Research On Alginate Lyase From Marine Vibrio Sp. QY103

Alginates are linear polysaccharides in whichβ-D-mannuronic acid (M) andα-L-guluronic acid (G) are (1,4)-linked to form blocks of consecutive G residues (polyG), consecutive M residues (polyM), and alternating M and G residues (polyMG). Alginates are synthesized as cell wall components by brown seaweeds and as exopolysaccharides by some bacteria belonging to the genera Azotobacter and Pseudomonas. In bacterial alginates, some of the M residues may be O-2- and/or O-3-acetylated. Acetylated alginate produced by Pseudomonas aeruginosa, which is an opportunistic pathogen of humans and other species, plays a crucial role in the adherence of the bacterium to target cells, biofilm development, protection of bacteria from phagocytes and prevention of antibiotic uptake. Once P. aeruginosa cells develop biofilm, they become more resistant to antibiotics (100-1000 folds) and to the innate and adaptive killing components of the host than their planktonic counterparts, resulting in severe chronic infections which persist despite aggressive antimicrobial therapy and a robust inflammatory response. Consequently, acetylated alginate is a major pathogenic factor in patients infected by P. aeruginosa.Alginate lyases catalyze the depolymerization of alginates byβ-elimination of the 4-O-glycosidic bond, with formation of 4-deoxy-L-erythro-hex-4-ene pyranosyluronate at the nonreducing end of the resulting oligosaccharide. There are several potential applications for alginate lyase, such as production of alginate-derived oligosaccharides, treatment of patients infected with mucoid P. aeruginosa biofilm and isolation of protoplasts from marine algae. It seems that alginate lyase may be used to degrade acetylated alginate surrounding mucoid P. aeruginosa cells and make them more susceptible to phagocytosis and antibiotic therapy. However, only a few alginate lyases which can degrade acetylated have been reported, which give low activity against acetylated alginate in the presence of several metal ions (such as Ca²⁺, Zn²⁺) found in purulent CF sputum.In this study, 32 strains with high production of alginate lyase are isolated from seawater or decaying thallus of Laminaria in Qingdao using medium containing alginate as the sole carbon source. Of them, strain QY103 yields the highest production of alginate lyase (5.87 U/mL against alginate and 4.22 U/mL against acetylated alginate). Strain QY103 is identified to belong to genus Vibrio using morphologic observation, physiological and biochemical methods, and molecular phylogenetic analysis based on the partial 16S rRNA gene sequence.The culture condition of alginate lyase production by Vibrio sp. QY103 is investigated. The optimized liquid fermentation medium (%, w/v) is: alginate 0.5, NH4NO3 0.2, KH2PO4 0.4, K2HPO4 0.6, NaCl 3.0, MgSO4 0.01, FeSO4 0.01, pH 5.5. After fermentation for 72 h at 150 r/min and at 28℃, the yield of alginate is up to 45.26 U/mL, which is 7.71-fold of that under the ordinary condition.An alginate lyase is purified 15.13-fold with a recovery yield of 29.81% from culture supernatants of Vibrio sp. QY103 to homogeneity using a combination of ammonium sulfate precipitation, DEAE-Sepharose FF anion-exchange chromatography and Superdex 75 gel filtration chromatography. The purified enzyme, which is named by AlyVII, gives a specific activity of 1863.45 U/mg and a single band on SDS-PAGE with a molecular mass of 35.6 kDa. AlyVII is most active at 40℃and at pH6.0 and is stable below 30℃and over a broad range of pH5.0-9.0. The activity of the enzyme is enhanced in the presence of Mg²⁺, Ca²⁺, Zn²⁺, Ba²⁺ and EDTA (0.1- to 0.5-fold at 1 mmol/L), Na+ (0.4-fold at 500 mmol/L) or K+ (0.2-fold at 100 mmol/L). Other compounds (Mn²⁺, Ni²⁺, Fe²⁺ and SDS tested at 1 mmol/L) inhibit the activity of the enzyme. AlyVII is more active against alginate, polyM and acetylated alginate from mucoid P. aeruginosa than against polyG.For approximately 86% of P. aeruginosa strains (6/7) tested in this study, alginat lyase AlyVII inhibits the formation of biofilm and disperses their formed biofilm in 96-well plate model and flow cell model. With the addition of AlyVII to a final concentration of 50μg/mL, formations of four strains'biofilms are inhibited by >80%, and formed biofilms of six strains are dispersed significantly (34.23%-64.72% of control biofilm remained). When treated with 100μg/mL AlyVII, both of inhibition efficiency and dispersion efficiency on biofilms of five strains exceed 80%. For 6/7 of P. aeruginosa strains tested in this study, addition of AlyVII decreases the MBEC of gentamicin to 1/16-1/64 of the control. However, AlyVII promotes slightly the growth of planktonic P. aeruginosa CS1.All together, alginate lyase AlyVII is the first acetylated alginate-degrading enzyme found in genus Vibrio. AlyVII exhibits anti-biofilm activity, and cations found in purulent CF sputum enhances the activity of AlyVII, therefore it may be used as therapeutic agent for the treatment of patients infected with mucoid P. aeruginosa biofilm.