Process for producing trans-4-hydroxy-L-proline
View Patent ↗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 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. 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.
1. A method for producing trans-4-hydroxy-L-proline, which comprises:
(a) cultivating in a medium a transformant obtained by introducing into host cell selected from the group consisting of microorganism and isolated animal cell a recombinant DNA comprising a DNA coding for L-proline-4-hydroxylase,
wherein the DNA is a DNA having any one of nucleotide sequences of SEQ ID NO: 3, 9, and 16, or is a DNA hybridizing to the DNA having any one of nucleotide sequences of SEQ ID NO: 3, 9 and 16 in a solution comprising 50% (v/v) formadide, 2% blocking reagent, 0.1% (w/v) N-laurylsarcosine, 0.02% (w/v) SDS, 750 mM sodium chloride and 75 mM sodium citrate at 42° C. for 1 hour,
wherein the hybridizing comprises additional steps of: washing twice in a solution containing 0.1% SDS, 300 mM sodium chloride and 30 mM sodium citrate at room temperature for 5 minutes, and washing twice with a solution comprising 0.1% SDS, 15 mM sodium chloride and 1.5 mM sodium citrate at 68° C. for 15 minutes each;
(b) producing and accumulating trans-4-hydroxy-L-proline; and
(c) collecting the trans-4-hydroxy-L-proline from the resulting culture.
2. The method for producing trans-4-hydroxy-L-proline according to claim 1 , wherein L-proline is added to the medium.
3. The method for producing trans-4-hydroxy-L-proline according to claim 1 , wherein L-proline, 2-ketoglutaric acid and divalent iron ions are added to the medium.
4. The method for producing trans-4-hydroxy-L-proline according to claim 1 , wherein the DNA codes for a protein having any one of amino acid sequences of SEQ ID NO: 2,19 and 20.
5. The method for producing trans-4-hydroxy-L-proline according to claim 1 , wherein the DNA is derived from a microorganism belonging to a genus selected from the group consisting of Dactylosporangium, Amycolatopsis and Streptomyces.
6. The method for producing trans-4-hydroxy-L-proline according to claim 1 , wherein the DNA is derived from a microorganism selected from the group consisting of Dactylosporangium sp. RH1 (FERM BP-4400), Amycolatopsis sp. RH2 (FERM BP-4581), Streptomyces griseoviridis JCM4250 and Streptomyces daghestanicus JCM4365.
7. The method of claim 1 , wherein the host cell is further transformed with the proA or proB genes from E. coli.
8. The method of claim 1 , wherein the host cell is further transformed with the proA or proB74 genes from E. coli.
9. A method for producing trans-4-hydroxy-L-proline, which comprises:
(a) adding to an aqueous medium
(i) cells of a transformant isolated from a culture of the transformant obtained by introducing into a host cell selected from the group consisting of microorganism and isolated animal cell a recombinant DNA comprising a DNA coding for L-proline-4-hydroxylase,
(ii) L-proline,
(iii) a divalent iron ion, and
(iv) 2-ketoglutaric acid,
wherein the DNA is a DNA having any one of nucleotide sequences of SEQ ID NO: 3, 9 and 16, or is a DNA hybridizing to the DNA having any one of nucleotide sequences of SEQ ID NO: 3, 9 and 16 in a solution comprising 50% (v/v) formamide, 2% blocking reagent, 0.1% (w/v) N-laurylsarcosine, 0.02% (w/v) SDS, 750 mM sodium chloride and 75 mM sodium citrate at 42° C. for 1 hour;
wherein the hybridizing comprises additional steps of: washing twice in a solution containing 0.1% SDS, 300 mM sodium chloride and 30 mM sodium citrate at room temperature for 5 minutes, and washing twice with a solution containing 0.1% SDS, 15 mM sodium chloride and 1.5 mM sodium citrate at 68° C. for 15 minute each,
(b) converting L-proline into trans-4-hydroxy-Lproline; and
(c) collecting the resulting trans-4-hydroxy-L-proline from the aqueous medium.
10. The method for producing trans-4-hydroxy-L-proline according to claim 9 , wherein the cells are immobilized cells.
11. The method for producing trans-4-hydroxy-L-proline according to claim 9 , wherein the DNA codes for a protein having any one of amino acid sequences of SEQ ID NO: 2, 19 and 20.
12. The method for producing trans-4-hydroxy-L-proline according to claim 9 , wherein the DNA is derived from a microorganism belonging to a genus selected from the group consisting of Dactylosporangium, Amycolatopsis and Streptomyces.
13. The method for producing trans-4-hydroxy-L-proline according to claim 9 , wherein the DNA is derived from the group consisting of Dactylosporangium sp. RH1 (FERM BP-4400), Amycolatopsis sp. RH2 (FERM BP-4581), Streptomyces griseoviridis JCM4250 and Streptomyces daghestanicus JCM4365.
14. The method of claim 9 , wherein the host cell is further transformed with the proA or proB genes from E. coli.
15. The method of claim 9 , wherein the host cell is further transformed with the proA and proB74 genes from E. coli .
16. The method for producing trans-4-hydroxyl-L-proline according to claim 5 , wherein the microorganisms are selected from those of Dactylosporangium sp. RH1 (FERM BP-4400), Amycolatopsis sp. RH2 (FERM BP-4581), Streptomyces griseoviridis JCM4250 and Streptomyces daghestanicus JCM4365.