Enzymatic Properties And Active Sites Of Chitosanase Of Bacillus Nakamurai

Chitosan is a natural cationic polymer with unique functional properties obtained by deacetylation of chitin,which is formed by linking D-glucosamine through ?-1,4-glycosidic bonds.Studies have shown that chitosan has a wide range of applications in every field of life,but because of its large molecular weight,chitosan is not easily soluble in water and can only be dissolved in certain acidic solutions,which seriously hinders the application value of chitosan.Chitosan hydrolyzed chitosan to produce low molecular weight chitosan oligosaccharides has a wide range of applications.The construction of chitosanase with high enzyme activity plays an important role in expanding chitosan in industrial,pharmaceutical and cosmetic applications.This experiment initially explored the enzymatic properties and active binding sites of Bacillus nakamurai chitosanase.The main contents and results are as follows:According to the amino acid sequence of Bacillus nakamurai chitosanase provided by the NCBI website GeneBank database?accession number: WP₀61527864.1?,the DNA sequence of the appropriate E.coli encoding gene was designed by codon optimization,and the restriction was added at both ends of the gene sequence.The dicer BamH I and restriction endonuclease Sall I restriction sites were sent to the company for synthesis of the gene.The fusion expression vector pGEX-4T-3 was ligated with the synthetic chitosanase gene by DNA T4 ligase and transformed into E.coli BL.-pGEX-4T-ChiA.The Bacillus nakamurai chitosanase was analyzed by bioinformatics software.The results showed that the chitosanase gene was 834 bp in length and encoded 278 amino acids.The encoded protein belongs to the GH46 family glycoside hydrolase family.The isoelectric point is 8.87 and the molecular weight is about 31.22 KDa.Secondary structure predictions revealed that the enzyme consisted of 16 alpha helices,5 beta sheets and some irregularly coiled shapes.Predicting the tertiary structure of chitosanase indicates that the main structure of the chitosanase is dumbbell-shaped,with two domains,upper and lower,connected by a piece of helix,one of which has a ?-fold and some irregular curls.The structure extends outwardly at the bottom of the structure.The expression of the recombinant protein was induced by the addition of IPTG,and a band of approximately 31 kDa was detected by SDS-PAGE electrophoresis.In order to increase the protein expression,the IPTG concentration,induction temperature and induction time were optimized.The results showed that the optimal protein expression conditions were obtained at 30 °C,0.07mmoL/L IPTG and induction for 6h.Then,it was purified by GST affinity chromatography,and the purified chitosanase pure enzyme solution concentration was 10.932 ?g/mL.The chitosan with deacetylation degree greater than 90% was used as the substrate,and the enzyme activity of the recombinant chitosanase was determined by DNS enzyme activity.The enzymatic properties were studied.The results showed that the optimum temperature for enzymatic reaction was 37 °C,and the residual enzyme activity was above 90% after treatment at 37 °C for one hour.The enzyme activity decreased sharply after treatment at 50 °C for 20 minutes,indicating that the enzyme had better temperature at normal temperature.Stability;the optimum reaction pH is 4.5,and the remaining enzyme activity in the range of 4⁵.5 pH is still more than 80%,indicating that the enzyme has good activity and stability under slightly acidic environment.The activity of chitosanase was completely inhibited in the enzymatic reaction of Ag+ and Hg+ ions;Cu2+,Zn2+,Ni+ and Fe3+ partially inhibited the activity of chitosanase in enzymatic reaction;Mn2+ and Ca2+ have an activation effect on the enzyme activity of chitosanase.K+,Na+ and EDTA had no significant effect on the enzymatic activity of chitosanase.Using chitosan as a substrate,the chitosan and chitosanase were docked by Autodock software to analyze the binding sites between substrate and chitosanase,and the best molecular docking conformation was determined according to the lowest free binding energy.Binding site.The results showed that ARG73,ASP71,THE236,ASP238,PHE54,SER243,GIU239,LEV69 amino acids were the optimal binding active sites.The amino acids involved in the active site were further mutated,followed by kinetic simulation of the complex by the Gromacs technique.The conformational change of the composite is judged by analyzing parameters such as the complex RMSD,the radius of gyration Rg,and the number of hydrogen bonds.The results showed that the mutation of ASP71 had a great influence on the conformation of the complex,which was not conducive to the binding of substrate and enzyme.