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US 4,304,864 AGranted
Long-chain acyl-coenzyme-A synthetase
AssigneeMitsubishi Chemical Industries Limited
InventorsShosaku Numa, Kohei Hosaka, Masayoshi Mishina, Takao Tanaka, Tatsuyuki Kamiryo
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Abstract
There are disclosed an enzyme, long-chain acyl-coenzyme-A synthetase which is specific for fatty acids having 14 to 18 carbon atoms and a process for the purification of the enzyme which involves solubilizing the enzyme with a surfactant and subjecting the solubilized enzyme to affinity chromatography.
Claims10
A process for the purification of a long-chain acyl CoA synthetase derived from microorganisms which comprises:
- disrupting microorganism cells containing long-chain acyl CoA synthetase to obtain the enzyme which is bound to the membranes and then solubilizing the enzyme with a surfactant;
- and
- subjecting the solubilized enzyme to affinity chromatography using a ligand which is a structural analog selected from the group consisting of ADP, ATP, CoA, and mixtures thereof, to separate it from contaminant proteins.
A process for the purification of long-chain acyl CoA synthetase, having the following physical and chemical properties:
- (i) substrate specificity:
- acting primarily on straight-chain fatty acids having 14 to 18 carbon atoms and essentially not acting on straight-chain fatty acids more than 18 or less than 14 carbon atoms;
- (ii) optimum pH:
- 7.1-9.6
- (iii) pH stability:
- 6.1-9.0 (at 4.degree. C., 36 hours)
- (iv) optimum temperature:
- 35.degree.-45.degree. C.
- (v) thermal stability:
- 45.degree. C. or below
- (vi)
- activator:
- Mg++
- inhibitor:
- Cu++ and EDTA
- (vii)
- molecular weight:
- approximately 84,000 (by SDS disk electrophoresis);
- approximately 105,000 (by gel filtration)
- (viii) amino acid composition:
- 13.2% lysine, 2.0% histidine, 4.6% arginine, 9.1% aspartic acid, 5.5% threonine, 7.8% serine, 10.5% glutamic acid, 7.5% proline, 8.1% glycine, 6.5% alanine, 6.0% valine, 1.6% methionine, 5.0% isoleucine, 7.0% leucine, 2.9% tyrosine and 2.6% phenylalanine (in molar percentages based on the total amount of the sixteen amino acids)
- comprising the steps of:
- disrupting microorganism cells containing long-chain acyl-CoA synthetase to obtain the enzyme which is bound to the membranes and then solubilizng the enzyme with a surfactant;
- and
- subjecting the solubilized enzyme to affinity chromatography using a ligand therefor which is a structural analog of a compound selected from the group consisting of ADP, ATP, CoA, and mixtures thereof, to separate said enzyme from contaminant proteins.
22 more elements
Dependencies are inferred from the claim text.