Expression and the Functional Study of Fusion Proteins α-Amylase and Hemolysin- αas an Application in Biofilm Polysaccharide Degradation

Authors

Gede Yuda Sugiarta, Faculty of Medicine, Universitas Indonesia, Depok 16424, Indonesia
Anggoro Wiseso, Research Centre for Biomedical Engineering, Department of Chemical Engineering, Faculty of Engineering, Universitas Indonesia, Depok 16424, Indonesia
Siska Yuliana Sari, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, Depok 16424, Indonesia
Etri Dian Kamila, Research Centre for Biomedical Engineering, Department of Chemical Engineering, Faculty of Engineering, Universitas Indonesia, Depok 16424, Indonesia
Vanessa Geraldine, Research Centre for Biomedical Engineering, Department of Chemical Engineering, Faculty of Engineering, Universitas Indonesia, Depok 16424, Indonesia
Diana Christina, Research Centre for Biomedical Engineering, Department of Chemical Engineering, Faculty of Engineering, Universitas Indonesia, Depok 16424, Indonesia
Muhammad Hanifi, Faculty of Medicine, Universitas Indonesia, Depok 16424, Indonesia
Dwiantari Satyapertiwi, Research Centre for Biomedical Engineering, Department of Chemical Engineering, Faculty of Engineering, Universitas Indonesia, Depok 16424, Indonesia
Robby Hertanto, Faculty of Medicine, Universitas Indonesia, Depok 16424, Indonesia
Budiman Bela, Department of Clinical Microbiology, Faculty of Medicine, Universitas Indonesia, Jakarta 10320, Indonesia
Masafumi Yohda, Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan
Muhamad Sahlan, Research Centre for Biomedical Engineering, Department of Chemical Engineering, Faculty of Engineering, Universitas Indonesia, Depok 16424, IndonesiaFollow

Abstract

Biofilm is an aggregate of consortium bacteria that adhere to each other on a surface. It is usually protected by the exopolysaccharide layer. Various invasive medical procedures, such as catheterization, endotracheal tube installation, and contact lens utilization, are vulnerable to biofilm infection. The National Institute of Health (NIH) estimates 65% of all microbial infections are caused by biofilm. Periplasmic α-amylase (MalS) is an enzyme that hydrolyzes α-1, 4- glicosidic bond in glycogen, starch, and others related polysaccharides in periplasmic space. Another protein called hemolysin-α (HlyA) is a secretion signal protein on C terminal of particular peptide in gram negative bacteria. We proposed a novel recombinant plasmid expressing α-amylase and hemolysin-α fusion in pSB1C3 which is cloned into E.coli to enable α-amylase excretion to extracellular for degrading biofilm polysaccharides content, as in starch agar. Microtiter assay was performed to analyze the reduction percentage of biofilm by adding recombinant E.coli into media. This system is more effective in degrading biofilm from gram positive bacteria i.e.: Bacillus substilis (30.21%) and Staphylococcus aureus (24.20%), and less effective degrading biofilm of gram negative i.e.: Vibrio cholera (5.30%), Pseudomonas aeruginosa (8.50%), Klebsiella pneumonia (6.75%) and E. coli (-0.6%). Gram positive bacteria have a thick layer of peptidoglycan, causing the enzyme to work more effectively in degrading polysaccharides.

Bahasa Abstract

Ekspresi dan Penelitian Fungsi Protein Fusi α-Amilase dan Hemolisin-α sebagai suatu Penerapan dalam Penurunan Polisakarida Biofilm. Biofilm adalah sekumpulan bakteria yang saling melekat satu sama lain pada suatu permukaan. Biofilm ini biasanya dilindungi oleh lapisan eksopolisakarida. Berbagai prosedur medis yang pro-aktif, seperti kateterisasi, instalasi alat bantuan pernafasan, dan penggunaan lensa kontak mata, rentan terhadap infeksi biofilm. NIH (National Institute of Health – Institusi Kesehatan Nasional) memperkirakan 65% dari semua infeksi mikroba disebabkan oleh biofilm. Enzim α-amilase periplasma (MalS) merupakan suatu enzim yang menghidrolisis α-1, ikatan 4-glikosidik dalam glikogen, zat tepung, dan lainnya terkait polisakarida pada ruang periplasma. Protein lainnya yang disebut hemolisin-α (HlyA) merupakan protein sinyal sekresi pada terminal C dari peptida tertentu dalam bakteria gram-negatif. C merupakan protein sinyal sekresi pada terminal C dari peptida tertentu dalam bakteria gram-negatif. Kami mengusulkan suatu plasmid rekombinan baru mengekspresikan fusi α-amilase and hemolisin-α dalam pSB1C3 yang diklon menjadi E. coli untuk memungkinkan ekskresi α-amilase ke luar sel tubuh (ekstraselular) untuk menurunkan isi polisakarida biofilm, seperti dalam agar zat tepung. Tes dengan tabung kecil dilakukan untuk menganalisis persentase pengurangan biofilm dengan menambahkan E. coli rekombinan ke dalam media. Sistem ini lebih efektif dalam menurunkan tingkat biofilm dari bakteria gram-positif, seperti Bacillus substilis (30.21%) dan Staphylococcus aureus (24.20%), dan kurang efektif menurunkan tingkat biofilm dari gram-negatif, Vibrio cholera (5.30%), Pseudomonas aeruginosa (8.50%), Klebsiella pneumonia (6.75%), dan E. coli (-0.6%). Bakteria gram-positif mempunyai lapisan peptidoglikan yang tebal, menyebabkan enzim untuk bekerja lebih efektif dalam menurunkan tingkat polisakarida.

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Recommended Citation

Sugiarta, Gede Yuda; Wiseso, Anggoro; Sari, Siska Yuliana; Kamila, Etri Dian; Geraldine, Vanessa; Christina, Diana; Hanifi, Muhammad; Satyapertiwi, Dwiantari; Hertanto, Robby; Bela, Budiman; Yohda, Masafumi; and Sahlan, Muhamad (2016) "Expression and the Functional Study of Fusion Proteins α-Amylase and Hemolysin- αas an Application in Biofilm Polysaccharide Degradation," Makara Journal of Technology: Vol. 20: Iss. 3, Article 4.
DOI: 10.7454/mst.v20i3.3067
Available at: https://scholarhub.ui.ac.id/mjt/vol20/iss3/4