# US 8,541,209 B2 · L-proline cis-4-hydroxylase and use thereof to produce cis-4-hydroxy-L-proline

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- Number: US 8,541,209 B2
- Document ID: US8541209B2
- Status: Granted
- Kind: B2
- Application number: 12/992,417
- Filed: 2009-05-12
- Published: 2011-04-21
- Granted: 2013-09-24
- Earliest claimed: 2008-05-12
- Assignees: Kyowa Hakko Bio Co., Ltd.
- Applicants: Kuniki Kino; Ryotaro Hara
- Inventors: Kuniki Kino; Ryotaro Hara
- Web page: https://patentgrep.com/patent/US8541209B2

## CPC

- C12P 13/24: Proline; Hydroxyproline; Histidine
- C12N 9/0071: {acting on paired donors with incorporation of molecular oxygen (1.14)}
- C12Y 114/11002: Procollagen-proline dioxygenase (1.14.11.2), i.e. proline-hydroxylase
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

Development of a method of economically and efficiently producing cis-4-hydroxy-L-proline. The present invention provides L-proline cis-4-hydroxylase. This enzyme may be derived from Lotus corniculatus rhizobia, Mesorhizobium loti or Medicago sativa rhizobia, Sinorhizobium meliloti. The present invention provides a method of producing cis-4-hydroxy-L-proline from L-proline by using this enzyme. The present invention provides a recombinant vector containing a polynucleotide encoding the enzyme and a transformant containing the vector.

## Claims

### Claim 1

A method of producing cis-4-hydroxy-L-proline, comprising:
(A) a step of providing L-proline and at least one protein selected from the group consisting of
(1) the protein consisting of SEQ ID NO: 1 or 2,
(2) a protein consisting of an amino acid sequence wherein one or several amino acids is/are deleted from, substituted in, or added to the amino acid sequence of SEQ ID NO: 1 or 2, wherein said protein has L-proline cis-4-hydroxylase activity, and
(3) a protein consisting of an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with the nucleic acid complement of SEQ ID NO: 3 or 4, wherein said protein has L-proline cis-4-hydroxylase activity and, wherein said stringent conditions comprise hybridization overnight in a solution of 6× saline-sodium citrate (SSC) and 0.2% sodium dodecyl sulfate (SDS) at 65° C., washing two times with a solution of 1×SSC and 0.1% SDS at 65° C. for 30 minutes, and washing two times with a solution of 0.2×SSC and 0.1% SDS at 65° C. for 30 minutes, and
(B) a step of reacting the at least one protein with the L-proline to give cis-4-hydroxyproline.

### Claim 2 (depends on 1)

The method of claim 1, wherein the protein is the protein consisting of SEQ ID NO: 1 or 2.

### Claim 3 (depends on 1)

The method of claim 1, wherein the protein is a protein consisting of an amino acid sequence wherein one or several amino acids is/are deleted from, substituted in, or added to the amino acid sequence of SEQ ID NO: 1 or 2, wherein said protein has L-proline cis-4-hydroxylase activity.

### Claim 4 (depends on 1)

The method of claim 1, wherein the protein is a protein consisting of an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with the nucleic acid complement of SEQ ID NO: 3 or 4, wherein said protein has L-proline cis-4-hydroxylase activity and, wherein said stringent conditions comprise hybridization overnight in a solution of 6× saline-sodium citrate (SSC) and 0.2% sodium dodecyl sulfate (SDS) at 65° C., washing two times with a solution of 1×SSC and 0.1% SDS at 65° C. for 30 minutes, and washing two times with a solution of 0.2×SSC and 0.1% SDS at 65° C. for 30 minutes.