Nanoparticle Enzymes Construction For Improving Escherichia Coli Alkaline Phosphatase Activity

Ferritin is the cellular organelle for free iron storage in animals,plants and microorganisms and plays an important physiological role.Due to its excellence in thermal stability,chemical stability,and special spherical nanostructures,ferritin has been widely used in the researches and applications of biosensors and vaccines.Recombinant heavy chain of human ferritin(rHF)has the property of self-assembly in vitro to form truncated tetramer-like nanoparticles.After forming the tetramer,the C-terminus of rHF is encapsulated in the cavity and the N-terminus is exposed outside the ferritin cavity.This structure provides favorable conditions for the subsequent modification and protein fusion of the ferritin protein shell.As a natural bio-nanomaterial,rHF has relatively simple components,and its modification and assembly techniques have also been built.It is applicable to display functional proteins in the nanocage shell of rHF by means of genetic engineering.This targeted high-aggregation may contribute the functions of proteins in nanoscale.Escherichia coli alkaline phosphatase(EAP)is often used as a model for the study of alkaline phosphatase with its own characteristics.At present,the researches on Escherichia coli alkaline phosphatase have reported its crystal structure and key function of residues.However,the activity of Escherichia coli alkaline phosphatase is much lower than that of bovine intestinal alkaline phosphatase,though its production is convenient and low-costing.Obtaining Escherichia coli alkaline phosphatase with activity equivalent to calf intestinal alkaline phosphatase is one of the main objectives that the industry has been striving to achieve.Although the point mutation for some residues can improve the enymatic activity of EAP to some extent,it is far from being able to meet the current requirements.Based on the previous research foundations of methyl parathion hydrolase and sitagliptin precursor transaminase ATA-117,the laboratory proposed the concept of biomatrix nanozymes.In this study,we mainly evaluated Escherichia coli alkaline phosphatase and the characteristics of ferritin fusion,with a view to clarify the feasibility and effectiveness of bio-matrix nanozymes,and is expected to obtain a biological method that can significantly improve the catalytic activity of the enzyme.We took alkaline phosphatase of Escherichia coli ATCC 25922 as the research object.After obtaining the EAP gene,we performed point mutation on the basis of existing research reports to obtain mutated genes EAP(D101S)and EAP(D101S/K167R/S374C)which produce high enzymatic EAP mutants.These genes were fused to the N-terminus of rHF to be the expression vectors,including pET28a-EAP-rHF,pET28a-EAP(D101S)-rHF and pET28a-EAP(D101S/K167R/S374C)-rHF.These vectors were extensively expressed in Escherichia coli BL21(DE3)pLysS system.After the separation and purification of the proteins,the physical and chemical properties of the nano-proteins were further studied.The study found that the fusion of EAP and its protein mutants to the N-terminus of rHF did not affect the self-assembly of rHF,when the purified protein was observed by transmission electron microscopy,EAP-rHF,EAP(D101S)-rHF and EAP respectively.The EAP(D101S/K167R/S374C)-rHF can form uniformly symmetrical hollow spherical structures,and they have the same enzymatic properties as the EAP that catalyzes the alkaline phosphatase substrate p-nitrophenyl phosphate(PNPP).Analysis of the enzyme kinetics of EAP and its mutant nanozymes using PNPP as a catalytic substrate revealed that the catalytic efficiency of EAP-rHF and EAP(D101S/K167R/S374C)-rHF were about 8 times higher than EAP and EAP(D101S/K167R/S374C);the catalytic efficiency of EAP(D101S)-rHF was 29 times higher than EAP(D101S).The study of the enzymatic activity of EAP nanozymes by physicochemical factors found that: EAP nanozymes have higher tolerance to temperature and pH,but did not change the optimal pH of EAP;nanometer enzymes show greater stability than free enzymes at the same concentration of EDTA treatment conditions.In this experiment,nano-ferritin was used as a carrier matrix to enhance the activity of alkaline phosphatase in Escherichia coli,and the change in EAP activity was also studied by changing the spatial distance between EAP and ferritin.We used EAP-rHF and EAP-(D101S/K167R/S374C)-rHF as research objects.By extending the connecting peptides between genes to change the spatial distance,we chose three different lengths of the connecting peptides,GGGGS(1 linker),GGGGSGGGGSGGGGS(3 linker)and GGGGSGGGGSGGGGSGGGGSGGGGS(5 linker).The experimental results also showed that the enzyme activity of EAP-rHF increased with the increase of the length of the linker peptide;the activity of EAP(D101S/K167R/S374C)-rHF was decreased with the increase of the length of the linker peptide.