Immobilization of phenylalanine dehydrogenase and its application in flow-injection analysis system for determination of plasma phenylalanine.
Phenylalanine dehydrogenase (L-PheDH) from Sporosarcina ureae was immobilized on DEAE-cellulose, modified initially with 2-amino-4,6-dichloro-s-triazine followed by hexamethylenediamine and glutaraldehyde. The highest activity of immobilized PheDH was determined as 95.75 U/g support with 56% retained activity. The optimum pH value of immobilized L-PheDH was shifted from pH 10.4 to 11.0. The immobilized L-PheDH showed activity variations close to the maximum value in a wider temperature range of 45-55 °C, whereas it was 40 °C for the native enzyme. The pH and the thermal stability of the immobilized L-PheDH were also better than the native enzyme. At pH 10.4 and 25 °C, K (m) values of the native and the immobilized L-PheDH were determined as K(m Phe) = 0.118, 0.063 mM and K(m NAD)(+) = 0.234, 0.128 mM, respectively. Formed NADH at the exit of packed bed reactor column was detected by the flow-injection analysis system. The conversion efficiency of the reactor was found to be 100% in the range of 5-600 μM Phe at 9 mM NAD(+) with a total flow rate of 0.1 mL/min. The reactor was used for the analyses of 30 samples each for 3 h per day. The half-life period of the reactor was 15 days.
Tarhan
Leman
L
Faculty of Science and Arts, Department of Chemistry, University of Dokuz Eylül, Buca, Izmir, Turkey. leman.tarhan@deu.edu.tr
Ayar-Kayali
Hulya
H
D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D019149:Bioreactors; D003636:DEAE-Cellulose / Q000737:chemistry; D003959:Diamines / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D017022:Flow Injection Analysis; D005976:Glutaral / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009243:NAD / Q000097:blood; D010649:Phenylalanine / Q000097:blood*; D010661:Phenylketonurias; D012680:Sensitivity and Specificity; D058644:Sporosarcina / Q000201:enzymology; D013696:Temperature
D003959:Diamines; D004800:Enzymes, Immobilized; D009243:NAD; D010649:Phenylalanine; D003636:DEAE-Cellulose; D000594:Amino Acid Oxidoreductases; C022342:phenylalanine oxidase; D005976:Glutaral; C007297:1,6-diaminohexane