁酸の3β−硫酸エステルの総量の測定法を確立す
ることを目的とする。DISCLOSURE OF THE INVENTION The present invention relates to a reagent for specifically detecting the total amount of 3β-sulfate of 3β-hydroxy bile acid, and a 3β-sulfate of 3β-hydroxy bile acid using the reagent. The aim is to establish a method for measuring the total amount of
【0006】[0006]
【課題を解決するための手段】本発明者は、上記課題を
解決するため鋭意研究を重ねた結果、シュードモナス属
に属する細菌から、ステロイド核を有する化合物の3β
−硫酸エステルを加水分解して、3α−ヒドロキシステ
ロイド化合物を生成する酵素を見出した。Means for Solving the Problems The inventors of the present invention have made intensive studies to solve the above-mentioned problems, and as a result, have found that a bacterium belonging to the genus Pseudomonas sp.
An enzyme that hydrolyzes a sulfate ester to produce a 3α-hydroxysteroid compound.
【0007】即ち、本発明は、次の性状を有するステロ
イドβ−硫酸サルファターゼに係る: (a) 作用:ステロイド核の3位にβ位で結合した硫酸エ
ステルに作用して硫酸エステル部分を加水分解し、その
際OH基の結合配置をβ配置からα配置に逆転させ、3
α−ヒドロキシステロイド核を生成する;および (b) 分子量:ゲル濾過カラムを用いた高速液体クロマト
グラフィー法により測定した結果、約70,000〜1
50,000ダルトンである。That is, the present invention relates to a steroid β-sulfate sulfatase having the following properties: (a) action: acting on a sulfate bound at the β-position to the 3-position of the steroid nucleus to hydrolyze the sulfate moiety; In this case, the bond configuration of the OH group is reversed from the β configuration to the α configuration,
produces an α-hydroxysteroid nucleus; and (b) molecular weight: about 70,000 to 1 as determined by high performance liquid chromatography using a gel filtration column.
50,000 daltons.
【0008】また、本発明は、シュードモナス属に属す
る細菌を、胆汁酸を含む培地にて培養し、その培養物か
ら本発明のステロイドβ−硫酸サルファターゼを採取す
ることを特徴とするステロイドβ−硫酸サルファターゼ
の製造法に係る。[0008] The present invention also provides a steroid β-sulfate which is obtained by culturing a bacterium belonging to the genus Pseudomonas in a medium containing bile acids and collecting the steroid β-sulfate sulfatase of the present invention from the culture. The present invention relates to a method for producing sulfate sulfatase.
【0009】さらに、本発明は、β−NADの存在下、
胆汁酸の3β−硫酸エステルを含む試料に、本発明のス
テロイドβ−硫酸サルファターゼおよび3α−ヒドロキ
システロイドデヒドロゲナーゼを同時にまたはこの順で
作用させ、その際生成するNADHを定量することから
なる胆汁酸の3β−硫酸エステルの定量法に係る。Further, the present invention provides a method for preparing
A steroid β-sulfate sulfatase and a 3α-hydroxysteroid dehydrogenase of the present invention are allowed to act on a sample containing 3β-sulfate of bile acid simultaneously or in this order, and the NADH produced at that time is quantified to determine bile acid. It relates to a method for determining 3β-sulfate.
【0010】本発明者は、鋭意研究の結果、胆汁酸3β
−硫酸の硫酸エステル部分を加水分解する、新規なステ
ロイドβ−硫酸サルファターゼを得ることに成功し、該
酵素と3α−ヒドロキシステロイド・デヒドロゲナーゼ
をβ−NAD存在下に共役させることにより、従来酵素
法では測定が不可能であった血液又は尿中の胆汁酸の3
β−硫酸エステルを容易に定量できることを見出した。The present inventors have conducted intensive studies and found that bile acid 3β
Succeeded in obtaining a novel steroid β-sulfate sulfatase which hydrolyzes a sulfate portion of sulfuric acid, and conjugated the enzyme with 3α-hydroxysteroid dehydrogenase in the presence of β-NAD to obtain a conventional enzyme method. Of bile acids in blood or urine that could not be measured by
It has been found that β-sulfate can be easily quantified.
【0011】本発明のステロイドβ−硫酸サルファター
ゼは、以下に示すように、胆汁酸の3β−硫酸エステル
のみならず、3β−硫酸エステル結合を有する他のステ
ロイド化合物にも作用する。[0011] The steroid β-sulfate sulfatase of the present invention acts not only on the 3β-sulfate ester of bile acid but also on other steroid compounds having a 3β-sulfate ester bond, as shown below.
【0012】本発明のステロイドβ−硫酸サルファター
ゼ(以下本酵素という)は、例えば、シュードモナス
(Pseudomonas )属に属する細菌から得られるが、上記
性質を有する限り、他の細菌、酵母、真菌類、植物また
は動物が産生するものであっても良い。その培養は、公
知の方法によって行うことができる。The steroid β-sulfate sulfatase of the present invention (hereinafter referred to as the present enzyme) can be obtained, for example, from a bacterium belonging to the genus Pseudomonas, but as long as it has the above properties, other bacteria, yeasts, fungi, It may be produced by plants or animals. The culturing can be performed by a known method.
【0013】本酵素は、好ましくは、例えば、シュード
モナス・テストステロニー(Pseudomonas testosteron
i)に属する細菌を培養することによって得られる。The present enzyme is preferably, for example, Pseudomonas testosteron
It is obtained by culturing the bacteria belonging to i).
【0014】シュードモナス・テストステロニー(以下
本菌種という)に属する細菌としては特に制限されず、
公知の細菌が使用できる。その中でも、例えば、シュー
ドモナス・テストステロニーATCC11996株等が
好ましい。この菌は市販品であり、入手可能である。The bacterium belonging to Pseudomonas testosteroni (hereinafter referred to as the present strain) is not particularly limited.
Known bacteria can be used. Among them, for example, Pseudomonas testosteroni ATCC11996 strain and the like are preferable. This fungus is a commercial product and is available.
【0015】一例として、シュードモナス・テストステ
ロニーに属する細菌から得られた本酵素の理化学的性質
をより詳細に記すと、以下の通りである。As an example, the physicochemical properties of the present enzyme obtained from a bacterium belonging to Pseudomonas testosterony are described in more detail below.
【0016】なお、本酵素の活性測定は以下のようにし
て行なわれる。すなわち3β−ヒドロキシ−5−コレン
酸3−硫酸(シグマ社製)の2.5mMメタノール溶液
0.1ml、β−NAD(オリエンタル酵母工業製)の1
5mM水溶液0.2ml、0.1Mトリス塩酸緩衝液(p
H8.0)1.0ml及び蒸溜水1.55mlを石英セルに
とり、30℃で平衡化した後、3α−ヒドロキシステロ
イド・デヒドロゲナーゼ(ナカライテスク製)溶液(1
0U/ml)0.05ml及び本酵素溶液0.1mlを順次添
加し、30℃で反応させ、340nmにおける反応初期
吸光度の増加を測定する。該条件下、1分間に1μmo
lのNADHを生成する酵素活性を1単位とする。The activity of the present enzyme is measured as follows. That is, 0.1 ml of a 2.5 mM methanol solution of 3β-hydroxy-5-cholenic acid 3-sulfuric acid (manufactured by Sigma) and 1 ml of β-NAD (manufactured by Oriental Yeast Co., Ltd.)
0.2 ml of 5 mM aqueous solution, 0.1 M Tris-HCl buffer (p
H8.0) and 1.55 ml of distilled water were placed in a quartz cell and equilibrated at 30 ° C., and then a 3α-hydroxysteroid dehydrogenase (Nacalai Tesque) solution (1
(0 U / ml) 0.05 ml and the present enzyme solution 0.1 ml are sequentially added and reacted at 30 ° C., and the increase in the initial absorbance at 340 nm is measured. Under these conditions, 1 μmo per minute
The enzyme activity for producing 1 NADH is defined as 1 unit.
【0017】(a) 作用:ステロイド核の3位にβ配置で
結合した硫酸エステルに作用して硫酸エステル部分を加
水分解し、その際、OH基の結合配置をβ配置からα配
置に逆転させ、3α−ヒドロキシステロイド核を生成す
る。(A) Action: Acts on a sulfate ester bonded to the 3-position of the steroid nucleus in a β-configuration to hydrolyze a sulfate ester moiety. At this time, the bond configuration of an OH group is reversed from a β-configuration to an α-configuration. Generates a 3α-hydroxysteroid nucleus.
【0018】(b) 基質特異性:ステロイド核を有する化
合物の3β硫酸エステルに作用する。詳細を、下記表1
に示す。(B) Substrate specificity: acts on 3β sulfate of a compound having a steroid nucleus. See Table 1 below for details
Shown in
【0019】[0019]
【表1】 [Table 1]
【0020】(c) 至適pH:至適pHは、8.5±0.
5であり、結果を図1に示す。本発明のサルファターゼ
は、至適pHは上記の通りであるが、pH約7〜9.5
の広い範囲で高い活性を示す。(C) Optimum pH: The optimum pH is 8.5 ± 0.5.
5 and the results are shown in FIG. The sulfatase of the present invention has an optimum pH as described above, but has a pH of about 7 to 9.5.
Shows high activity in a wide range of
【0021】(d) 安定pH:安定pH範囲は、30℃、
16時間処理で、pH6.5〜9.5である。結果を図
2に示す。(D) Stable pH: The stable pH range is 30 ° C.
The pH is 6.5 to 9.5 after 16 hours of treatment. The results are shown in FIG.
【0022】(e) 至適温度および熱安定性:至適温度は
35℃±3℃。pH7.5、10分間処理条件下で、3
5℃以下では活性は100%残存するが、それ以上の温
度では急速に活性は低下し、45℃で0%となる。結果
を図3及び図4に示す。(E) Optimum temperature and thermal stability: The optimum temperature is 35 ° C. ± 3 ° C. pH 7.5, under treatment conditions for 10 minutes, 3
At 5 ° C. or lower, 100% of the activity remains, but at higher temperatures, the activity rapidly decreases to 0% at 45 ° C. The results are shown in FIGS.
【0023】(f) 分子量:ゲル濾過カラムのシム−パッ
クジオール−300(Shim-pack Diol-300)(島津製作
所製)を用いた高速液体クロマトグラフィー法により測
定した結果、分子量約70,000〜150,000ダ
ルトン程度である。(F) Molecular weight: As a result of a high-performance liquid chromatography method using Shim-pack Diol-300 (manufactured by Shimadzu Corporation) of a gel filtration column, a molecular weight of about 70,000- It is about 150,000 daltons.
【0024】(g) Km値:3β−ヒドロキシ−5−コレ
ン酸3−硫酸に対するKm値:1×10-5M以下であ
る。(G) Km value: Km value for 3β-hydroxy-5-cholenic acid 3-sulfuric acid: 1 × 10 −5 M or less.
【0025】(h) 阻害及び活性化:Hg2+、Cu2+、Z
n2+により阻害を受けるが、金属キレート剤(EDT
A、O−フェナンスロリン等)によっては影響を受けな
い。また、p−クロロマーキュリーベンゾエート、モノ
ヨード酢酸等のSH試薬によっては、ほとんど阻害され
ない。詳細を、下記表2に示す。(H) Inhibition and activation: Hg 2+ , Cu 2+ , Z
Inhibited by n 2+ , metal chelators (EDT
A, O-phenanthroline, etc.). Further, it is hardly inhibited by SH reagents such as p-chloromercury benzoate and monoiodoacetic acid. Details are shown in Table 2 below.
【0026】[0026]
【表2】 [Table 2]
【0027】本酵素は、例えば、上述したように、シュ
ードモナス属に属する細菌を培養することによって得ら
れる。The enzyme can be obtained, for example, by culturing a bacterium belonging to the genus Pseudomonas, as described above.
【0028】本発明のサルファターゼを産生する生物の
培養に使用する培地は、特に制限されず、各種の由来生
物に合わせて適当な培地が使用できる。The medium used for culturing the organism producing the sulfatase of the present invention is not particularly limited, and an appropriate medium can be used according to various origin organisms.
【0029】以下に、本菌種の場合の培養条件を例示と
して記載する。Hereinafter, culture conditions for the present strain will be described as examples.
【0030】培地としては、シュードモナス属細菌用の
培地がいずれも使用できるが、例えば、有機栄養源とし
て酵母エキス、ペプトン、肉エキス等、無機栄養源とし
てリン酸アンモニウム、硝酸アンモニウム、リン酸カリ
ウム、リン酸ナトリウム、塩化マグネシウム、塩化マン
ガン等を含むものが用いられる。更に本酵素を生産させ
るためには、胆汁酸を培地に添加する。胆汁酸として
は、例えば、コール酸、デオキシコール酸、ケノデオキ
シコール酸、リトコール酸、これらの硫酸エステル及び
これらの塩等を挙げることができ、1種又は2種以上を
使用できる。胆汁酸の添加量は特に制限されないが、通
常0.05〜1.0重量%程度とすればよい。As the medium, any medium for bacteria of the genus Pseudomonas can be used. For example, yeast extract, peptone, meat extract and the like as organic nutrients, and ammonium phosphate, ammonium nitrate, potassium phosphate and phosphorus as inorganic nutrients. Those containing sodium acid, magnesium chloride, manganese chloride and the like are used. To further produce the enzyme, bile acids are added to the medium. Examples of bile acids include cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, sulfates thereof, salts thereof, and the like, and one or more kinds can be used. The amount of bile acid added is not particularly limited, but may be generally about 0.05 to 1.0% by weight.
【0031】培養時間及び温度は特に制限されないが、
通常好気的条件下に24〜34℃程度で12〜24時間
程度培養するのが好ましい。この程度の時間培養する
と、酵素活性が最大になる。The culture time and temperature are not particularly limited.
Usually, it is preferable to culture at about 24-34 ° C. for about 12-24 hours under aerobic conditions. Cultivation for such a period maximizes the enzyme activity.
【0032】培養によって得られる菌体から、本酵素が
抽出される。抽出は、通常の菌体内酵素の抽出法に従っ
て行なうことができる。例えば超音波処理、各種機械的
処理、酵素処理等の方法により菌体を破砕し、不溶物を
遠心分離した上清に酵素を回収することができる。この
粗酵素を、除核酸処理、硫安塩析、イオン交換クロマト
グラフィー、疎水性クロマトグラフィー、ゲル濾過等の
一般的な酵素精製法を適宜選択、組み合わせて精製する
ことにより、本酵素を得ることができる。The present enzyme is extracted from the cells obtained by culturing. The extraction can be performed according to a usual method for extracting enzymes in cells. For example, the cells can be disrupted by a method such as ultrasonic treatment, various mechanical treatments, and enzyme treatments, and the enzyme can be recovered in the supernatant obtained by centrifuging insolubles. By purifying the crude enzyme by appropriately selecting and combining common enzyme purification methods such as nucleic acid removal treatment, ammonium sulfate salting out, ion exchange chromatography, hydrophobic chromatography, gel filtration, etc., the present enzyme can be obtained. it can.
【0033】このようにして得られる本酵素と3α−ヒ
ドロキシステロイド・デヒドロゲナーゼとを、β−NA
Dの存在下、胆汁酸の3β−硫酸エステルを含む試料に
作用させることにより、胆汁酸3β−硫酸エステルの定
量を行なうことができる。即ち、本酵素が胆汁酸3β−
硫酸エステルを3α−ヒドロキシ胆汁酸に変換し、次い
で3α−ヒドロキシステロイド・デヒドロゲナーゼが3
α−ヒドロキシ胆汁酸を3−オキソ胆汁酸に変換すると
ともに、該デヒドロゲナーゼの補酵素であるNADをN
ADHに還元する。従って、NADからNADHへの変
換量を定量することにより、尿、血液などの生体由来の
試料中の胆汁酸3β−硫酸エステルを定量することがで
きる。The thus obtained enzyme and 3α-hydroxysteroid dehydrogenase are combined with β-NA
By acting on a sample containing bile acid 3β-sulfate in the presence of D, bile acid 3β-sulfate can be quantified. That is, the enzyme is bile acid 3β-
The sulfate ester is converted to 3α-hydroxy bile acid, which is then converted to 3α-hydroxy steroid dehydrogenase.
While converting α-hydroxy bile acid to 3-oxo bile acid, NAD, which is a coenzyme of the dehydrogenase, is converted to N
Reduce to ADH. Therefore, by determining the amount of conversion from NAD to NADH, bile acid 3β-sulfate in a sample derived from a living body such as urine or blood can be determined.
【0034】3α−ヒドロキシステロイド・デヒドロゲ
ナーゼとしては特に制限されず、公知のものがいずれも
使用できる。The 3α-hydroxysteroid dehydrogenase is not particularly limited, and any known one can be used.
【0035】酵素の使用量は特に制限されないが、通常
本酵素を0.1〜3.0単位/ml程度、及び3α−ヒ
ドロキシステロイド・デヒドロゲナーゼを0.1〜3.
0単位/ml程度使用すればよい。The amount of the enzyme to be used is not particularly limited. Usually, the present enzyme is used at about 0.1 to 3.0 units / ml, and 3α-hydroxysteroid dehydrogenase is used at 0.1 to 3.
What is necessary is just to use about 0 unit / ml.
【0036】β−NADの使用量も特に制限されず適宜
選択すればよいが、通常反応系中のβ−NAD濃度が
0.5〜5mM程度の濃度になるように添加すればよ
い。酵素反応は、通常25〜40℃程度の温度下に、5
〜30分程度行なわれる。The amount of β-NAD to be used is not particularly limited and may be appropriately selected, but may be usually added so that the β-NAD concentration in the reaction system is about 0.5 to 5 mM. The enzymatic reaction is usually performed at a temperature of about 25 to 40 ° C for 5 minutes.
This is performed for about 30 minutes.
【0037】またNADHの定量は、公知の方法に従っ
て行なうことができる。その具体例としては、UV(3
40nm)吸収測定法、蛍光強度測定法、ジアホラーゼ
或いはフェナジンメトサルフェート等を介して酸化還元
色素反応と共役させる比色定量法(還元発色比色法)、
NADHオキシダーゼを作用させてNADHから過酸化
水素を定量的に生成させ、該過酸化水素を酸化縮合発色
系と共役させることによって比色定量(酸化発色)する
方法等が挙げられる。NADH can be quantified according to a known method. As a specific example, UV (3
40 nm) absorption measurement method, fluorescence intensity measurement method, colorimetric quantification method (reduction colorimetric colorimetry method), which is conjugated with a redox dye reaction via diaphorase or phenazine methosulfate, etc.
A method in which NADH oxidase is allowed to act to quantitatively generate hydrogen peroxide from NADH, and the hydrogen peroxide is conjugated with an oxidative condensation coloring system to perform colorimetric determination (oxidative coloring), and the like.
【0038】[0038]
【発明の効果】本発明によれば、新規なステロイドβ−
硫酸サルファターゼを提供できる。また、β−NADの
存在下に本酵素と3α−ヒドロキシステロイド・デヒド
ロゲナーゼを共役させることにより、従来酵素法では不
可能であった尿または血液中の胆汁酸3β−硫酸エステ
ルを容易に定量できるようになり、各種の疾患の診断に
も応用できる。According to the present invention, a novel steroid β-
Sulfate sulfatase can be provided. In addition, by conjugating the present enzyme with 3α-hydroxysteroid dehydrogenase in the presence of β-NAD, bile acid 3β-sulfate in urine or blood, which was not possible by the conventional enzymatic method, can be easily quantified. It can be applied to diagnosis of various diseases.
【0039】また、本発明の酵素は、例えば24以下の
炭素数のステロイド骨格を有する化合物の3β−硫酸エ
ステルを広く加水分解する活性を有するため、従来測定
の難しかった、各種のステロイド系化合物の硫酸エステ
ルの定量に有用である。Further, the enzyme of the present invention has an activity of widely hydrolyzing 3β-sulfate ester of a compound having a steroid skeleton having 24 or less carbon atoms. Useful for the determination of sulfate.
【0040】[0040]
【実施例】以下に実施例を挙げるが、本発明はこれらに
制限されるものではない。The following examples are given, but the present invention is not limited to these examples.
【0041】なお、以下の実施例において「%」とある
のは、「重量%」を意味する。In the following examples, “%” means “% by weight”.
【0042】実施例1 リン酸一アンモニウム0.1%、リン酸二アンモニウム
0.1%、リン酸一カリウム0.2%、塩化マグネシウ
ム0.01%、塩化マンガン0.01%及び酵母エキス
1.0%より成る培地(pH6.9)300mlを、2L
容三角フラスコに入れ、オートクレーブで滅菌した。こ
れに、シュードモナス・テストステロニーATCC11
996を植菌し、28℃で24時間振盪培養した。この
種培養液を、50L容ジャーファーメンター中にて、上
記と同組成の滅菌済培地30Lに植菌し、同時に別途滅
菌した10%コール酸ナトリウム水溶液1.2Lを無菌
的に加え、28℃で通気撹拌しながら15時間培養し
た。Example 1 Monoammonium phosphate 0.1%, diammonium phosphate 0.1%, monopotassium phosphate 0.2%, magnesium chloride 0.01%, manganese chloride 0.01% and yeast extract 1 300 ml of a medium (pH 6.9) containing 2.0%
The flask was placed in an Erlenmeyer flask and sterilized in an autoclave. This includes Pseudomonas testosterony ATCC11
996 was inoculated and cultured with shaking at 28 ° C. for 24 hours. This seed culture solution was inoculated into 30 L of a sterilized medium having the same composition as above in a 50 L jar fermenter, and at the same time, 1.2 L of a 10% aqueous sodium cholate solution which had been separately sterilized was added aseptically. And cultured for 15 hours with aeration and stirring.
【0043】培養液を遠心分離し、得られた菌体を30
mMリン酸緩衝液(pH7.2)に懸濁し、この懸濁液
をミニ・ラボ細胞破壊装置(ラニイー社製)にかけて、
菌体を破砕した。これを遠心分離して沈殿物を除き、粗
酵素液を得た。この粗酵素液をプロタミン硫酸で除核酸
処理した後、硫酸アンモニウムにより塩析処理し、硫酸
アンモニウム35〜70%飽和沈澱画分を集め、10m
Mトリス−塩酸緩衝液(pH8.0)に透析した。この
透析液を、10mMトリス−塩酸緩衝液(pH8.0)
で平衡化したDEAEセファロースCL−6B(ファル
マシア社製)・カラムに通液した。得られた非吸着画分
を、硫酸アンモニウム70%飽和塩析し、沈殿画分を集
め、50mMリン酸緩衝液(pH6.8)に溶解し、硫
酸アンモニウム濃度を30%飽和に調整して、硫酸アン
モニウムを30%飽和濃度に含む50mMリン酸緩衝液
で平衡化したオクチルセファロースCL−4B(ファル
マシア社製)・カラムに通液した。得られた非吸着画分
を、硫酸アンモニウム70%飽和塩析し、沈殿画分を5
0mM塩化ナトリウムを含む10mMリン酸緩衝液(p
H6.4)に溶解、同緩衝液に透析し、同緩衝液に平衡
化したCMセファロース・ファーストフロー(ファルマ
シア社製)・カラムに通液して活性画分を吸着させた。
このカラムを、10mMリン酸緩衝液(pH6.4)中
の塩化ナトリウム濃度を50mMから300mMまで連
続的に上昇させるグラジエント溶出法で展開し、活性画
分を溶出した。これを、硫酸アンモニウムによる塩析処
理で濃縮し、0.15M塩化ナトリウムを含む50mM
リン酸緩衝液(pH6.8)で平衡化したウルトロゲル
ACA34(LKB社製)・カラムを通してゲル濾過し
た。活性画分を限外濾過により脱塩及び濃縮し、ステロ
イドβ−硫酸サルファターゼ250単位を得た。該精製
標品は、ポリアクリルアミド電気泳動用PAGプレート
4/15(第一化学薬品製)によるスラブゲル電気泳動
において単一バンドを示した。The culture solution was centrifuged, and the obtained cells
mM phosphate buffer (pH 7.2), and this suspension was applied to a mini-lab cell disrupter (manufactured by Lanie) to obtain a suspension.
The cells were disrupted. This was centrifuged to remove the precipitate to obtain a crude enzyme solution. The crude enzyme solution was subjected to a nucleic acid removal treatment with protamine sulfate, and then subjected to salting out treatment with ammonium sulfate.
It was dialyzed against M Tris-HCl buffer (pH 8.0). This dialysate is added to a 10 mM Tris-HCl buffer (pH 8.0)
The solution was passed through a column of DEAE Sepharose CL-6B (manufactured by Pharmacia) equilibrated in the above. The obtained non-adsorbed fraction was subjected to 70% saturated salting out with ammonium sulfate, and the precipitated fraction was collected and dissolved in a 50 mM phosphate buffer (pH 6.8). The solution was passed through an octyl sepharose CL-4B (Pharmacia) column equilibrated with 50 mM phosphate buffer containing 30% saturation. The obtained non-adsorbed fraction was subjected to 70% saturated salting out with ammonium sulfate, and the precipitated fraction
10 mM phosphate buffer containing 0 mM sodium chloride (p
H6.4), dialyzed against the same buffer, passed through a CM Sepharose Fast Flow (Pharmacia) column equilibrated with the same buffer to adsorb the active fraction.
This column was developed by a gradient elution method in which the concentration of sodium chloride in a 10 mM phosphate buffer (pH 6.4) was continuously increased from 50 mM to 300 mM, and the active fraction was eluted. This was concentrated by a salting-out treatment with ammonium sulfate, and 50 mM containing 0.15 M sodium chloride.
Gel filtration was performed through an Ultrogel ACA34 (manufactured by LKB) column equilibrated with a phosphate buffer (pH 6.8). The active fraction was desalted and concentrated by ultrafiltration to obtain 250 units of steroid β-sulfate sulfatase. The purified sample showed a single band in slab gel electrophoresis using a polyacrylamide electrophoresis PAG plate 4/15 (Daiichi Pure Chemicals).
【0044】また、該精製標品の分子量を、ゲル濾過カ
ラムのシム−パックジオール−300(島津製作所製)
を用いた高速液体クロマトグラフィー法により測定した
結果、約95,000ダルトンであった。The molecular weight of the purified sample was determined by measuring the molecular weight of Shim-Pakdiol-300 (manufactured by Shimadzu Corporation) in a gel filtration column.
As a result of measurement by a high performance liquid chromatography method using, it was about 95,000 daltons.
【0045】実施例2 実施例1で得られたステロイドβ−硫酸サルファターゼ
を用い、ステロイド核に二重結合を有する代表的な異常
胆汁酸である3β−ヒドロキシ−5−コレン酸の硫酸エ
ステル(3β−ヒドロキシ−5−コレン酸3−硫酸)の
定量を行った。β−NAD(オリエンタル酵母工業製)
3μmol、3α−ヒドロキシステロイド・デヒドロゲ
ナーゼ(ナカライテスク製)0.5単位、ステロイドβ
−硫酸サルファターゼ0.4単位及び0.5%ヒドラジ
ン一水和物を含む35mMピロリン酸緩衝液(pH8.
9)2.9mlに、基質として、濃度の異なる3β−ヒド
ロキシ−5−コレン酸3−硫酸(シグマ社製)メタノー
ル溶液を0.1ml加え、30℃で10分間反応させた
後、340nmにおける吸光度を測定した。結果を図5
に示す。図5から、基質濃度と吸光度が正の相関関係に
あり、従って3β−ヒドロキシ−5−コレン酸3−硫酸
の定量が可能であることが判る。Example 2 Using the steroid β-sulfate sulfatase obtained in Example 1, a sulfate ester of 3β-hydroxy-5-cholenic acid, a typical abnormal bile acid having a double bond in the steroid nucleus ( 3β-hydroxy-5-cholenic acid 3-sulfuric acid) was quantified. β-NAD (Oriental Yeast Industry)
3 μmol, 0.5 unit of 3α-hydroxysteroid dehydrogenase (manufactured by Nacalai Tesque), steroid β
-35 mM pyrophosphate buffer containing 0.4 units of sulfate sulfatase and 0.5% hydrazine monohydrate (pH 8.
9) To 2.9 ml of a substrate, 0.1 ml of a methanol solution of 3β-hydroxy-5-cholenic acid 3-sulfuric acid (manufactured by Sigma) having a different concentration was added as a substrate, reacted at 30 ° C. for 10 minutes, and then subjected to absorbance at 340 nm. Was measured. Fig. 5 shows the results.
Shown in From FIG. 5, it is found that the substrate concentration and the absorbance have a positive correlation, and therefore, the quantification of 3β-hydroxy-5-cholenic acid 3-sulfuric acid is possible.
【0046】実施例3 試薬1:ステロイドβ−硫酸サルファターゼ50単位、
牛血清アルブミン20mg、サッカロース400mg、およ
びTween20 600mgを100mMトリス−塩酸
緩衝液(pH7.5)20mlに溶解した。Example 3 Reagent 1: 50 units of steroid β-sulfate sulfatase,
20 mg of bovine serum albumin, 400 mg of saccharose, and 600 mg of Tween 20 were dissolved in 20 ml of 100 mM Tris-HCl buffer (pH 7.5).
【0047】試薬2:β−NAD(オリエンタル酵母工
業製)130mg、ニトロブルーテトラゾリウム(ナカラ
イテスク製)30mg、3α−ヒドロキシステロイド・デ
ヒドロゲナーゼ100単位、ジアホラーゼ(オリエンタ
ル酵母工業製)400単位、牛血清アルブミン250m
g、およびサッカロース1gを200mMリン酸緩衝液
(pH7.0)50mlに溶解した。Reagent 2: 130 mg of β-NAD (manufactured by Oriental Yeast), 30 mg of nitroblue tetrazolium (manufactured by Nacalai Tesque), 100 units of 3α-hydroxysteroid dehydrogenase, 400 units of diaphorase (manufactured by Oriental Yeast), 250 m of bovine serum albumin
g and 1 g of saccharose were dissolved in 50 ml of 200 mM phosphate buffer (pH 7.0).
【0048】試薬3:2Mクエン酸水溶液 測定:各種濃度の3β−ヒドロキシ−5−コレン酸3−
硫酸の水溶液0.2mlに 試薬1 0.4mlを加えて30℃で10分間保持し、脱
硫酸反応を行わせた後、 試薬2 0.5mlを添加、混合して30℃で10分間保
持し、発色させ、次いで 試薬3 0.1mlを加えて反応を停止後、540nmの
吸光度を測定した。その結果、図6に示すように、良好
な結果が得られた。Reagent 3: 2 M aqueous citric acid solution Measurement: Various concentrations of 3β-hydroxy-5-cholenic acid 3-
0.4 ml of reagent 1 was added to 0.2 ml of an aqueous solution of sulfuric acid, and the mixture was kept at 30 ° C. for 10 minutes. After desulfation reaction was performed, 0.5 ml of reagent 2 was added, mixed, and kept at 30 ° C. for 10 minutes. After the color was developed, 0.1 ml of Reagent 3 was added to stop the reaction, and the absorbance at 540 nm was measured. As a result, good results were obtained as shown in FIG.
【0049】実施例4 3β−ヒドロキシ−5−コレン酸3−硫酸(3β−CA
S)を添加した尿を、無添加尿で種々の濃度に希釈し、
試験用尿とした。Example 4 3β-hydroxy-5-cholenic acid 3-sulfuric acid (3β-CA
Diluting the urine to which S) has been added to various concentrations with no urine added;
Test urine was used.
【0050】試験用尿0.2mlに実施例3の試薬1
0.4mlを加え、30℃で10分間保持した。これに、
実施例3の試薬2 0.5mlを添加、混合して30℃で
正確に10分間保持し、発色させ、実施例3の試薬3
0.1mlを加えて反応を停止後、540nmの吸光度を
測定した。結果を表3に示す。Reagent 1 of Example 3 in 0.2 ml of test urine
0.4 ml was added and kept at 30 ° C. for 10 minutes. to this,
0.5 ml of the reagent 3 of Example 3 was added, mixed, and kept at 30 ° C. for exactly 10 minutes to develop a color.
After stopping the reaction by adding 0.1 ml, the absorbance at 540 nm was measured. Table 3 shows the results.
【0051】[0051]
【表3】 [Table 3]
【0052】この結果より、基質濃度と吸光度が正の相
関関係にあることが判った。From the results, it was found that the substrate concentration and the absorbance had a positive correlation.
【0053】実施例5 3β−ヒドロキシ−5−コレン酸3−硫酸(3β−CA
S)を添加した血清を、無添加血清で種々の濃度に希釈
し、試験用血清とした。Example 5 3β-hydroxy-5-cholenic acid 3-sulfuric acid (3β-CA
The serum to which S) was added was diluted to various concentrations with serum without addition, and used as a test serum.
【0054】試験用血清0.2mlに実施例3の試薬1
0.4mlを加え、以後実施例4と同様にして3β−CA
Sを定量した。結果を表4に示す。Reagent 1 of Example 3 in 0.2 ml of test serum
0.4 ml, and then 3β-CA
S was quantified. Table 4 shows the results.
【0055】[0055]
【表4】 [Table 4]
【0056】この結果より、基質濃度と吸光度が正の相
関関係にあることが判った。From the results, it was found that the substrate concentration and the absorbance had a positive correlation.
【図1】本酵素の至適pHを示すグラフである。FIG. 1 is a graph showing the optimum pH of the present enzyme.
【図2】本酵素の安定pHを示すグラフである。FIG. 2 is a graph showing a stable pH of the present enzyme.
【図3】本酵素の至適温度を示すグラフである。FIG. 3 is a graph showing the optimum temperature of the present enzyme.
【図4】本酵素の熱安定性を示すグラフである。FIG. 4 is a graph showing the thermostability of the present enzyme.
【図5】本発明方法を実施した場合の基質(胆汁酸3β
−硫酸)濃度と吸光度の関係を示すグラフである。FIG. 5 shows a substrate (bile acid 3β) when the method of the present invention is carried out.
-Graph showing the relationship between (sulfuric acid) concentration and absorbance.
【図6】本発明方法を実施した場合の基質(胆汁酸3β
−硫酸)濃度と吸光度の関係を示すグラフである。FIG. 6 shows a substrate (bile acid 3β) when the method of the present invention is carried out.
-Graph showing the relationship between (sulfuric acid) concentration and absorbance.
───────────────────────────────────────────────────── フロントページの続き (58)調査した分野(Int.Cl.7,DB名) C12N 9/00 - 9/99 C12Q 1/00 - 1/70 BIOSIS(DIALOG) WPI(DIALOG)────────────────────────────────────────────────── ─── Continued on the front page (58) Fields surveyed (Int. Cl. 7 , DB name) C12N 9/00-9/99 C12Q 1/00-1/70 BIOSIS (DIALOG) WPI (DIALOG)
Claims (3)
ルファターゼ: (a) 作用:ステロイド核の3位にβ位で結合した硫酸エ
ステルに作用して硫酸エステル部分を加水分解し、その
際OH基の結合配置をβ配置からα配置に逆転させ、3
α−ヒドロキシステロイド核を生成する;および (b) 分子量:ゲル濾過カラムを用いた高速液体クロマト
グラフィー法により測定した結果、約70,000〜1
50,000ダルトンである。1. A steroid β-sulfate sulfatase having the following properties: (a) action: acting on a sulfate bound at the β-position to the 3-position of the steroid nucleus to hydrolyze the sulfate moiety, whereby OH The bond configuration of the group is reversed from the β configuration to the α configuration, and 3
produces an α-hydroxysteroid nucleus; and (b) molecular weight: about 70,000 to 1 as determined by high performance liquid chromatography using a gel filtration column.
50,000 daltons.
酸を含む培地にて培養し、その培養物から請求項1に記
載のステロイドβ−硫酸サルファターゼを採取すること
を特徴とするステロイドβ−硫酸サルファターゼの製造
法。2. A steroid β-sulfate, wherein a bacterium belonging to the genus Pseudomonas is cultured in a medium containing bile acids, and the steroid β-sulfate sulfatase according to claim 1 is collected from the culture. A method for producing sulfatase.
酸エステルを含む試料に、請求項1に記載のステロイド
β−硫酸サルファターゼおよび3α−ヒドロキシステロ
イドデヒドロゲナーゼを同時にまたはこの順で作用さ
せ、その際生成するNADHを定量することからなる胆
汁酸の3β−硫酸エステルの定量法。3. A sample containing bile acid 3β-sulfate in the presence of β-NAD, the steroid β-sulfate sulfatase and 3α-hydroxysteroid dehydrogenase according to claim 1 act simultaneously or in this order. And a method for quantifying 3β-sulfate of bile acid, which comprises quantifying NADH produced at that time.
Priority Applications (1)
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JP3674893A JP3035685B2 (en) | 1993-02-25 | 1993-02-25 | Steroid β-sulfate sulfatase, method for producing the same, and method for determination of bile acid 3β-sulfate |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP3674893A JP3035685B2 (en) | 1993-02-25 | 1993-02-25 | Steroid β-sulfate sulfatase, method for producing the same, and method for determination of bile acid 3β-sulfate |
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Publication Number | Publication Date |
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JP3035685B2 true JP3035685B2 (en) | 2000-04-24 |
Family
ID=12478359
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