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

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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.

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Acknowledgements

This study was granted by the TR Prime Ministry State Planning Organization (DPT). We greatly appreciate Dokuz Eylul Hospital University for blood samples and Tanyalcin Laboratory for the analysis of the l-Phe with amino acid analyzer.

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  1. Faculty of Science and Arts, Department of Chemistry, University of Dokuz Eylül, 35160, Buca, Izmir, Turkey

    Leman Tarhan & Hulya Ayar-Kayali

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  1. Leman Tarhan
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  2. Hulya Ayar-Kayali
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Correspondence to Leman Tarhan.

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Tarhan, L., Ayar-Kayali, H. Immobilization of Phenylalanine Dehydrogenase and Its Application in Flow-Injection Analysis System for Determination of Plasma Phenylalanine. Appl Biochem Biotechnol 163, 258–267 (2011). https://doi.org/10.1007/s12010-010-9035-8

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  • DOI: https://doi.org/10.1007/s12010-010-9035-8

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