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

Diserahkan: 4 April 2021/Diterima: 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

RUJUKAN

Aguayo, M.D.C.L., Burgos, M.J.G., Pulido, R.P., Gálvez, A. & López, R.L. 2016. Effect of different activated coatings containing enterocin AS-48 against Listeria monocytogenes on apple cubes. Innovative Food Science & Emerging Technologies 35: 177-183.

Ananou, S., Maqueda, M., Martínez-Bueno, M. & Gálvez, A. 2005. Control of Staphylococcus aureus in sausages by enterocin AS-48. Meat Science 71(3): 549-556.

Arbulu, S., Jiménez, J.J., Gútiez, L., Cintas, L.M., Herranz, C. & Hernández, P.E. 2015. Cloning and expression of synthetic genes encoding the broad antimicrobial spectrum bacteriocins SRCAM 602, OR-7, E-760, and L-1077, by recombinant Pichia pastoris. BioMed Research International 2015: 767183.

Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. & Struhl, K. 1994. Current Protocols in Molecular Biology.  New York:  John Wiley & Sons.

Bali, V., Panesar, P.S., Bera, M.B. & Kennedy, J.F. 2016. Bacteriocins: Recent trends and potential applications. Critical Reviews in Food Science and Nutrition 56(5): 817-834.

Borrero, J., Kunze, G., Jiménez, J.J., Böer, E., Gútiez, L., Herranz, C., Cintas, L.M. & Hernández, P.E. 2012. Cloning, production, and functional expression of the bacteriocin enterocin A, produced by Enterococcus faecium T136, by the yeasts Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, and Arxula adeninivorans. Applied and Environmental Microbiology 78(16): 5956-5961.

Carrique-Mas, J.J. & Bryant, J. 2013. A review of foodborne bacterial and parasitic zoonoses in Vietnam. Ecohealth 10(4): 465-489.

Dimitrijević, R., Stojanović, M., Živković, I., Petersen, A., Jankov, R.M., Dimitrijević, L. & Gavrović-Jankulović, M. 2009. The identification of a low molecular mass bacteriocin, rhamnosin A, produced by Lactobacillus rhamnosus strain 68. Journal of Applied Microbiology 107(6): 2108-2115.

Dortu, C., Huch, M., Holzapfel, W., Franz. C.M.A.P. & Thonart, P. 2008. Anti‐listerial activity of bacteriocin‐producing Lactobacillus curvatus CWBI‐B28 and Lactobacillus sakei CWBI‐B1365 on raw beef and poultry meat. Letters in Applied Microbiology 47(6): 581-586.

Gram, L., Ravn, L., Rasch, M., Bruhn, J.B., Christensen, A.B. & Givskov, M. 2002. Food spoilage - Interactions between food spoilage bacteria. International Journal of Food Microbiology 78(1-2): 79-97.

Grande, M.J., Lucas, R., Abriouel, H., Valdivia, E., Omar, N.B., Maqueda, M., Martínez-Bueno, M., Martínez-Cañamero, M. & Gálvez, A. 2006. Inhibition of toxicogenic Bacillus cereus in rice-based foods by enterocin AS-48. International Journal of Food Microbiology 106(2): 185-194.

Grande, M.J., Lucas, R., Valdivia, E., Abriouel, H., Maqueda, M., Omar, N.B., Martínez-Cañamero, M. & Gálvezi, M. 2005. Stability of enterocin AS-48 in fruit and vegetable juices. Journal of Food Protection 68(10): 2085-2094.

Gutiérrez, J., Larsen, R., Cintas, L.M., Kok, J. & Hernández, P.E. 2006. High-level heterologous production and functional expression of the sec-dependent enterocin P from Enterococcus faecium P13 in Lactococcus lactis. Applied Microbiology and Biotechnology 72(1): 41-51.

Invitrogen. 2010. User Manual - EasySelect^TM Pichia Expression Kit for Expression of Recombinant Proteins using pPICZ and pPICZα in Pichia pastoris. United States: Thermo Fisher Scientific.

Karbalaei, M., Rezaee, S.A. & Farsiani, H. 2020. Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins. Journal of Cellular Physiology 235(9): 5867-5881.

Krainer, F.W., Dietzsch, C., Hajek, T., Herwig, C., Spadiut, O. & Glieder, A. 2012. Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway. Microbial Cell Factories 11(1): 1-14.

Li, Z., Cheng, Q., Guo, H., Zhang, R. & Si, D. 2020. Expression of hybrid peptide EF-1 in Pichia pastoris, its purification, and antimicrobial characterization. Molecules 25(23): 5538.

Line, J.E., Svetoch, E.A., Eruslanov, B.V., Perelygin, V.V., Mitsevich, E.V., Mitsevich, I.P., Levchuk, V.P., Svetoch, O.E., Seal, B.S., Siragusa, G.R. & Stern, N.J. 2008. Isolation and purification of enterocin E-760 with broad antimicrobial activity against Gram-positive and Gram-negative bacteria. Antimicrobial Agents and Chemotherapy 52(3): 1094-1100.

Lucas, R., Grande, M.A.J., Abriouel, H., Maqueda, M., Omar, N.B., Valdivia, E., Martínez-Cañamero, M. & Gálvez, A. 2006. Application of the broad-spectrum bacteriocin enterocin AS-48 to inhibit Bacillus coagulans in canned fruit and vegetable foods. Food and Chemical Toxicology 44(10): 1774-1781.

 Lv, X., Ma, H., Sun, M., Lin, Y., Bai, F., Li, J. & Zhang, B. 2018. A novel bacteriocin DY4-2 produced by Lactobacillus plantarum from cutlassfish and its application as bio-preservative for the control of Pseudomonas fluorescens in fresh turbot (Scophthalmus maximus) fillets. Food Control 89: 22-31.

Meade, E., Slattery, M.A. & Garvey, M. 2020. Bacteriocins, potent antimicrobial peptides and the fight against multi drug resistant species: Resistance is futile? Antibiotics 9(1): 32.

Mesa-Pereira, B., Rea, M.C., Cotter, P.D., Hill, C. & Ros, R.P. 2018. Heterologous expression of biopreservative bacteriocins with a view to low cost production. Frontiers in Microbiology 9: 1654.

Nelson, M., Raschke, E. & McClelland, M. 1993. Effect of site-specific methylation on restriction endonucleases and DNA modification methyltransferases. Nucleic Acids Research 21(13): 3139.

Odeyemi, O.A., Alegbeleye, O.O., Strateva, M. & Stratev, D. 2020. Understanding spoilage microbial community and spoilage mechanisms in foods of animal origin. Comprehensive Reviews in Food Science and Food Safety 19(2): 311-331.

Olejnik-Schmidt, A.K., Schmidt, M.T., Sip, A., Szablewski, T. & Grajek, W. 2014. Expression of bacteriocin divercin AS7 in Escherichia coli and its functional analysis. Annals of Microbiology 64(3): 1197-1202.

Pedro, A., Oppolzer, D., Bonifacio, M. & Maia, C. 2015. Evaluation of Mut^S and Mut⁺ Pichia pastoris strains for membrane-bound catechol-O-methyltransferase biosynthesis. Applied Biochemistry and Biotechnology 175(8): 3840-3855.

Quezada-Rivera, J., Soria-Guerra, R., Pérez-Juárez, F., Martínez-González, L., Valdés-Rodríguez, S.E., Vasco-Méndez, S.E. & Morales-Domínguez, J.F. 2019. Heterologous expression of bacteriocin E-760 in Chlamydomonas reinhardtii and functional analysis. Phyton 88(1): 25.

Reis, J., Paula, A., Casarotti, S. & Penna, A.L.B. 2012. Lactic acid bacteria antimicrobial compounds: Characteristics and applications. Food Engineering Reviews 4(2): 124-140.

Schägger, H. 2006. Tricine–SDS-PAGE. Nature Protocols 1(1): 16-22.

Shin, J.M., Gwak, J.W., Kamarajan, P., Fenno, J.C., Rickard, A.H. & Kapila, Y.L. 2016. Biomedical applications of nisin. Journal of Applied Microbiology 120(6): 1449-1465.

Silva, C.C., Silva, S.P. & Ribeiro, S.C. 2018. Application of bacteriocins and protective cultures in dairy food preservation. Frontiers in Microbiology 9: 594.

Todorov, S.D. & Dicks, L.M. 2007. Bacteriocin production by Lactobacillus pentosus ST712BZ isolated from boza. Brazilian Journal of Microbiology 38(1): 166-172.

Thuan, H.V.,  Nguyen, N.T.T., Vinh, L., Ninh, H.L., Huy, Q.N., Tuan, V.L. & Nuorti, P.J. 2017. Epidemiologic characteristics of foodborne outbreaks in southern Vietnam, 2009-2013. Journal of Microbiology and Infectious Diseases 7(1): 13-20.

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