Sains Malaysiana 45(7)(2016): 1057–1062

Purification of Glutathione Transferases (GSTs) from Identified Rhizospheric Bacteria

(Penulenan Glutation Transferases (GSTs) daripada Bakteria Rizosfera Dikenal Pasti)

KHANOM SIMARANI*, WAN HAIZATUL AKMAL CHE YUSOFF & ZAZALI ALIAS

Institute of Biological Sciences, Faculty of Science, University of Malaya, 56100 Kuala Lumpur

Malaysia

Received: 12 March 2015/Accepted: 27 January 2016

ABSTRACT

The glutathione S-Transferase (GST) enzyme plays an important role in cellular detoxification. This multifunctional enzyme is involved in Phase II detoxification pathways that protect cellular macromolecules from being attacked by harmful compound. The study is an attempt to isolate glutathione transferase-expressing bacteria from the rhizospheric soil of selected herbal plants. Screening showed nine positive isolates out of twelve bacterial samples from a large microbial population in our soil collection. Crude extract from strain E1 which was isolated from Piper sarmentosum (Kadok) showed the highest specific activity against 1-chloro-2, 4-dinitrobenzene substrates (5.78 × 10-06 μmol/min/mg). Based on the carbon utilization of E1 assessed using Biolog system, the strain was identified as Comamonas testosterone E1. Glutathione S-transferase purification using GST trap yielded two distinct subunits with molecular weights of 23 and 24 kDa as visualized on 1D SDS-polyacrylamide gel electrophoresis. The purified GST showed reactivity towards 1-chloro-2, 4-dinitrobenzene, 1, 2-dichloro-4-nitrobenzene and ethacrynic acid with specific activity of 0.264 ± 0.038 nmol/min/mg and 0.056 ± 0.002 nmol/min/mg and 10.500 ± 3.130 nmol/min/mg, respectively. However, no activity was detected against p-Nitrobenzyl chloride, Sulfobromophthalein, trans-4-phenyl-3-butene-2-one, hexa-2, 4- dienal, trans-hepta-2, 4-dienal and trans-oct-2-enal in the study.

Keywords: Comamonas testosterone; GST-expressing bacteria; Piper sarmentosum; rhizospheric soil; xenobiotic

ABSTRAK

Enzim glutation S-Transferase (GST) memainkan peranan yang penting dalam detoksifikasi bersel. Enzim pelbagai fungsi ini terlibat dalam Fasa II laluan detoksifikasi yang melindungi sel makromolekul daripada diserang oleh sebatian berbahaya. Kajian ini merupakan cubaan untuk memencilkan bakteria glutation transferase-nyata dari tanah rizosfera beberapa tanaman herba. Daripada keseluruhan dua belas sampel bakteria dari populasi mikroorganisma yang terdapat dalam sampel tanah terkumpul yang diuji, sembilan sampel didapati berpotensi. Ekstrak mentah daripada strain E1 yang telah diasingkan daripada pokok herba Piper sarmentosum (Kadok) menunjukkan jumlah aktiviti spesifik yang tinggi terhadap substrat 1-chloro-2, 4-dinitrobenzena (5.78 × 10-06 μmol/min/mg). E1 dikenal pasti sebagai Comamonas testosterone berdasarkan penggunaan karbon yang dinilai menggunakan sistem Biolog. Penulenan glutation S-transferase (GST) menggunakan perangkap GST menghasilkan dua subunit berbeza dengan berat molekul masing-masing 23 dan 24 kDa seperti ditunjukkan dalam 1D SDS-poliakrilamida gel elektroforesis. GST tulen menunjukkan reaktiviti terhadap 1-kloro-2, 4-dinitrobenzena (CDNB), 1, 2-dikloro-4-nitrobenzena (DCNB) dan asid etakrinik (EA) iaitu dengan jumlah aktiviti spesifik masing-masing sebanyak 0.264 ± 0.0382, 0.056 ± 0.002 dan 10.500 ± 3.130 nmol/min/mg. Walau bagaimanapun, aktiviti terhadap p-Nitrofenil klorida (NBC), sulfobromoftalein (BSP), trans-4-fenil-3-butena-2-satu (PBO), hexa-2, 4- dienal, trans-hepta-2, 4-dienal dan trans-oct-2-enal tidak dikesan dalam kajian ini.

Kata kunci: Comamonas testosterone; bakteria GST-nyata; Piper sarmentosum; tanah rizosfera; xenobiosis

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*Corresponding author; email: hanom_ss@um.edu.my