Characterization And Functional Studies Of Lytic Polysaccharide Monooxygenases From Different Sources

Lytic polysaccharide monooxygenases(LPMOs)are new type of single copper oxidase recently discovered.LPMOs enhance the hydrolysis efficiency of glycoside hydrolases(GH)to depolymerize the polysaccharide by catalyzing the oxidative cleavage of recalcitrant polysaccharide.LPMOs are widely distributed and have been reported in bacteria,archaea,fungi and insects.However,comparison of LPMOs derived from microorganisms in different habitats on properties and function is rarely reported.In this paper,the AA10 family LPMO of actinomycetes,gut bacteria and archaea were focused and obtained by heterologous expression,then the comparative analysis of enzymatic properties of three LPMOs were assessed and the enzymatic activity on chtin was discovered.On the basis,the function of LPMOs in chitin degradation by Streptomyces coelicolor was homologous verified.Firstly,three AA10 family LPMO genes from Streptomyces coelicolor(actinomycetes),Enterococcus faecalis(gut bacteria)and Natrialbaceae archaeon(archaea)were isolated.then the three recombinant proteins named ScLPMO10G,Ef LPMO10B and NaLPMO10A,respectively,were obtained in E.coli expression system.Bioinformatics analysis was also carried out,finding the three LPMOs sharing considerable homology with typical AA10 LPMOs in amino acid sequence and protein structure.Secondly,comparative analysis of enzymatic properties of three LPMOs were assessed.All three LPMOs showed oxidation activity on chitin.Sc LPMO10G,Ef LPMO10B,and NaLPMO10A significantly enhanced the hydrolysis efficiency of chitinase,the efficiency was increased by 50.93%,48.74%,50.11%,respectively.The three LPMOs showed differences in enzymatic properties,including optimum reaction temperature,pH and stability.Among which,NaLPMO10A from N.archaeon showed the best stability at high temperature(60 C),high pH(10.0)and high concentration of Na~+(1mol/L),which kept more than 70%of relative activity while incubated in the 1 mol/L of Na~+solution for 8 h.Finally,when Streptomyces coelicolor strains Sc?10E(+)and Sc?10G(+)overexpressing Sc LPMO10E and ScLPMO10G,respectively,as well as the wild-type strain were cultured with chitin as the sole carbon source,the extracellular reducing sugar content,extracellular chitinase activity and the hydrolysis efficiency of extracellular enzymes to crystalline chitin of strains Sc?10E(+)and Sc?10G(+)were significantly higher than those of the wild-type strains,the chitin hydrolysis efficiency was increased by81.79%and 224.44%,respectively.All the results indicated that LPMOs played an important role in the chitin degradation by Streptomyces coelicolor.In this paper,comparative analysis of enzymatic properties of LPMOs derived from Streptomyces coelicolor,Enterococcus faecalis and Natrialbaceae archaeon in different habitats were assessed,the function of LPMOs in chitin degradation by Streptomyces coelicolor was homologous verified.This results provides guidance for the comprehensive utilization of LPMOs as well as the efficient degradation of biomass,and lays a foundation for revealing the biological functions of LPMOs.