Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Patent
1990-07-26
1992-02-25
Robinson, Douglas W.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
435233, 435196, C12N 916
Patent
active
050913050
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to bile acid sulfate sulfatase, its preparation and methods of quantitatively determining bile acid.
The enzyme of the invention is a useful novel enzyme for clinical examinations, and is useful for the determination of sulfated bile acids such as bile acids in which the OH group at 3-position is sulfated (hereinafter referred to as bile acid 3.alpha.-sulfate), and for the determination of bile acid in blood or urine.
PRIOR ART
It is well known that the bile acid in blood or urine markedly increases due to hepatobiliary diseases. It is therefore an important item to determine the bile acid in blood or urine for evaluating the hepatic function in clinical examinations. Conventional method of determining bile acid adopts the enzymatic analysis method wherein 3.alpha.-hydroxysteroid dehydrogenase is employed. Said dehydrogenase oxidizes bile acids wherein the 3-position OH group is in .alpha.-configuration (3.alpha.-hydroxy bile acid) to 3-oxo bile acids, and concurrently reduces the coenzyme .beta.-NAD to NADH. By determining the NADH, the 3.alpha.-hydroxy bile acids are determined. In the conventional clinical examination, the quantity of 3.alpha.-hydroxy bile acids is regarded as the total amount of bile acids.
However, bile acids in blood are present in part as sulfated bile acids which have been sulfated, and the hydrophilic property is enhanced by the sulfation and the excretion thereof is facilitated, so that the ratio of sulfated bile acids, in particular bile acid 3.alpha.-sulfates, in urine is extremely increased. The dehydrogenase in the prior art method acts only on the 3.alpha.-hydroxy bile acids, and hence in the prior art method it is impossible to determine the bile acid 3.alpha.-sulfates wherein the 3.alpha.-hydroxyl group has been sulfated. In clinical examinations, it is necessary to determine the total bile acid 3.alpha.-sulfates or the total bile acids including it in urine or blood, but bile acid sulfatase capable of specifically hydrolyzing the sulfate ester moiety of the bile acid 3.alpha.-sulfates is not known yet.
Under the circumstances, in order to determine the bile acid 3.alpha.-sulfates, a sample therefor must be subjected to a column chromatography to separate bile acid 3.alpha.-sulfates, and then the sulfate moiety of the bile acid 3.alpha.-sulfates must be chemically hydrolyzed by solvolysis. Such process is, however, very cumbersome and cannot be used easily in the daily clinical examinations.
DISCLOSURE OF THE INVENTION
It is an object of the invention to provide a novel bile acid sulfate sulfatase capable of specifically hydrolyzing the sulfate ester moiety of bile acid 3.alpha.-sulfates.
It is another object of the invention to provide a method for the preparation of such bile acid sulfate sulfatase.
It is another object of the invention to provide a method capable of easily determining the total bile acid 3.alpha.-sulfates in blood or urine which were impossible to measure in the conventional enzymatic method.
It is a further object of the invention to provide a method capable of easily determining the total bile acids, including the bile acid 3.alpha.-sulfates, in blood or urine.
Other objects and features of the invention will become apparent from the following description.
The present invention provides a bile acid sulfate sulfatase, its preparation, and methods of determining bile acids, as defined in 1 to 4 below.
1. A bile acid sulfate sulfatase having the following properties: sulfate ester moiety thereof and to invert the bonding configuration of the resulting OH group from .alpha.-configuration to .beta.-configuration, thereby producing 3.beta.-hydroxy bile acids; acids, and 3.alpha.-sulfates each of glycine-conjugated and taurine-conjugated bile acids; and
2. A method for the preparation of a bile acid sulfate sulfatase comprising culturing Pseudomonas testosteroni in a culture medium containing bile acid, and recovering the bile acid sulfate sulfatase of claim 1 from the resulting culture.
3. A method
REFERENCES:
patent: 4889801 (1989-12-01), Ushizawa
Takagi et al., Vitamins (Japan) 60:165-172 (1986).
Imperato et al., Clin Res 25:109A (1977).
Huijghebaert et al., App. and Env. Micro. 44:1030-34 (1982).
Sugimori Tsunetake
Tatsuke Yasuhiko
Tsukada Yoji
Marukin Shoyu Co., Ltd.
Robinson Douglas W.
Saucier S.
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