Process for production of cis-4-hydroxy-L-proline
View Patent ↗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.
1. A method of producing cis-4-hydroxy-L-proline, comprising the steps of:
(A) providing a prokaryote that produces an enzyme 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 the 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 the protein has L-proline cis-4-hydroxylase activity and, wherein the 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,
(B) contacting the prokaryote with L-proline in a reaction system to produce cis-4-hydroxy-L-proline, and
(C) recovering cis-4-hydroxy-L-proline from the reaction system.
2. The method of claim 1 , wherein the protein consists of SEQ ID NO: 1 or 2.
3. The method of claim 1 , wherein the protein consists 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 the protein has L-proline cis-4-hydroxylase activity.
4. The method of claim 1 , wherein the protein consists 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 the protein has L-proline cis-4-hydroxylase activity and, wherein the stringent conditions comprise hybridization overnight in a solution of 6×SSC and 0.2% 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.
5. A method of producing cis-4-hydroxy-L-proline, comprising the steps of:
(A) providing a host organism that produces an enzyme 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 the 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 the protein has L-proline cis-4-hydroxylase activity and, wherein the stringent conditions comprise hybridization overnight in a solution of 6×SSC and 0.2% 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,
(B) culturing the host organism in a culture medium to produce cis-4-hydroxy-L-proline, and
(C) recovering cis-4-hydroxy-L-proline from the culture medium.
6. The method of claim 5 , wherein the protein consists of SEQ ID NO: 1 or 2.
7. The method of claim 5 , wherein the protein consists 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 the protein has L-proline cis-4-hydroxylase activity.
8. The method of claim 5 , wherein the protein consists 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 the protein has L-proline cis-4-hydroxylase activity and, wherein the stringent conditions comprise hybridization overnight in a solution of 6×SSC and 0.2% 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.
9. The method of claim 5 , wherein the host organism is a prokaryote.
10. The method of claim 9 , wherein the protein consists of SEQ ID NO: 1 or 2.
11. The method of claim 9 , wherein the protein consists 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 the protein has L-proline cis-4-hydroxylase activity.
12. The method of claim 9 , wherein the protein consists 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 the protein has L-proline cis-4-hydroxylase activity and, wherein the stringent conditions comprise hybridization overnight in a solution of 6×SSC and 0.2% 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.