Claims
- 1. An isolated and purified L-proline-3-hydroxylase having the following physicochemical properties:
- (1) Action and Substrate Specificity:
- The enzyme catalyzes hydroxylation of L-proline at the 3-position of L-proline in the presence of 2-ketoglutaric acid and a divalent iron ion to produce cis-3-hydroxy-L-proline,
- (2) Optimum pH Range:
- The enzyme has a n optimum pH range of 6.5 to 7.5 for its reaction at 30.degree. C. for 20 minutes,
- (3) Stable pH Range:
- The enzyme is stable at pH values of 6.5 to 8.0, when it is allowed to stand at 4.degree. C. for 23 hours,
- (4) Optimum Temperature Range:
- The optimum temperature range is 35 to 40.degree. C. when it is allowed to stand at pH 7.0 for 15 minutes,
- (5) Stable Temperature Range:
- The enzyme is inactivated, when it is allowed to stand at pH 7.5 and at 50.degree. C. for 30 minutes,
- (6) Inhibitors:
- The enzyme is inhibited by metal ions of Zn.sup.++, Cu.sup.++ Co.sup.++ and Ba.sup.++ and ethylenediaminetetraacetic acid,
- (7) Activation:
- The enzyme does not need any cofactor for its activation; L-Ascorbic acid accelerates the activity of the enzyme,
- (8) Km Value:
- Km Value is 0.49 mM for L-proline and is 0.11 mM for 2-ketoglutaric acid, when determined in a 100 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) buffer (pH 7.0) containing 5 mM L-ascorbic acid, 1 mM ferrous sulfate and a predetermined amount of the enzyme,
- (9) Isoelectric point:
- The enzyme has an isoelectric point of 4.3 by Phast system,
- (10) Molecular Weight:
- The enzyme has a molecular weight of 35,000.+-.5,000 daltons by sodium dodecylsulfate-polyacrylamide gel electrophoresis
- (11) N-terminal Amino Acid Sequence:
- The enzyme has an N-terminal amino acid sequence illustrated by Sequence No. 5:
- Sequence No. 5:
- (N-terminal) 1 MetArgSerHisIleLeuGlyArgIleGlu
- 11 LeuAspGlnGluArgLeuGlyArgAspLeu
- 21 GluTyrLeuAlaThrValProThrVal.
- 2. An isolated and purified protein comprising the amino acid sequence of residues 2 to 290 as defined in SEO ID NOs: 1 or 2.
- 3. The isolated and purified L-proline-3-hydroxylase according to claim 1, which is derived from a microorganism belonging to the genus Streptomyces or Bacillus.
- 4. An isolated and purified protein according to claim 2, wherein said protein is from a microorganism of the genus Streptomyces or Bacillus.
- 5. An isolated and purified protein according to claim 2, wherein said protein is L-proline-3-hydroxylase.
- 6. An isolated and purified protein according to claim 2 having the amino acid sequence of residues 2 to 290 as defined in SEQ ID NOs: 1 or 2.
Priority Claims (1)
Number |
Date |
Country |
Kind |
5-221941 |
Sep 1993 |
JPX |
|
Parent Case Info
This application is a continuation-in-part of Ser. No. 08/474,135, filed Jul. 6, 1995, now abandoned, which is continuation-in-part of Ser. No. 08/301,654 filed Sep. 7, 1994, now abandoned.
Continuation in Parts (2)
|
Number |
Date |
Country |
Parent |
474135 |
Jul 1995 |
|
Parent |
301654 |
Sep 1994 |
|