Study On The Mechanism Of Action Of Lytic Polysaccharide Monooxygenases CxLPMOA From Arthrobotrys Sp.CX1

With the rapid development of the global economy,fossil energy on the planet is nearly exhausted,and it is imminent to solve the energy crisis.Renewable resources have attracted people's attention.Lignocellulose is the most abundant renewable resource on the planet,but its conversion has always been Issues of great concern.With the rise of molecular biology and proteomics,it provides a new way of degradation.In the early stage of this project,a lytic polysaccharide monooxygenases(LPMOs)protein,named cxLPMOA,was excavated from the data of the differential protein transcriptome of a cellulose-degrading bacteria Arthrobotrys sp.CX1.Through bioinformatics analysis,cxLPMOA contains two domains,namely the N-terminal glycoside hydrolase 61 family catalytic domain and the C-terminal carbohydrate-binding modules(CBM).In order to study its mechanism of action,the cxLPMOA holoenzyme gene and its N-terminal glycoside hydrolase 61 family catalytic domain cxLPMOA-core gene were recombinantly constructed and expressed to construct recombinant plasmids p PICZ?A-cxLPMOA,p PICZ?A-cxLPMOA-core,and then imported In Pichia pastoris X-33,recombinant bacteria p PICZ?A-cxLPMOA/X-33 and p PICZ?A-cxLPMOA-core/X-33 were obtained and heterologously expressed.Purified by Ni column,pure cxLPMOA and cxLPMOA-core enzymes were obtained and their corresponding enzymatic properties were studied.It was found that cxLPMOA produced cellotriose,cellotetraose and a small amount of cellopentose after acting on substrate PASC.The optimum temperature of cxLPMOA and cxLPMOA-core is 60 ?,and the optimum p H value is about 5,which has high thermophilicity.However,when the CBM structure is lacking,the temperature stability and p H stability of cxLPMOA-core both decrease,and the enzyme activity decreases rapidly when the temperature exceeds 60 ° C,and the enzyme activity is basically lost when the p H is 3.Under the condition of adding appropriate concentration of metal ions,the activity of cxLPMOA-core lacking CBM structure is higher than that of cxLPMOA.Through software,it was found that the 19 th and 103 th histidine in cxLPMOA protein were the substrate catalytic sites.The two site amino acids were mutated to arginine and alanine by site-directed mutagenesis technology,respectively constructed and named 19H-A /X-33,19H-R / X-33,103H-A / X-33,103H-R / X-33 four mutant strains were expressed and purified.The enzyme activity of the mutant protein was measured.From the results,we can know that after the enzyme activity measurement,no enzyme activity was detected.Finally,we analyzed that CBM is one of the factors that affect the mechanism of action of cxLPMOA,and key amino acid sites are also factors that affect its mechanism of action.It laid the foundation for its further research.