Molecular Modification And Domain Function Of Staphyloccccus Aureus Phage Lysin LysDZ25

Staphylococcus aureus is a common pathogenic microorganism and an important nosocomial infection bacterium.With the continuous emergence and spread of resistant and multidrug resistant S.aureus,traditional antibiotic treatment has gradually lost its effectiveness.Therefore,there is an urgent need to develop new and effective biological green inhibitors to control S.aureus.Phage lysin is an enzyme that can digest bacterial cell walls expressed in the late stage of infection with double stranded DNA bacteriophages.It can destroy the peptidoglycan of bacterial cell wall from the outside,leading to bacterial osmotic lysis,thereby achieving a bactericidal effect.Currently,many studies have confirmed that lysins have great bactericidal potential.In this study,we screened a phage lysin from the phages of S.aureus that can efficiently cleave S.aureus,named LysDZ25.We used the Escherichia coli expression system to express and purify LysDZ25,studied its basic enzymatic properties,and found that LysDZ25 has good tolerance to both serum and NaCl solution,so it has good application potential.However,LysDZ25 has poor thermal stability and basically loses its activity when it is kept at 37℃ or above for 20 minutes.In order to improve the cracking activity and thermal stability of LysDZ25,we rationally designed the mutant using the machine learning methods(MDL)independently developed by our laboratory.Through two rounds of point mutation and combined mutation,we obtained the mutant S333V/N245R/D299L with improved activity and thermal stability.The cleavage activity of this mutant protein was 7 folds higher than that of LysDZ25,and its activity hardly decreases at 37℃ for 20 minutes,while its activity remains one-third at 40℃for 20 minutes.At 37℃,the half-life of the mutant protein was increased from 15 min of LysDZ25 to 55-60 min.Using the RoseTTAFold software tool to construct a three-dimensional(3D)structure of LysDZ25,it was found that LysDZ25 contains three structural domains,the N-terminal cysteine and histidine dependent amide hydrolase/peptidase catalytic domain(CHAP),the central amidase catalytic domain(AmiC),and the C-terminal cell wall binding domain(SH3b).The domains are connected by two linkers,L1 and L2,respectively.We explored the function of each domain by constructing fusion proteins of AmiC and SH3b with GFP and domain recombinant proteins.The results show that the CHAP domain plays a major role in LysDZ25,and SH3b plays the function of binding to the cell wall of S.aureus.The ability of AmiC binding to the cell wall of S.aureus is first discovered,and the lysate activity is improved after absence.And through the combination of domains and linkers,We obtain recombinant proteins CHAP+L1+SH3b and CHAP+L2+SH3b with higher cleavage activity,which were 2 and 1.7 folds as active as LysDZ25,respectively.Further truncation of the linkers of the recombinant protein in these two domains revealed that the cleavage activity of the recombinant protein was further improved by truncating 7-8 amino acids from Nterminal to C-terminal.In summary,this article successfully expressed LysDZ25,a phage lysin from S.aureus,using an E.coli expression system,and studied its properties.Through rational design and experimental verification,we have obtained a mutant protein with improved cleavage activity and thermal stability,which will contribute to the application of this enzyme in practical production,and have a positive guiding significance for the molecular modification of phage lysin.In addition,we also split the domain of LysDZ25 and explored the function of each domain.After combining different domains and linkers,we obtained a recombinant protein with higher cleavage activity.This work has positive guiding significance for the molecular modification of phage lyase,and also lays a solid foundation for the development of efficient and novel bioinhibitors of S.aureus.