Characterization Of Alginate Lyase Aly08 With Its Anti-biofilm Property Toward Pseudomonas Aeruginosa

Pseudomonas aeruginosa(P.aeruginosa),an opportunistic human pathogen,is one of the three most common conditional pathogens in clinic.It is a major causative agent of mortality and morbidity in immunocompromised individuals and those with cystic fibrosis(CF).Once invaded the pulmonary epithelium,the genetic transformation of P.aeruginosa from non-alginate producing(nonmucoid)to alginate producing strain(mucoid)facilitated bacterial adherence in lung mucosa,stabilization of biofilm and immune escape,resulting in clinically persistent and refractory infection.Alginate is an acidic heteropolysaccharide,which is a key extracellular polysaccharide(EPS)component in the biofilm of P.aeruginosa.Meanwhile,alginate lyase can specifically target and destroy the alginate in the biofilm via ?-eliminating reaction,bypassing drug resistance issues.It has emerged as an efficient novel therapeutic strategy for the treatment of P.aeruginosa infections and attracted increasing attentions.Although various alginate lyases have been characterized,low activities/stabilities limited their clinical application.Therefore,the discovery of alginate lyases with better activities/stabilities and further improvement of their enzymatic properties via corresponding methods are highly desirable.In this study,a polysaccharide lyase family 7(PL7)alginate lyase-encoding gene,aly08,was cloned from the marine bacterium Vibrio sp.SY01,expressed and fermented in Escherichia coli.The enzyme was purified from the fermentation supernatant with the Ni-NTA column affinity purification method.Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis(SDS-PAGE)analysis confirmed the purity and molecular weight of the enzyme,the 35 kDa enzyme was then named Aly08.At its optimal pH(8.35)and temperature(45°C),Aly08 exhibited a specific activity of approximately 841 U/mg.Meanwhile,good pH-stability and thermo-tolerance was observed as well.High Performance Liquid Chromatography(HPLC)and negative-ion Electrospray Ionization Mass Spectrometry(ESI-MS)showed that Aly08 degraded alginates into disaccharides and trisaccharides highly efficiently in an endo-manner.As demonstrated with the 96-well plate model and flow cell model,Aly08 may inhibit and eliminate P.aeruginosa biofilm.In order to further improve its biological properties and anti-biofilm activities of alginate lyase,the purified Aly08 was immobilized on chitosan nanoparticles(CS-NPs)by ion cross-linking.The resulting immobilized enzyme exhibited significantly enhanced biological properties,such as thermal stability and reusability.The biofilm mass and thickness of P.aeruginosa was determined with confocal laser scanning microscopy(CLSM).Following treatment with immobilized and free Aly08 enzyme,the thickness of biofilm was 21.7 ± 5.1 ?m and 49.6 ± 7.3 ?m,respectively.Similar results were observed in the pre-formed biofilms.Therefore,comparing with free Aly08,immobilized Aly08 exhibited higher efficiency in inhibiting biofilm formation and interrupting the established mature biofilm of P.aeruginosa.Additionally,when the immobilized Aly08 was used in combination with different antibiotics against P.aeruginosa PAO1,the biofilm dispersion mediated by Aly08 enhanced the directtargeting and eliminating ability of antibiotics,thereby significantly increasing the sensitivities of P.aeruginosa to antibiotics.In summary,alginate lyase Aly08 has high activity,good thermo-stability and pHstability.Meanwhile,Aly08-immobilized chitosan nanoparticles will facilitate further developments of alginate lyases as anti-biofilm agents.