Abstract
Enzymatic properties of immobilized pyridoxaminephosphate oxidase (EC 1. 4. 3. 5) and the continuous production of pyridoxal 5'-phosphate (PLP) by the immobilized enzyme column were studied The enzyme partially purified from dry baker's yeast was immobilized to agarose matrix via phenyl-mercury ligand, and then lyophilized. The relative rate of activity for pyridoxine 5'-phosphate (PNP) to that for pyridoxamine 5'-phosphate (PMP) apparently increased (ratio of PNP oxidase activity per PMP oxidase activity was more than 1), where biphasic Lineweaver-Burk plots for two substrates were revealed, indicating that the immobilized, lyophilized preparation could efficiently oxidize PNP and PMP at high concentration. By combining the immobilized enzyme and aminoethyl-Sephadex columns, the sufficient production of PLP and its removal from the solution were achieved, when the solution (100 ml, 10 nmol substrate) was recycled in a closed-like system. β-Alanine-NaOH buffer (0.2 M, pH 8.0 or 9.0) was sufficient to allow the continuous production of PLP (conversion ratio; 80%) as well as its removal by adding substrate and by adjusting pH for several cycles.
