Immobilization of Phenylalanine Dehydrogenase and Its Application in Flow-Injection Analysis System for Determination of Plasma Phenylalanine

Abstract

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.

Fe3O4@SiO2@NiAl-LDH microspheres implication in separation, kinetic and structural properties of phenylalanine dehydrogenase

“…Magnetic gold mesoporous silica nanoparticle core shells (mAu@PSNs) have been able to increase the activity and thermal stability of cellulase so that enzyme separation could be easily carried out by a magnet despite covalent bonds [ 110 ]. A cellulase treated with Fe 3 O 4 nanoparticles has revealed extended thermal stability in the range of 50–60 °C up to 12 h [ 111 ]. It seems that regardless of the type of connection, nano magnetite particles can cause significant changes in the thermal stability of the enzyme.…”

Preparation of Molecularly Imprinted Electrochemical L-Phenylalanine Sensor with p-Toluene Sulfonic Acid Modified Pt Electrode

“…NIR, chemiluminescence, spectroscopy, amperometry, voltammetry and HPLC techniques are used in the literature for the determination of L-Phe. [32][33][34][35][36][37][38][39][40][41] Some important results of these commonly used techniques are shown in Table I. In addition, for the determination of L-Phe, Amperometry has been successfully performed with voltammetry, DPV and CV techniques.…”

Assembly of peptide linker to amino acid dehydrogenase and immobilized with metal–organic framework

“…Therefore, the improvement of the catalytic activity and thermostability of PheDHs is a focus of research. 21,22 Enhancing the stability of oxidoreductases is a critical challenge in biotechnology. Inspired by natural biomineralization processes, a novel method for immobilization of PheDHs by encapsulating them within MOFs is proposed.…”