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

An isolated and purified L-proline-4-hydroxylase 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 at 30.degree. 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.degree. for 24 hours,
• (4) Optimum Temperature Range:
• The optimum temperature range is 30.degree. to 40.degree. 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.sup.++ and Cu.sup.++ 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.degree. 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.degree. C. for 20 minutes,
• (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 as in Sequence No. 1,
• ______________________________________ SequenceNo. 1:
• ______________________________________ (N-terminal) 1 MetLeuthrProThrGluLeuLysGlnTyr 11 ArgGluAlaGlyTyrLeuLeuIleGluAsp 21 GlyLeuGlyProArgGluVal. ______________________________________