Characterization Of A New Bifunctional,Cold-Adapted,Exolytic Alginate Lyase,and The Functional Analysis Of Key Amino Acids

Alginate is the most abundant structural polysaccharide in brown algae and the major component of Pseudomonas aeruginosa biofilm.Alginate lyases could degrade alginate to functional AOs with unsaturated bonds or monosaccharides,which can facilitate the biorefinery of brown algae and expand the scope of pharmaceutical application.Bifunctional alginate lyase could eradicate P.aeruginosa biofilm,which is a potential drug-resistant bacteria preparation and has important clinical significance for eliminating intractable bacterial infection.In order to meet the needs of biorefinery of brown algae and clinical anti-biofilm medication,screening of alginate lyases with new catalytic properties has gradually become a hotspot for development.However,the lack of research on the mechanism of alginate lyase greatly hindered its directional evolution and application.The purpose of this study was to screen the cold-adapted and alginate lyases,and predict the key amino acids that may be involved in the reaction according to bioinformatics,providing basic data for the cold-adapted and the bifunctional mechanism of the alginate lyase.The results of this study were as follows:1-Screening the strains producing alginate lyase and the exogenous expression of alginate lyase Alys1.37 strains producing alginate lyase were isolated and distributed in 17 genera of 4 phyla.In addition,five potential new species were screened.Sequence analysis of alginate lyase Alys1 showed that it belonged to subfamily 5 of the PL7 family.It was found that the enzyme activity of rAlysl showed optimal activity at 35℃,higher activity at low temperatures,and that rAlysl has low Tm and poor thermal stability.These results indicated that rAlysl has some characteristics of a cold-adapted enzyme.Moreover,rAlysl could depolymerize alginates,PolyM and PolyG,and produced a monosaccharide and prefers M to G.Therefore,rAlysl is a coldadapted,bifunctional,and exolytic alginate lyase.2.The function analysis of the key amino acid residues that may affect biofunctional and cold-adapted alginate lyase Alys1.The site-directed mutagenesis of alginate lyase Alysl was performed by muting residues on the active center and lid-loops.The substrate preference of mutants E135G,F142G,T78G,and Y273G changed from PolyM to PolyG,indicating that these are key amino acids related to the substrate specificity of rAlys1.Based on the characteristics of cold-adapted enzymes in the paper reported,amino acids that may be related to coldadapted amino acids were mutated,and the temperature properties of mutants P22A and P195G were changed.It is speculated that amino acids P22 and P195 play an important role in the cold-adapted properties of enzymes.This provides basic data for futher elucidating the broad substrate mechanism and cold-adapted mechanism of rAlysl.3.The effects of alginate lyase Alysl and mutants on biofilms.The rates of degrading the biofilms of alginate lyase rAlysl and mutant E135G for P.aeruginosa biofilm were 55.6%and 77.4%,respectively.The rates of inhibition biofilm formation were 55.4%and 63.9%,respectively.Moreover,in the presence of high glucose concentration,mutant E135G still inhibits biofilm formation.The rAlysl and mutant E135G have synergistic or additive effects with antibiotics.These results showed that rAlysl and mutant E135G were pharmaceutical preparation to reduce the drug resistance of P.aeruginosa,and had application prospects in the treatment of refractory P.aeruginosa infection diseases.