Sains Malaysiana 49(3)(2020): 527-536

http://dx.doi.org/10.17576/jsm-2020-4903-07

Metalotionein 1 daripada Eleusine indica L. Memberikan Toleransi terhadap Logam Berat dalam Escherichia coli

(Metallothionein 1 from Eleusine indica L. Confers Heavy Metal Tolerance in Escherichia coli)

ROOHAIDA OTHMAN^1,2* & NORUL HUDA MOHD NASIR¹

¹Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

²Institute of Systems Biology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

Received: 20 May 2019/Accepted: 5 December 2019

ABSTRAK

Metalotionein (MT) berpotensi besar dalam pemuliharaan alam sekitar disebabkan peranannya yang penting dalam metabolisme dan penyahtoksikan ion logam melalui proses pengkelatan ion logam. cDNA MT1 daripada Eleusine indica L. (eiMT1) telah diamplifikasi dan diekspreskan dalam Escherichia coli. Pengekspresan protein rekombinan diaruh menggunakan 1 mM IPTG pada 37°C selama 4 jam. Protein rekombinan dengan berat molekul 12 kDa telah diperoleh dan ditulenkan menggunakan dua kaedah penulenan iaitu kromatografi penukaran ion dan kromatografi penurasan gel. Analisis jujukan protein rekombinan telah mengesahkan bahawa protein eiMT yang diekspreskan mengandungi jujukan asid amino sebagaimana dijangkakan bagi protein yang dikodkan oleh cDNA eiMT1. Untuk menilai keupayaan protein eiMT ini memberikan toleransi terhadap logam berat bagi E. coli, sel bakteria yang ditransformasikan dengan plasmid yang membawa cDNA eiMT1 telah dikulturkan dalam media yang mengandungi ion tembaga (Cu), zink (Zn), besi (Fe), kromium (Cr) dan kadmium (Cd). Sel yang mengekspres eiMT didapati menunjukkan tahap pertumbuhan yang lebih tinggi berbanding sel jenis liar di dalam media yang mengandungi kesemua ion logam serta mampu bermandiri di dalam Cu, Zn, Fe dan Cr sehingga kepekatan 500 µM manakala bagi Cd sehingga kepekatan logam 400 µM. Hasil kajian ini menunjukkan bahawa eiMT membolehkan peningkatan toleransi sel E. coli terhadap logam berat dan memainkan peranan penting dalam menyingkirkan ion logam berlebihan. Hal ini mencadangkan bahawa eiMT juga kemungkinan mampu melakukan penyahtoksikan ion logam berlebihan dalam E. indica. Oleh itu, sel E. coli yang membawa cDNA eiMT di samping tumbuhan E. indica sendiri boleh diaplikasikan dalam proses remediasi dengan menyerap logam berat dalam persekitaran yang tercemar.

Kata kunci: Eleusine indica L.; logam berat; metalotionein; protein rekombinan; rumput sambau

Abstract

Metallothionein (MT) has a huge potential in environmental conservation due to its important role in metal ion metabolism and detoxification by chelating the metal ions. MT1 cDNA from Eleusine indica L. (eiMT1) was amplified and expressed in Escherichia coli cells. Recombinant protein expression was induced using 1 mM IPTG at 37^oC for 4 h. Recombinant protein with the molecular mass of 12 kDa was obtained and purified through two purification methods involving ion exchange chromatography and gel filtration chromatography. Sequence analysis of the recombinant protein confirmed that the expressed eiMT protein contained amino acid sequence as expected for the protein encoded by the eiMT1 cDNA. To evaluate the ability of this eiMT protein to confer heavy metal tolerance in E. coli, the bacterial cells transformed with the recombinant plasmids containing the eiMT1 cDNA were grown in media containing copper (Cu), zinc (Zn), iron (Fe), chromium (Cr) and cadmium (Cd) ions. The eiMT-expressing cells showed higher growth level compared to wild type cells in media containing all metal ions and was able to survive in Cu, Zn, Fe and Cr until 500 µM in concentration while for Cd until 400 µM. These results showed that eiMT allowed enhanced heavy metal tolerance in E. coli cells and played an important role in removing excess metal ions. This suggested that the eiMT protein might also be capable of detoxifying metal ions in E. indica. Hence, both the E. coli cells containing the eiMT cDNA as well as the E. indica plant itself may be applied in remediation process by adsorbing heavy metals in polluted environment.

Keywords: Eleusine indica L.; goose grass; heavy metal; metallothionein; recombinant protein

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*Corresponding author; email: roohaida@ukm.edu.my