Preparation and properties of immobilized flavokinase.
Flavokinase (ATP: riboflavin 5'-phosphotransferase, EC 2.7.1.26) purified from rat liver by affinity chromatography, has been immobilized by amide linkage to omega-aminoalkyl-agarose beads. The immobilized enzyme differs from the soluble enzyme in having greater stability, slightly higher Km for the substrates, riboflavin and ATP, a broader pH optimum, and a lower energy of activation. These results suggest that the immobilized enzyme is influenced by the microenvironment of the bead and is subject to some degree of internal diffusional limitation. A small (3 ml), continuous, plug-flow reactor prepared with immobilized flavokinase effects 50% conversion of riboflavin to riboflavin 5'-phosphate (FMN) with a flow rate of 0.16 ml/min, which corresponds to an output of 5 nmol FMN/min. Immobilized flavokinase is effective for phosphorylating riboflavin and numerous riboflavin analogs and provides a facile method for preparing exclusively, unlike other synthetic methods, the 5'-phosphates.
D055598:Chemical Phenomena; D002621:Chemistry; D004800:Enzymes, Immobilized / Q000378:metabolism*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D012256:Riboflavin / Q000031:analogs & derivatives / Q000378:metabolism; D012685:Sepharose
D004800:Enzymes, Immobilized; D012685:Sepharose; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C022532:riboflavin kinase; D012256:Riboflavin