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

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. AbstrakEkspresi 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 subst...

A brief overview on the application and sources of α‐amylase and expression hosts properties in order to production of recombinant α‐amylase

“…This secretory system transports the protein to outer space in one step without entering the periplasmic space. Bacteria use this pathway mostly for the secretion of exoenzymes and toxins with a high molecular weight 47,48 …”

“…Bacteria use this pathway mostly for the secretion of exoenzymes and toxins with a high molecular weight. 47,48 Type II secretory pathway. This secretory pathway transports protein to the extracellular space in two steps.…”