Study On Inorganic Mineralization Combined With Organic Polymer Network For Protein Stability Improvement

Proteins and other biological products,due to their structural instability,are easily affected by external factors such as temperature,acidity,and organic solvents,leading to the loss of biological activity.This is also an important factor limiting the wider application of biological drugs.This thesis attempted to improve protein stability by constructing mineralized shells using calcium phosphate(CaP)inorganic materials.In addition,the organic protection network formed by chitosan(CS)and sodium alginate(SA)was also studied to promote the improvement of protein stability.By combining the above inorganic mineralization with the organic molecular network,the organicinorganic composite protection system containing chitosan/sodium alginate-calcium phosphate was prepared,providing a new method for the study of protein stability improvement.This thesis first used bovine serum albumin(BSA)as a model protein to study the changes in protein encapsulation efficiency by changing factors such as protein concentration,reaction time,calcium phosphorus ratio,and the addition of Mg2+.The experimental results showed that when the concentration of protein was 1 mg/mL,the ratio of calcium to phosphorus was 10:3,the reaction time was 1 h,and Mg2+ was not added to the system,the coprecipitation of protein molecules and calcium phosphate minerals could be achieved,and the encapsulation efficiency of protein was up to 99%.Further analysis using SEM,XRD,and FTIR revealed that calcium phosphate minerals successfully embedded protein molecules.In addition,protein calcium phosphate complex products could be released under mild conditions,with an average release rate of 70%in a composite system without Mg2+ After heating the composite system at 65℃ for 1 h,the UV absorption spectrum of the composite system remained basically unchanged,indicating that calcium phosphate minerals effectively improved the protein thermal stability.When the system was heated within the range of 25-95℃,the protein activity retention rate of the composite system without Mg2+ was 90%,indicating that the calcium phosphate system had a high-temperature protective effect on BSA.In order to further study the effect of organic polymer system on the stability of protein,chitosan and sodium alginate were used as two polymer systems to investigate the stability of horseradish peroxidase(HRP)and glucose oxidase(GOx).Exploring the changes in the initial catalytic activity of composite enzymes by changing different enzyme reaction concentrations and substrate concentrations,the results showed that when pyrogallic acid was used as a hydrogen donor,the initial catalytic activity of CSHRP increased by 1.5-2.5 times,and SA-HRP increased by 1-1.5 times.The experiment evaluated the influence of organic polymer systems on enzyme stability by analyzing the influence of factors such as temperature,pH value,and organic solvents on enzyme catalytic activity.After heating at 70℃ for 30 min,the relative free enzyme activity of CS-GOx remained at 105%.The results showed that the introduction of polysaccharide could effectively improve the stability of protein.In combination with the above inorganic phosphate and organic molecular network systems,the protection system composed of organic and inorganic systems was prepared through chitosan/sodium alginate and calcium phosphate systems.By changing the addition order of Ca2+/PO43-,calcium phosphorus ratio,and different systems(Mg2+,CS,SA),the results showed that when PO43-was first added to the reaction system with Ca/P=5:6,the encapsulation efficiency of CaP-CS-GOx was as high as 90%.After heating at 90℃ for 30 min,the activities of free GOx,CaP-GOx,and CS-GOx were basically lost,and the residual activity of CaP-CS-GOx relative to free enzyme was 100%,indicating that the combination of polysaccharide and calcium phosphate could promote each other to enhance their protective effects on protein thermal stability.This thesis explored the effectiveness of combining inorganic mineralization and organic molecular networks to improve protein stability.The preparation method was simple and did not damage the original biological activity of proteins,effectively improving protein protection effects,and had important significance in the research of organic inorganic phase composite protection systems.