Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing heterocyclic carbon compound having only o – n – s,...
Reexamination Certificate
2000-03-13
2001-10-16
Lilling, Herbert J. (Department: 2721)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing heterocyclic carbon compound having only o, n, s,...
C435S197000, C435S280000
Reexamination Certificate
active
06303349
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel esterase which is useful as a catalyst for the hydrolyses of esters, methods for the isolation of said esterase, methods for the kinetic resolution of a mixture of a (R)- form and a (S)-form of an ester compound using said esterase, and methods using said esterase for producing optically active chroman-3-acetic acids and esters thereof, which are useful as medicinal materials.
Furthermore, the present invention relates to methods for producing optically active chroman-3-acetic acids and esters thereof, wherein a mixture of (3R)- and (3S)-chroman-3-acetic acid esters represented by the general formula (I) described below is treated with an ester hydrolase which has an optically selective hydrolyzing activity or microorganisms which carry said hydrolase, or a preparation therefrom.
2. Description of the Related Art
A number of attempts have been made in recent years to apply enzymes in organic syntheses. In particular, the use of esterases, which have high substrate specificities for the optically selective hydrolysis of various ester compounds selectively to thereby obtain optically active compounds, or for producing chiral compounds from prochiral ester compounds, has high industrial efficacy.
An esterase derived from porcine liver is generally used in such purposes as mentioned above but its high cost and limited availability precludes its industrial usage. The use of esterases from microbial sources in place of the esterase derived from porcine liver has been tried. Since enzymes derived from different organisms are characteristically different in their substrate specificities, the selectivity and reaction rate of the enzymes markedly vary depending on the compounds to which the enzymes are applied.
A 6-aminochroman-3-acetic acid ester disclosed in European Patent Publications, EP 0709370 and EP 0760364, is a useful intermediate of an antiplatelet. However, no esterase which shows high optical selectivity for 6-aminochroman-3-acetic acid esters and which can effectively hydrolyze them has been reported.
An objective of the present invention is to find an esterase which is optically selective and useful as a catalyst for producing optically active chroman-3-acetic acids and esters thereof, which are useful as medicinal materials.
Another objective of the present invention is to provide methods for the simple and efficient production of optically active chroman-3-acetic acids and esters thereof.
SUMMARY OF THE INVENTION
As a result of intensive studies to search for a method for the simple and efficient production of optically active chroman-3-acetic acids and esters thereof, the present inventors found that optically active chroman-3-acetic acids and esters thereof can be obtained by treating a mixture of (3R)- and (3S)-chroman-3-acetic acid esters with an ester hydrolase which has an optically selective hydrolyzing activity or microorganisms which carry said hydrolase, or an enzyme preparation therefrom. Furthermore, the present inventors found a novel esterase, which has an optically selective hydrolyzing activity and shows excellent heat and pH stability, from a microorganism of genus Pseudonocardia using a variety of purification techniques and thus came to complete the present invention.
Namely, the present invention relates to a novel esterase found in a microorganism of genus Pseudonocardia which is useful as a catalyst for hydrolyses of esters, methods for the isolation of said esterase, methods for the kinetic resolution of a mixture of (R) and (S) ester compounds using said esterase, and methods using said esterase for producing optically active chroman-3-acetic acids and esters thereof, which are useful as medicinal materials.
Furthermore, the present invention relates to methods for producing optically active chroman-3-acetic acids and esters thereof, wherein a mixture of (3R)- and (3S)-chroman-3-acetic acid esters represented by the formula (I)
[wherein R
1
is a straight or branched alkyl group having 1-5 carbon atoms and R
2
is a hydrogen atom or substituted or unsubstituted amino group]
is treated with an ester hydrolase which has an optically selective hydrolyzing activity or microorganisms which carry said hydrolase, or a preparation therefrom.
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
The present invention will be explained in greater detail as follows:
(1) Enzyme-carrying microorganisms and method for culturing the same:
The target novel esterase is contained in cells of
Pseudonocardia thermophila
FERM-BP-6275 (deposited under Acceptance Number for Deposit FERM-BP-6275 on Mar. 2, 1998 at the National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan). These cells can be cultured according to any known method. Any medium known as a nutrient medium for general microorganisms can be used. Organic nutrient sources such as meat extract, yeast extract, malt extract, peptone and NZ amine; carbon sources such as glucose, maltose, sucrose, starch andorganic acids; nitrogen sources such as ammonium sulfate, urea and ammonium chloride; inorganic nutrient sources such as phosphates, magnesium, potassium and iron; and vitamins can be used in appropriate combinations. The cells are cultured aerobically in a medium at a pH between 6 and 9 at a temperature between 30° C. and 60° C., preferably between 45° C. and 55° C. Culturing is carried out for 1-7 days until the target esterase content reaches its maximum.
(2) Purification of the enzyme:
The enzyme can be purified using a conventional enzyme purification method. Cells are harvested from the completed culture by centrifugation, and mechanically decomposed using an sonication, FRENCH® PRESSURE CELL PRESS, DYNO®-MILL or the like. Solids such as cell debris are removed by centrifugation to obtain a crude enzyme solution. The enzyme is then purified by salting out with ammonium sulfate or other salts, gel filtration, ion exchange chromatography, hydrophobic chromatography, crystallization or the like. An example will be described in Example 1.
(3) Measurement of enzyme activity:
A reaction mixture (1 ml) containing 10 &mgr;mol of racemic 6-aminochroman-3-acetic acid methyl ester, 0.1 mmol potassium phosphate buffer (pH 7.2) and an appropriate amount of esterase was reacted at 55° C. for 30 minutes. After the reaction, a portion of the reaction mixture was diluted with a 10 mM sodium phosphate buffer solution (pH 6.0) −30% acetonitrile solution, and 6-aminochroman-3-acetic acid methyl ester and 6-aminochroman-3-acetic acid were each quantitatively measured by high performance liquid chromatography. Elution was carried out on an inertsil ODS-2 column (a product of GL Science) using a 10 mM sodium phosphate buffer solution (pH 6.0) −30% acetonitrile as the carrier at a flow rate of 0.7 ml/minute. Absorbance at 254 nm was measured for detection. Next, an equivalent volume of chloroform was added to the remaining reaction solution to extract the remaining 6-aminochroman-3-acetic acid methyl ester. The chloroform was then removed by distillation under vacuum and the resulting residue was dissolved in ethanol and further diluted with a hexane/ethanol solution (3/2,v/v) (3S)-6-aminochroman-3-acetic acid methyl ester and (3R)-6-aminochroman-3-acetic acid methyl ester in the solution thus prepared were each quantitatively measured to determine optical purity. Elution was carried out on a CHIRALCEL OD-H optical resolution column (a product of DAICEL CHEMICAL INDUSTRIES, LTD) using a hexane/ethanol solution (3:2 in the volume ratio) as a carrier at a flow rate of 0.5 ml/minute.
Absorbance at 254 nm was measured for detection.
The amount of enzyme which hydrolyzes 1 &mgr;mol of 6-aminochroman-3-acetic acid ethyl ester per minute was defined as one unit.
(4) Homogeneity of the enzyme
SDS polyacrylamide gel electrophoresis was carried out by the Laemmli method on a 10-20% gel using a tris-glyci
Fukuhara Nobuhiro
Matsuba Yasuko
Oikawa Toshihiro
Burns Doane Swecker & Mathis L.L.P.
Lilling Herbert J.
Mitsui Chemicals Inc.
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