Activity Analysis Of Zearalenone Hydrolase Nanoparticles Based On Ferritin

Zearalenone(ZEN)is a non-steroidal estrogen mycotoxin secreted by the fungi of the genus Fusarium.Its molecule contains a macrolide structure.Considered to have side effects,especially in animal reproduction.It is one of the three most polluted mycotoxins in the world.Biological treatment is a promising method to degrade ZEN.Lactose hydrolase is one of the most widely studied ZEN-degrading enzymes.Rm ZHD,a lactitol hydrolase,converts ZEN’s ester bonds to form non-toxic products.Increasing the activity of zearalenone degrading enzymes in the form of discovering new enzymes and point mutations is limited by various conditions,and the increase in activity is relatively low,which cannot meet the current needs of efficient degradation.Macrolide antibiotics,which have the same ester bond structure as ZEN,are named because their structure contains macrolides.They have high inhibitory activity against Gram-positive bacteria and mycoplasma,and are weakly basic lipophilic antibiotics.Macrolide antibiotics are often added to animal feeds as additives,and a large amount of antibiotics that are not absorbed are excreted in the form of prototypes or metabolites,polluting the ecological environment such as soil and water.Compost is often used to remove antibiotic pollution,but it is limited by many conditions,including moisture content,carbon-nitrogen ratio,and ventilation.Simple composting cannot effectively remove antibiotic residues in feces,and the method that can easily and efficiently degrade antibiotics needs further study.Recombinant human ferritin is a biological nanostructure scaffold widely used as a24-mer protein cage.It has self-assembly in vivo and in vitro.Compared with other biological macromolecules,the nano-elements of ferritin are relatively It is simple,and the modification and assembly technology is also relatively mature,and it has the characteristics of connecting functional proteins at the N-terminus without affecting the integrity of the nanoparticles.Due to its unique molecular structure,ferritin has been widely used in the development of biosensors,vaccines and targeted drug delivery systems.Ferritin can withstand high temperatures and a wide range of p H levels for a limited time without causing significant damage to the cage-like quaternary structure formed by 24 subunits.In this study,we applied ferritin-based self-assembly to the construction of Rm ZHD nanoparticle enzymes to improve the catalytic activity of Rm ZHD.Without disrupting ferritin self-assembly,Rm ZHD and its mutant Rm ZHD(Y160A)were fused to the N-terminus of ferritin,and expressed and purified in vitro in an E.coli expression system to obtain high-purity Rm ZHD-r HF and Rm ZHD(Y160A)-r HF protein.The diameters of Rm ZHD-r HF and Rm ZHD(Y160A)-r HF measured by transmission electron microscope are about 12 nm.Using free enzyme as a control,the ability of two nanoparticle enzymes to degrade ZEN was studied,and the results showed that Rm ZHD-r HF and Rm ZHD(Y160A)-r HF increased the hydrolysis efficiency of ZEN by 3.5 times and 4.6 times,respectively.At the same time,the two nanoparticle enzymes have better catalytic ability in high temperature and wide p H range.In addition,we selected Rm ZHD nanoparticle enzymes and studied their ability to degrade two representative macrolide antibiotics,erythromycin and tylosin.The results show that the nanoenzyme Rm ZHD-r HF has a comparative effect on these two antibiotics.High hydrolytic capacity,Kcat is 2.75 times and 3.4 times higher than the free enzymes of erythromycin and tylosin,respectively.After zearalenone degrading enzyme is fused with ferritin,it can self-assemble to form a 24-mer nanoenzyme protein,which can enhance its catalytic activity on the substrate and make the enzyme more resistant to temperature and p H..This method is suitable for the fusion expression of human ferritin and various enzymes,and is used to improve the characteristics of the target enzyme.It can also further increase the enzyme activity by extending the length of the linker peptide.This method has high research value and scientific significance in scientific research and applied basic research.