Sains Malaysiana 40(11)(2011): 1263–1269

Bioanalisis Berasaskan Sistem Glutamat Dehidrogenase-Diaporase

untuk Pengesanan Ammonium

(Bioanalysis Based on Glutamate Dehydrogenase/diaphorase System

for Ammonium Determination)

Nur Ellina Azmi, Jaafar Abdullah*, Hamidah Sidek & Samsulida Abd Rahman

Pusat Penyelidikan Bioteknologi Perindustrian, SIRIM Berhad, No.1, Persiaran Dato’ Menteri

Seksyen 2, P.O. Box 7035, 40911 Shah Alam, Selangor D.E., Malaysia

Musa Ahmad & Lee Yook Heng

Pusat Pengajian Sains Kimia dan Teknologi Makanan, Fakulti Sains dan Teknologi

Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor D.E., Malaysia

Received: 2 September 2010 / Accepted: 2 February 2011

ABSTRACT

Bioanalysis for ammonium detection based on dual enzyme system Glutamate dehydrogenase/ Diaphorase (GLDH-Dph) and thiazolyl blue tetrazolium bromide (MTT) reagent has been described. In this work, GLDH will catalyze the conversion of α-ketoglutaric acid to L-glutamate and β-nicotinamide adenine dinucleotide (NADH) will be oxidized to NAD+ in the presence of ammonium. The excess of unreacted NADH was then oxidized to NAD+ by Dph and MTT reagent was reduced to purple color formazan. The intensity of formazan product formed was observed at the wavelength of 563 nm by using spectrophotometer. Bioanalysis showed optimum response for ammonium detection at phosphate buffer of pH 8, GLDH, Dph and MTT concentrations at 13.2 unit/mL, 1.17 unit/mL and 0.2 mM, respectively. Bioanalysis showed linear response towards ammonium in the concentration range of 3 - 50 μM with the detection limit of 1 μM.

Keywords: Ammonium; bioanalysis; Dph; GLDH

ABSTRAK

Bioanalisis bagi pengesanan ammonium berdasarkan penggunaan sistem dua enzim Glutamat dehidrogenase-Diaporase (GLDH-Dph) dan reagen tiazolil biru tetrazolium bromida (MTT) diterangkan. Dalam kajian ini, GLDH memangkinkan tindak balas penukaran asid α-ketoglutarik membentuk L-glutamat dan kofaktor β-nikotinamida adenina dinukleotida (NADH) akan dioksidakan kepada NAD+ dengan kehadiran ammonium. Seterusnya, baki NADH yang tidak digunakan telah dioksidakan kepada NAD+ oleh Dph dan MTT dan diturunkan kepada formazan yang berwarna ungu. Keamatan formazan yang terbentuk boleh dicerap menggunakan spektrofotometer pada panjang gelombang 563 nm. Bioanalisis ini memberikan rangsangan yang optimum bagi pengesanan ammonium pada pH larutan penimbal fosfat bersamaan 8, kepekatan GLDH, Dph dan reagen MTT pada 13.2 unit/mL, 1.17 unit/mL dan 0.2 mM, masing-masing. Bioanalisis ini memberikan rangsangan linear terhadap ammonium dalam julat kepekatan 3 - 50 μM dengan had pengesanan 1 μM.

Kata kunci: Ammonium; bioanalisis; Dph; GLDH

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*Corresponding author; email: jaafar@sirim.my