公开(公告)号：US20230265475A1

公开(公告)日：2023-08-24

申请号：US18157226

申请日：2023-01-20

Applicant: API CORPORATION

Inventor： Ryoma MIYAKE ,  Hiroshi KAWABATA

IPC: C12P17/12 ,  C12N9/02

CPC classification number: C12P17/12 ,  C12N9/0071

Abstract: 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.

公开(公告)号：US20180282709A1

公开(公告)日：2018-10-04

申请号：US15947220

申请日：2018-04-06

Applicant: API CORPORATION

Inventor： Makoto HIBI ,  Jun OGAWA ,  Ryoma MIYAKE ,  Hiroshi KAWABATA

IPC: C12N9/02 ,  C12P17/10 ,  C12P13/04 ,  C12P17/12

Abstract: 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 α-amino acid to produce 4-hydroxy amino acid: (A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO:2, 4, 6, 8, 10, 12, 16, or 18; (B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO:2, 4, 6, 8, 10, 12, 16, or 18 except that one or several amino acids are deleted, substituted, and/or added, and having pipecolic acid 4-hydroxylase activity; and (C) a polypeptide having an amino acid sequence that is not less than 80% identical to the amino acid sequence represented by SEQ ID NO:2, 4, 6, 8, 10, 12, 16, or 18, and having pipecolic acid 4-hydroxylase activity.

公开(公告)号：US20150252393A1

公开(公告)日：2015-09-10

申请号：US14637429

申请日：2015-03-04

Applicant: API CORPORATION

Inventor： Hiroshi KAWABATA ,  Ryoma MIYAKE ,  Kuniko ASADA ,  Ryouhei KATOU

IPC: C12P7/62 ,  C12P41/00

CPC classification number: C12P7/62 ,  C12N9/14 ,  C12N9/18 ,  C12P41/005 ,  Y02P20/52

Abstract: 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.

Abstract translation: 本发明的一个目的是提供一种新的水解酶，其在2-乙烯基环丙烷-1,1-二羧酸二烷基酯用酶水解时使用，以有效地获得（1S，2S）-1-烷氧基羰基-2 - 乙烯基环丙烷羧酸，其可用作合成丙型肝炎治疗剂的中间体。根据本发明，提供了水解酶蛋白质，其由SEQ ID NOS中任一项所示的氨基酸序列组成。 2至5，并且其具有以比由SEQ ID NO。所示的氨基酸序列组成的蛋白质更高的选择性催化的活性。 1，由2-乙烯基环丙烷-1,1-二羧酸二乙酯生成（1S，2S）-1-乙氧基羰基-2-乙烯基环丙烷羧酸的反应。