Sains Malaysiana 51(6)(2022): 1667-1676

http://doi.org/10.17576/jsm-2022-5106-05

Expression of the Recombinant Enterocin E-760 in Pichia pastoris X33 and Its Antimicrobial Activity towards Listeria monocytogenes

(Pengekspresan Rekombinan Enterocin E-760 pada Pichia pastoris X33 dan Aktiviti Antimikrob terhadap Listeria monocytogenes)

KHANH HOANG VIET NGUYEN^1,*, LAN ANH TO¹ & KIM PHUONG LUONG²

¹Department of Molecular Biotechnology, Institute of New Biotechnology,

Academy of Military of Science and Technology, 17 Hoang Sam, Cau Giay, Hanoi, Vietnam

² Faculty of Biology, Hanoi VNU University of Science,

 Vietnam National University, 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam

Received: 4 April 2021/Accepted: 25 November 2021

Abstract

Bioactive compounds such as bacteriocins have become potent and promising alternatives to chemical food preservatives for extending food shelf-life and eliminating food loss from microbial spoilage. Enterocin E-760 is a specific bacterocin belonging to class II that possesses broad spectrum antibacterial activity against both Gram-negative and Gram-positive bacteria. In this study, the enterocin E-760 gene was fused to a His-tag and cloned into the expression vector, pPICzαA, and transformed into Escherichia coli DH5α cells. The recombinant plasmid was isolated, linearised and transformed into competent Pichia pastoris X33 cells using electroporation. The Pichia transformants were determined using PCR and expressed under methanol induction with the highest antibacterial activity of culture supernatants reaching 40 AU/mL. Enterocin E-760 exhibited a molecular weight of approximately 5.5 kDa and was detected directly on a Tricine SDS-PAGE gel containing Listeria monocytogenes ATCC 35152 after ethanol precipitation at a concentration ranging from 30% to 70%. This study represented the initial stages of research into using enterocin as a biopreservative in food processing.

Keywords: Antibacterial peptide; bacteriocin; enterocin E-760; food preservation

Abstrak

Sebatian bioaktif seperti bakteriosin telah menjadi alternatif yang kuat dan berpotensi menggantikan pengawet makanan kimia untuk memanjangkan jangka hayat makanan dan menghapuskan kerosakan makanan daripada tindakan mikrob. Enterosin E-760 ialah bakteriosin khusus yang tergolong dalam kelas II yang mempunyai aktiviti antibakteria dengan spektrum yang luas terhadap kedua-dua bakteria Gram-negatif dan Gram-positif. Dalam kajian ini, gen enterosin E-760 telah digabungkan dengan penanda His lalu diklonkan ke dalam vektor pengekspresan, pPICzαA dan ditransformasi ke dalam sel Escherichia coli DH5α. Plasmid rekombinan telah dipencil, dilinear dan ditransformasikan ke dalam sel kompeten Pichia pastoris X33 melalui elektroporasi. Transforman Pichia ditentukan menggunakan PCR dan diekspreskan melalui induksi menggunakan metanol menghasilkan aktiviti antibakteria bagi supernatan kultur yang tertinggi mencapai 40 AU/mL. Enterosin E-760 mempamerkan berat molekul kira-kira 5.5 kDa dan dikesan secara langsung pada gel SDS-PAGE Trisin yang mengandungi Listeria monocytogenes ATCC 35152 selepas pemendakan etanol pada kepekatan antara 30% hingga 70%. Kajian ini merupakan penyelidikan pada peringkat awal menggunakan enterosin sebagai biopengawet dalam pemprosesan makanan.

Kata kunci: Bakteriosin; enterosin E-760; pengawetan makanan; peptida antibakteria

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^*Corresponding author; email: hoangviet1015@gmail.com