Determination of L-phenylalanine in serum by flow-injection analysis using immobilized phenylalanine dehydrogenase and fluorimetric detection

A flow-injection system with an immobilized enzyme reactor is proposed for the determination of L-phenylalanine, Phenylalanine dehydrogenase from Rhodoccus sp. M4 was immobilized on tresylated poly (vinyl alcohol) beads (13 mu m) and packed into a stainless-steel column (5 cm x 4 mm i.d.). Serum sample was deproteinized with tungstic acid and filtered through an ultrafiltration membrane. The sample solution (30 mu l) was injected into the carrier stream (water). The NADH formed was detected at 465 nm (excitation at 340 nm). The calibration graph was linear for 0.9-600 mu m L-phenylalanine; the detection limit was 0.3 mu m. The sample throughout was 25 h(-1) without carryover. The half-life period of the immobilized enzyme was 23 days.