DhaA Based On Arabinogalacatan Modification And Self-assembly And The Study On Its Stability

DhaA can catalyze hydrolytic cleavage of the carbon-chlorine bond of mustard gas,along with production of thiodiglycol,a proton and a chloride ion.In the catalytic process,any toxic product and intermediate product have not been generated with high efficiency,which makes environment-friendly degradation of mustard gas possible.However,the environment tolerance of DhaA is poor,which restricts its application as enzymatic decontaminants.The catalytic activity of DhaA rapidly decreased at high temperature,low pH and hypersaline.In order to solve this problem,we designed two schemes to improve the stability of DhaA in this thesis.The first scheme is chemical modification of DhaA with arabinogalactan(AG).AG is highly branched and rich in hydroxyl moieties,which has the potential to protect proteins.The apparent molecular weight of AG-DhaA is 150 kDa,and the AG/DhaA molar ratio of it is 4.5±1.1.The volume of AG-DhaA reaches 70 times that of DhaA.The modification of AG does not alter the secondary structure of protein,only part ofα-helix is converted into β-sheet.AG-DhaA still remains 91.1%of the catalytic activity of DhaA,and the kinetic parameters are also largely preserved,as the Km of AG-DhaA and DhaA are 1.90±0.44 and 1.64±0.39.Compared with free DhaA,the stability of AG-DhaA has been significantly improved.The half-life at 50℃ is prolonged from 1.17h to 2.01h;After incubation at pH 3.0 for 1h,AG-DhaA can retain 35.2%of its original activity,while DhaA only remains about 10.6%of its original activity;The half-life at 37℃ is prolonged from 27.61h to 56.35h;The half-life at hypersaline solution is prolonged from 12.12h to 47.48h.In order to further improve the stability of DhaA,we developed another scheme based on modification of polysaccharides.Negatively charged at the optimum pH,AG-DhaA can interact with positively charged polymers in aqueous solution and form the nanoparticles by self-assembly.Two positively charged polymers,chitosan(CTS)and polyethyleneimine(PEI)were selected.They interacted with AG-DhaA through the electrostatic force in aqueous solution and formed the nanoparticles.Both PEI and CTS interacted with AG-DhaA and formed stable nanoparticles driven by electrostatic force.The size of PEI-AG-DhaA was 82.97±24.77nm,while the size of CTS-AG-DhaA was 151.36±69.22nm.Compared with AG-DhaA,the enzyme kinetic parameters and catalytic activity of PEI-AG-DhaA and CTS-AG-DhaA particles were slightly altered.The stabilities of PEI-AG-DhaA and CTS-AG-DhaA were improved significantly.The half-life at 50℃ is prolonged to 8.55h and 5.66h;After incubation at pH 3.0 for 1h,PEI-AG-DhaA and CTS-AG-DhaA can retain 35.0%and 33.4%of its original activity,while DhaA only remains 22.8%;After incubation at pH 4.0 for 1h,PEI-AG-DhaA and CTS-AG-DhaA can retain 56.8%and 86.2%of its original activity,while DhaA only remains 36.6%;The half-life at 37℃ is prolonged from 2.51d to 7.39d and 5.38d.PEI-AG-DhaA presents more prominent improvement of thermal stability and storage stability than CTS-AG-DhaA,while CTS-AG-DhaA shows better protection ability at low pH than PEI-AG-DhaA.