Api Corporation

Ube Corporation

Ryoma Miyake

Hiroshi Kawabata

Kuniko Asada

Jun Ogawa

Makoto Hibi

Yasumasa Dekishima

Takanobu Iura

Toyokazu Yoshida

Koichi Ishida

Yuuki Asuma

Ryouhei Katou

Tatsuya Suzuki

Kuniki Kino

Tomoko Maeda

Jun Takehara

Hisatoshi Uehara

Tsugihiko Hidaka

Yasuyo Saito

Ryotaro Hara

Masaharu Mizutani

An object of the present invention is to provide a method of industrially producing a high-purity L-cyclic amino acid more inexpensively and with a high efficiency, from a cyclic amino acid having a double bond at the 1-position. The present invention provides a method in which an L-cyclic amino acid is produced by allowing a cyclic amino acid having a double bond at the 1-position to react with a specific enzyme having a catalytic ability to reduce a cyclic amino acid having a double bond at the 1-position to produce an L-cyclic amino acid.

Production method for L-cyclic amino acids

The present invention provides a novel hydrolase that can industrially produce optically highly pure (1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropane carboxylic acid with high efficiency at low costs, and a production method using the hydrolase.

Hydrolase and method for producing (1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropane carboxylic acid using same

A method of producing high-purity hydroxy-L-pipecolic acids that includes allowing an L-pipecolic acid hydroxylase, a microorganism or cell having the ability to produce the enzyme, a processed product of the microorganism or cell, and/or a culture liquid comprising the enzyme and obtained by culturing the microorganism or cell, to act on L-pipecolic acid as a substrate in the presence of 2-oxoglutaric acid and ferrous ion, wherein the L-pipecolic acid hydroxylase (1) acts on L-pipecolic acid in the presence of 2-oxoglutaric acid and ferrous ion to add a hydroxy group to the carbon atom at positions 3, 4, and/or 5 of L-pipecolic acid; and (2) has a catalytic efficiency (kcat/Km) with L-proline that is equal to or less than 7 times the catalytic efficiency (kcat/Km) with L-pipecolic acid.

Method for producing hydroxy-L-pipecolic acid

A novel method of producing high-purity hydroxy-L-pipecolic acids in an efficient and inexpensive manner while suppressing the production of hydroxy-L-proline is provided. The method includes allowing an L-pipecolic acid hydroxylase, a microorganism or cell having the ability to produce the enzyme, a processed product of the microorganism or cell, and/or a culture liquid comprising the enzyme and obtained by culturing the microorganism or cell, to act on L-pipecolic acid as a substrate in the presence of 2-oxoglutaric acid and ferrous ion, wherein the L-pipecolic acid hydroxylase has the properties:

The present invention provides a pipecolic acid 4-hydroxylase protein exemplified by the following (A), (B), and (C), having activity to react with L-pipecolic acid in the presence of 2-oxoglutaric acid and iron(II) ions to produce trans-4-hydroxy-L-pipecolic acid, and a method for producing 4-hydroxy amino acid, which method comprises reacting the pipecolic acid 4-hydroxylase protein, cells containing the protein, a treated product of the cells, and/or a culture liquid obtained by culturing the cells, with &#x3b1;-amino acid to produce 4-hydroxy amino acid:

Pipecolinic acid 4-hydroxylase and method for producing 4-hydroxy amino acid using same

A method for producing cis-5-hydroxy-L-pipecolic acid, the method comprising allowing a 2-oxoglutarate-dependent L-pipecolic acid hydroxylase to act on L-pipecolic acid to generate cis-5-hydroxy-L-pipecolic acid, wherein the 2-oxoglutarate-dependent L-pipecolic acid hydroxylase comprises the polypeptide (A), (B) or (C) below: (A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4 or 11; (B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4 or 11 except that one or several amino acids are deleted, substituted, and/or added, which polypeptide has 2-oxoglutarate-dependent L-pipecolic acid hydroxylase activity; or (C) a polypeptide comprising an amino acid sequence with an identity of not less than 60% to the amino acid sequence represented by SEQ ID NO: 4 or 11, which polypeptide has 2-oxoglutarate-dependent L-pipecolic acid hydroxylase activity.

Method for manufacturing cis-5-hydroxy-L-pipecolic acid

According to the present invention, it becomes possible to perform a process for converting into an &#x3b1;-substituted cysteine represented by general formula (1) or a salt thereof at low cost and on an industrial scale by employing a process that is routed through a compound represented by general formula (3) to a compound represented by general formula (6). Particularly, by employing a process that is routed through a compound represented by general formula (7-2), it becomes possible to detach a tert-butyl protection group in a simple manner and to produce the compound represented by general formula (1) with high purity. Furthermore, by employing a process that is routed through tert-butylthiomethanol or a process that is routed through a compound represented by general formula (9), it becomes possible to produce a compound represented by general formula (2) without generating bischloromethylether that is an oncogenic substance. In the production of an &#x3b1;-substituted-D-cysteine or a salt thereof, it becomes possible to perform a process for converting the compound represented by general formula (2) into a compound represented by general formula (3S) in one step by allowing an enzyme or the like to act on the compound represented by general formula (2).

Method for producing alpha-substituted cysteine or salt thereof or synthetic intermediate of alpha-substituted cysteine

The present invention aims to provide a method for efficiently producing hydroxy-L-lysine. The present invention provides a method for producing hydroxy-L-lysine, the method comprising allowing 2-oxoglutarate-dependent L-lysine hydroxylase, a cell containing 2-oxoglutarate-dependent L-lysine hydroxylase, a processed product of the cell, and/or a culture broth obtained by culturing the cell, to act on L-lysine to produce hydroxy-L-lysine represented by the following General Formula (I) (wherein each of R, Rand Rrepresents a hydrogen atom or hydroxyl group, with the proviso that at least one of R, Rand Rrepresents a hydroxyl group). 

L-lysine hydroxylase and production method for hydroxy-L-lysine and hydroxy-L-pipecolic acid using same

It is an object of the present invention to provide a novel hydrolase, which is used when dialkyl 2-vinylcyclopropane-1,1-dicarboxylate is hydrolyzed with an enzyme, so as to efficiently obtain (1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid that is useful as an intermediate for synthesizing therapeutic agents for hepatitis C. According to the present invention, there is provided a hydrolase protein, which consists of the amino acid sequence shown in any one of SEQ ID NOS. 2 to 5 and which has activity of catalyzing, at higher selectivity than the protein consisting of the amino acid sequence shown in SEQ ID NO. 1, a reaction of producing (1S,2S)-1-ethoxycarbonyl-2-vinylcyclopropanecarboxylic acid from diethyl 2-vinylcyclopropane-1,1 -dicarboxylate.

Hydrolase protein

It is an object of the present invention to provide a novel method for producing (1R,2S)/(1S,2R)-1-amino-1-alkoxycarbonyl-2-vinylcyclopropane which is useful as a synthetic intermediate of therapeutic agents for hepatitis C and a synthetic intermediate thereof. According to the present invention, when a trans-2-butene derivative having a leaving group at each of the 1- and 4-positions is reacted with a malonic ester in the presence of a base, a specific amount of an alkali metal alkoxide or an alkali metal hydride is used as the base, and further a specific amount of a malonic ester is used to produce a cyclopropane diester, and further, chiral or achiral 1-amino-1-alkoxy-carbonyl-2-vinylcyclopropane and a salt thereof are synthesized using the cyclopropane diester.

Method for producing 1-amino-1-alkoxycarbonyl-2-vinylcyclopropane

It is an object of the present invention to provide a novel hydrolase, which is used when dialkyl 2-vinylcyclopropane-1,1-dicarboxylate is hydrolyzed with an enzyme, so as to efficiently obtain (1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid that is useful as an intermediate for synthesizing therapeutic agents for hepatitis C. According to the present invention, there is provided a hydrolase protein, which consists of the amino acid sequence shown in any one of SEQ ID NOS. 2 to 5 and which has activity of catalyzing, at higher selectivity than the protein consisting of the amino acid sequence shown in SEQ ID NO. 1, a reaction of producing (1S,2S)-1-ethoxycarbonyl-2-vinylcyclopropanecarboxylic acid from diethyl 2-vinylcyclopropane-1,1-dicarboxylate.

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Kanagawa, Japan

Ryoma Miyake