Preparation of (-)-5,6,7,8-tetrahydrofolate using immobilized dihydrofolate reductase.
Dihydrofolate reductase from methotrexate-resistant Lactobacillus casei was immobilized on carbodiimide-activated CH-Sepharose. The immobilized enzyme was utilized in the synthesis of (-)-5,6,7,8-tetrahydrofolate from dihydrofolate and NADPH in a batchwise reaction system. The products of the reaction, (-)-tetrahydrofolate and NADP+, were separated on a Sephadex G-10 column equilibrated with 50 mM NH4HCO3 containing beta-mercaptoethanol and ethanol. The tetrahydrofolate was then characterized by ultraviolet and circular dichroic spectra and its reactivity as a cofactor in the thymidylate synthetase reaction.
D001426:Bacterial Proteins; D002942:Circular Dichroism; D004800:Enzymes, Immobilized; D005492:Folic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D009249:NADP / Q000032:analysis; D013056:Spectrophotometry, Ultraviolet; D013237:Stereoisomerism; D013762:Tetrahydrofolate Dehydrogenase; D013763:Tetrahydrofolates / Q000032:analysis / Q000096:biosynthesis*
D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; C010920:dihydrofolate; C030371:5,6,7,8-tetrahydrofolic acid; D009249:NADP; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase