Exploration Of The Product Specificity Of Chitosanase CsnMY002 And Its Mutant Using Molecular Dynamics Simulations

Infection has been a large problem in clinical medicine until the discovery of vaccines and antibiotics provided us with powerful tools to overcome it.However,long-term abuse of antibiotics has led to many public health problems including drug residues,bacterial resistance,environmental pollution and so on.Therefore,It is an hot target to search for safe and effective natural antimicrobial substances.Chitosan oligosaccharide is a functional biopolymer derived from the partial deacetylation of Chitosan.It is widely used in food,agriculture and other fields because of its good antibacterial effect.At present,making chito-oligosaccharides are mainly divided into three categories: physical method,chemical method and enzymatic method.Compared with physical method and chemical method,enzymatic method has the advantages of mild reaction conditions,simple and safe experimental process,simple final product,non-toxic by-product and low environmental pollution.Chitosanase CsnMY002 is a new type of enzyme substance for the preparation of chitosan oligosaccharide isolated from Bacillus subtilis.In the study of its enzymatic activity characteristics,it was found that chitosanase CsnMY002 had a “3 + 3” symmetric fragmentation mode for Chitosan(Glc N)6,and the two mutants successfully transformed the cleavage mode into “4 + 2” asymmetric cleavage,which could effectively increase the yield of Chitosan and thus increase the commercial value of the final product.While,we do not know how this the cause of the change in cleavage patterns and the conformational changes caused by the mutants.In this study,molecular dynamics simulations were performed to explored the conformational changes the substrates binding to the WT and mutants.The results were as fellow:Firstly,the binding of substrate can activate the active conformation of protein,and stretch and deform the active region and the catalytic region.For example,the wild type Chitosanase CsnMY002,it has an active region that allows rapid recruitment of Chitosan by means of stretching and deformation,when either Chitosan or Chitosan is continuously docking and reacting,at the same time,the tight interaction network was formed rapidly,and Chitosan was degraded by the catalysis of catalytic residue.Secondly,when the mutant CsnMY002 will cause different binding modes and catalysis in the original catalytic residue sites,resulting in different conformational changes in the original degradation sites,therefore,the degree of polymerization of the final chitooligosaccharide degradation product is affected.Thirdly,Arg37,Ile145 Gly148 and Trp204 are important catalytic residues of the Chitosanase CsnMY002.Arg37 can form two stable hydrogen bonds to the-1 site,the results show that the complex structure of Chitosan is more compact,and the Ile145 Gly148 sites are the important binding and catalytic sites,the catalytic degradation of the ?-(1,4)-glycosidic bond between +1 and-1 sites can be carried out in the cooperation of Glu19 sites.The Trp204 site in the ?9 Helix is also an important catalytic degradation site,which can catalyze the degradation of the ?-(1,4)-glycosidic bonds between the +1 and +2 sites in the presence of Lys21 or Arg21.Our study will provide a reliable way to obtain excellent mutant Chitosanase in the future.