Process for producing trans-4-hydroxy-L-proline
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 microorganism belonging to the genus Dactylosporangium or Amycolatopsis having an enzymatic activity of catalyzing hydroxylation of L-proline at the 4-position of L-proline.
2 . A microorganism selected from a group consisting of Dactylosporangium sp. RH1 (FERM BP-440) and Amycolatoisis sp. RH2 (FERM BP-4581).
3 . A microorganism which is capable of producing enzyme having the following physicochemical properties:
(1) Action and Substrate Specificity:
It 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,
(2) Optimum pH Range:
The enzyme has an optimum pH range of 6.0 to 7.0 for its reaction of 30° C. for 20 minutes,
(3) Stable pH Range:
The enzyme is stable at pH values of 6.5 to 10.0, when it is allowed to stand 40° C. for 24 hours,
(4) Optimum Temperature Range:
The optimum temperature range is 30° C. to 40° C. when it is allowed to stand at pH 6.5 for 15 minutes,
(5) Stable Temperature Range:
The enzyme is inactivated, when it is allowed to stand at pH 9.0 and at 500 for 30 minutes,
(6) Inhibitors:
The enzyme is inhibited by metal ions of Zn ++ and Cu ++ and ethylenediaminetetraacetic acid, and not inhibited by 2-ketoglutaric acid,
(7) Activation:
The enzyme does not need any cofactor for its activation, L-ascorbic acid accelerates the reaction and catalase does not accelerate the reaction,
(8) Km Value:
Km value is 0.27 mM for L-proline when determined in a 80 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 6.5) containing 8 mM 2-ketoglutaric acid, 4 mM L-ascorbic acid, 2 mM ferrous sulfate and a predetermined amount of the enzyme at 30° C. for 20 minutes, Km value is 0.55 mM for 2-ketoglutaric acid, when determined in a 80 mM 2-(N-morpholino) ethanesulfonic acid (MES) buffer (pH 6.5) containing 4 mM L-proline, 4 mM L-ascorbic acid, 2 mM ferrous sulfate and a predetermined amount of the enzyme at 30° C. for 20 minutes,
(9) Molecular Weight:
The enzyme has a molecular weight of 32,000±5,000 daltons by sodium dodecysulfate-polyacrylamide gel electrophoresis and of 43,800±5,000 daltons by gel filtration,
(10) N-Terminal Amino Acid Sequence:
The enzyme has an N-terminal amino acid sequence as in Sequence No. 1.
4 . The microorganism according to claim 3 , which belongs to the genus Dactylosporangium or Amycolatopsis.