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

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

2001-04-13

2003-09-09

Nashed, Nashaat T. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing alpha or beta amino acid or substituted amino acid...

C435S252300, C435S252330, C435S189000, C536S023200

Reexamination Certificate

active

06617140

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a process for producing trans-4-hydroxy-L-proline. Trans-4-hydroxy-L-proline is useful as a starting compound for medicines and an additive to foods. The present invention also relates to a novel enzyme capable of catalyzing the hydroxylation of L-proline at the 4-position of L-proline (hereinafter referred to as L-proline-4-hydroxylase). The novel enzyme is used in the above-mentioned process.
The present invention also relates to a process for industrially producing trans-4-hydroxy-L-proline, a gene encoding a protein having an activity of L-proline-4-hydroxylase (hereinafter referred to as “L-proline-4-hydroxylase gene”) which is useful for the above-mentioned process, a transformant containing the gene, and a process for producing L-proline-4-hydroxylase using the transformant.
In addition, this invention relates to a process for industrially producing trans-4-hydroxy-L-proline using a transformant which contains L-proline-4-hydroxylase gene and has a reinforced proline biosynthesis activity.
BACKGROUND OF THE INVENTION
The following processes are known as a method for producing trans-4-hydroxy-L-proline using microorganisms.
1) A process in which trans-4-hydroxy-L-proline is produced from 4-hydroxy-2-oxoglutaric acid using microorganisms of the genus Escherichia (Japanese Published Unexamined Patent Application No. 266,995/91)
2) A process in which trans-4-hydroxy-L-proline is produced directly through fermentation using bacteria or fungi (European (EP 0 547 898 A2, and Japanese Published Unexamined Patent Application Nos. 236,980/93 and 245,782/94)
3) A process in which trans-4-hydroxy-L-proline is produced from L-proline using microorganisms of the genus Streptomyces [J. Biol. Chem., 254, 6684 (1979), Biochem. Biophys. Res. Comm., 120, 45, (1984), Tetrahedron Letters, 34, 7489 (1993), and Tetrahedron Letters, 35, 4649 (1994)].
The conventional processes can, however, hardly be performed on an industrial scale for the following reasons:
1) A substrate for producing trans-4-hydroxy-L-proline, such as 4-hydroxy-2-oxoglutaric acid is too expensive and is difficult to obtain.
2) The productivity of trans-4-hydroxy-L-proline is low.
3) The activity of the enzymes that relate to the production of trans-4-hydroxy-L-proline is quite weak.
Heretofore, L-proline-4-hydroxylase has not been isolated. A process for producing trans-4-hydroxy-L-proline, using an enzyme source which is isolated from a microorganism belonging to the genus Dactylosporangium or Amycolatopsis and which catalyzes the hydroxylation of L-proline into trans-4-hydroxy-L-proline, has not been known. Although there was a paper reporting that L-proline-4-hydroxylase was isolated from a microorganism belonging to the genus Streptomyces (Tetrahedron Letters, 34, 7489-7492, 1993), the report is silent about steps for isolating the enzyme, the enzyme purity, the physicochemical properties of the enzyme, etc.
With respect to the enzyme that catalyzes the production of trans-4-hydroxy-L-proline, it was reported in a paper that L-proline-4-hydroxylase is purified from a microorganism of the genus Streptomyces. However, a method for obtaining the enzyme and physicochemical properties of the enzyme are not described therein. Further, no paper reported that a gene encoding L-proline-4-hydroxylase having the activity of converting free L-proline into trans-4-hydroxy-L-proline in the presence of 2-ketoglutaric acid and a divalent iron ion had been cloned.
A process in which trans-4-hydroxy-L-proline is produced industrially advantageously using L-proline-4-hydroxylase having a high level of activity has been in demand.
The object of the present invention is to provide an efficient process for the production of trans-4-hydroxy-L-proline on the industrially applicable basis, and the additional object of the present invention is to provide a novel enzyme which catalyzes the hydroxylation of L-proline at the 4-position of L-proline and which is useful in the above process.
SUMMARY OF THE INVENTION
The present invention provides a process for the production of trans-4-hydroxy-L-proline which comprises allowing L-proline to coexist with 2-ketoglutaric acid, a divalent iron ion and an enzyme source which catalyzes hydroxylation of L-proline at the 4-position of L-proline in an aqueous medium to convert L-proline into trans-4-hydroxy-L-proline, and recovering the trans-4-hydroxy-L-proline from the aqueous medium.
The present invention further provides a novel hydroxylase (L-proline-4-hydroxylase)having the following physicochemical properties:
(1) Action and Substrate Specificity
The enzyme 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 at 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 at 4° C. for 24 hours.
(4) Optimum Temperature Range
The optimum temperature range is 30 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 50° C. for 30 minutes.
(6) Inhibitors
The activity of the enzyme is inhibited by metal ions of Zn
++
and Cu
++
and ethylenediaminetetraacetic acid.
(7) Activation
The enzyme does not need any cofactors for its activation.
L-Ascorbic acid accelerates the activity of the enzyme.
(8) Km Value
Km value is 0.27 mM for L-proline and 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-ascorbic acid, 2 mM ferrous sulfate and the enzyme preparation.
(9) Molecular Weight
The enzyme has a molecular weight of 32,000±5,000 daltons by sodium dodecylsulfate-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 illustrated by Sequence No. 1 mentioned below.
Sequence No. 1:
(N-terminal)
 1 MetLeuThrProThrGluLeuLysGlnTyr

11 ArgGluAlaGlyTyrLeuLeuIleGluAsp

21 GlyLeuGlyProArgGluVal
The present invention also provides an L-proline-4-hydroxylase gene and a transformant containing the above-mentioned gene for producing trans-4-hydroxy-L-proline efficiently and industrially advantageously using L-proline-4-hydroxylase from L-proline that is available at low cost, a process for mass-producing the L-proline-4-hydroxylase using the gene and the transformant, and a process for producing trans-4-hydroxy-L-proline industrially at low cost using the transformant or the L-proline-4-hydroxylase.
In addition, the present invention provides a transformant, which contains the above-mentioned gene for producing trans-4-hydroxy-L-proline and has reinforced proline biosynthesis activity, and a process for producing trans-4-hydroxy-L-proline using the transformant industrially at low cost.


REFERENCES:
patent: 5344923 (1994-09-01), Verma et al.
patent: 0547898 (1993-06-01), None
patent: 0555475 (1993-08-01), None
T. Shibasaki, et al., “3Ta4 Cloning and Expression of L-proline 4-hydroxylase gene”, Japan Society for Bioscience, Biotechnology, and Agrochemistry, Mar. 1996, p. 257.
Onishi et al, Biochemical and Biophysical Research Communications, 120, 45-51, (1984).
Baldwin et al, Tetrahedron Letters, 34, 7,489-7,492 (1993).
Lawrence et al. “Purification and initial characterization of proline 4-hydroxylase from Streptomyces griseoviridus P8648: a 2-oxyacid, ferrous-dependent dioxygenase involved in etamycin biosynthesis” Biochem. J. 313, 185-191.
Webster's Third New International Dictionary, Gove, ed. G. &C. Merriam Company, Springfield, Mass.,.
Bergey's Manual of Systematic Bacteriology, vol. 4 Williams & Wilkins(1989), pp 2440-2442.

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