Study On The Nanoproteases Of LaCoO₃,Zn-doped TiO₂ And ZnCO₂O₄

In proteomics and biotechnology,the selective cleavage of amide bonds is crucial for the determination of protein structures.However,peptide bonds are very stable,with a half-life of more than 600 years in the natural environment.Natural trypsin can selectively hydrolyze peptide bonds,but it has problems such as high cost,sensitivity to pH,temperature and surfactant,and short length of hydrolyzed peptides.Nanoproteases can overcome the shortcomings of natural proteases,which have the advantages of high stability,low price,and large-scale preparation.Most of the existing nanoproteases have a weak proteolytic activity,which can only catalyse peptide-bond hydrolysis only at high temperature(60 ℃),and the reaction time is relatively long.Therefore,it is necessary to develop highly efficient and reusable nanoproteases with high protease activity at room temperature.Most of the reported nanoproteases contain quadrivalent metal ions(MⅣ),in which the amide carbonyl carbon of peptide bonds are nucleophilically attacked by MⅣ-bound hydroxyl(MⅣ-OH),resulting in peptide bond breakage.In natural carboxypeptidase A,the amide carbonyl carbon of peptide bonds are nucleophilically attacked by Zn2+-bound hydroxyl(Zn2+-OH).Low-valent Zn2+has lower positive charge than MⅣ,therefore,Zn2+-OH has stronger nucleophilicity than MⅣ-OH,which may be the main reason for the high catalytic efficiency of carboxypeptidase A.It is speculated that nanomaterials containing lowvalent metal ions may have strong proteolytic activity.In this paper,several high activity nanoproteases were screened from nanomaterials containing low-valent metal ions,and their interaction with proteins,the effect of buffer solution on hydrolysis,recyclability,hydrolysis mechanism,and the effect of doping of other metal ions on the protease activity were studied.The content of this thesis includes the following three parts:Part 1:Eight low-valent metal oxide nanoparticles were screened,and only perovskite LaCoO3 nanoparticles(LaCoO3 NPs)selectively hydrolyze bovine serum albumin(BSA)under weakly acidic conditions at 20℃,producing fragments of 10-45 kDa,which were suitable for middle-down proteomics.The effect of pH and concentration of phosphate buffer solution(PB)on hydrolysis was studied,and it was found that BSA could be hydrolyzed by LaCoO3 NPs under pH 4.0-6.0 conditions,and the buffer concentration had no effect on the hydrolysis of BSA.Zeta potential data showed that the adsorption capacity of BSA was positively correlated with protease activity.The desorption process of peptides from the nanoparticle surface is a ratecontrolling step.LaCoO3 NPs have good stability and reusability,and their hydrolytic activity remains unchanged and their structure remains stable after 5 cycles of catalytic hydrolysis of BSA.LaCoO3 NPs can also hydrolyze heat-denatured and surfactantdenatured BSA.Initial analysis of mechanism indicated that the high Lewis acidity of La3+ and the low-valent of Co2+played key roles in the high protease activity of LaCoO3 NPs.In addition,y-Globulins from bovine blood can be also hydrolyzed by LaCoO3 NPs.Findings provide a new effective strategy to design and discover highly efficient nanoproteases from low-valent metal oxide nanoparticles.Part 2:Zn,Co,and Ni-doped TiO2 NPs were synthesized and found that only Zndoped TiO2 NPs could significantly improve the protease activity of TiO2 NPs.The effect of different Zn doping ratios and the pH of PB buffer solution on hydrolysis was studied,and it was found that the hydrolysis activity of 4.0-10.0 mol%Zn-doped TiO2 NPs was significantly enhanced compared with that of pure TiO2 NPs under pH 4.0-7.0 conditions.The perovskite ZnTiO3 NPs containing with both Zn and Ti elements had low activity,indicating that the high catalytic activity of Zn-doped TiO2 NPs was not only related to the low-valent Zn element,but also affected by the catalyst structure and Zn content.Comparing the hydrolysis activity with adsorption capacity of several titanium oxides,it was found that the Ti valence state only affected the protein adsorption capacity.XPS data showed that the doping of Zn atoms led to a decrease in the electron density of Ti and an increase in the electron density of Zn,which was conducive to the nucleophilic attack of Zn2+-OH on the Ti4+polarized carbonyl C atom,and made the peptide bond break easier.The above results indicated that the doping of 4.0-10.0 mol%Zn atoms was beneficial to improve the protease activity of TiO2 NPs(pH4.0-7.0).Part 3:We investigated the protease activities of ZnCo2O4 NPs and ZIF-67 and found that they are potential nanoproteases.It was found by SDS-PAGE that ZnCo2O4 NPs had significant protease activity under acidic conditions,and BSA could be selectively hydrolyzed by ZIF-67 under neutral conditions.The problems that they are unstable and unrecyclable need further study.