Kyowa Hakko Kogyo Co., Ltd.

Takeshi Shibasaki

Akio Ozaki

Katsuhiko Ando

Shigeru Chiba

Hideo Mori

Keiko Ochiai

Yoichi Uosaki

The present invention is directed to a process for producing trans-4-hydroxy-L-proline, which is useful as a raw material for medicines or as an additive to foods. In the process, L-proline is converted into trans-4-hydroxy-L-proline in the presence of an enzyme source which is derived from a microorganism belonging to the genusorand which catalyzes the hydroxylation of L-proline into trans-4-hydroxy-L-proline, a divalent iron ion and 2-ketoglutaric acid, in an aqueous medium, and the produced trans-4-hydroxy-L-proline is collected from the aqueous medium. In addition, the present invention is directed to a process for producing trans-4-hydroxy-L-proline, wherein the L-proline biosynthesis activity of the host cell of the transformant is reinforced.

Process for producing trans-4-hydroxy-L-proline

An isolated gene encodes for a L-proline-3-hydroxylase having the amino acid sequence of SEQ ID NOS: 1, 2, 15, 16 and 17, or for a protein having enzymatic activity to hydroxylate the 3-position of L-proline and to act on free L-proline in the presence of 2-ketoglutaric acid and divalent iron ions to produce cis-3-hydroxy-L-proline. The gene is inserted into a vector and the resulting recombinant DNA is used to create a transformant. L-proline-3-hydroxylase is produced by culturing the transformant in a medium.

Process for producing cis-3-hydroxy-L-proline

Provided is an industrially applicable process for producing trans-4-hydroxy-L-proline, which is useful as a raw material for medicines or as an additive to foods. In the process, L-proline is converted into trans-4-hydroxy-L-proline in the presence of an enzyme source which is derived from a microorganism belonging to the genus Dactylosporangium, Amycolatopsis or Streptomyces and which catalyzes the hydroxylation of L-proline into trans-4-hydroxy-L-proline, a divalent iron ion and 2-ketoglutaric acid, in an aqueous medium, and the produced trans-4-hydroxy-L-proline is collected from the aqueous medium. Also provided is a novel enzyme L-proline-4-hydroxylase which is useful for the process, a gene of L-proline-4-hydroxylase which is useful for the process, a transformant containing the gene, and a process for producing L-proline-4-hydroxylase using the transformant. In addition, provided is a process for producing L-proline-4-hydroxylase using the transformant which contains the gene and has a reinforced proline biosynthesis activity.

An industrially applicable process for producing cis-3-hydroxy-L-proline, which is useful as a raw material for medicines or as an additive to foods. In the process, L-proline is converted into cis-3-hydroxy-L-proline in the presence of an enzyme source which is derived from a microorganism belonging to the genus Streptomyces or Bacillus and which catalyzes hydroxylation of L-proline into cis-3-hydroxy-L-proline, a divalent iron ion and 2-ketoglutaric acid, in an aqueous medium, and the produced cis-3-hydroxy-L-proline is collected from the aqueous medium. A novel enzyme L-proline-3-hydroxylase, a gene of L-proline-3-hydroxylase which is useful for the process, a transformant containing the gene, and a process for producing L-proline-3-hydroxylase using the transformant.

Process for producing CIS-3-hydroxy-L-proline

An industrially applicable process for producing trans-4-hydroxy-L-proline, which is useful as a raw material for medicines or as an additive to foods. In the process, L-proline is converted into trans-4-hydroxy-L-proline in the presence of an enzyme source which is derived from a microorganism belonging to the genus Dactylosporangium, Amycolatopsis or Streptomyces and which catalyzes the hydroxylation of L-proline into trans-4-hydroxy-L-proline, a divalent iron ion and 2-ketoglutaric acid, in an aqueous medium, and the produced trans-4-hydroxy-L-proline is collected from the aqueous medium. Also provided is a novel enzyme L-proline-4-hydroxylase which is useful for the process, a gene of L-proline-4-hydroxylase which is useful for the process, a transformant containing the gene, and a process for producing L-proline-4-hydroxylase using the transformant. In addition, provided is a process for producing L-proline-4-hydroxylase using the transformant which contains the gene and has a reinforced proline biosynthesis activity.

An industrially applicable process for producing cis-3-hydroxy-L-proline, which is useful as a raw material for medicines or as an additive to foods. In the process, L-proline is converted into cis-3-hydroxy-L-proline in the presence of an enzyme source which is derived from a microorganism belonging to the genus Streptomyces or Bacillus and which catalyzes hydroxylation of L-proline into cis-3-hydroxy-L-proline, a divalent iron ion and 2-ketoglutaric acid, in an aqueous medium, and the produced cis-3-hydroxy-L-proline is collected from the aqueous medium. A novel enzyme L-proline-3-hydroxylase, a gene of L-proline-3-hydroxylase which is useful for the process, a transformant containing the gene, and a process for producing L-proline-3-hydroxylase using the transformant, is also provided.

Process for producing Cis-3-hydroxy-L-proline

Isolated and purified L-proline-4-hydroxylase which catalyzes hydroxylation of L-proline at the 4-position of L-proline in the presence of 2-ketoglutaric acid and a divalent iron ion to produce trans-4-hydroxy-L-proline. The L-proline-4-hydroxylase may be isolated from a microorganism selected from the group consisting of Dactylosporanlium sp. RHI deposited with the National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology, Japan, under the designation PERM BP-4400 and Amycolatopsis sp. RH2 deposited as FERM BP-4581.

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Machida, Japan

Takeshi Shibasaki