| Abstract: | 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. |
