Modification Of Amelogenin And Its Regulation On Bioprocess-inspired Synthesis

It's fantastic that nature organisms are capable of creating subtle structures with outstanding functions in mild environment,such as the layered organic-inorganic structure and high toughness of nacre,the multi-scale structure and reversible solid-solid adhesion of gecko setae,and the directional water collection of spider silk.In recent decades,scientists have designed and synthesized bio-inspired structures or bio-inspired functions by studying and mimicking the structures or functions of biomaterials.However,the natural formation processes of these functional structures are also worth studying in developing advanced synthesis techniques for materials.This research field is referred as bioprocess-inspired synthesis and less studied.Owing to the participation of organic matrix,cells and metabolism,biomineralization is a typical biological process for producing organic-inorganic composite biomaterials?e.g.bones,enamel and mollusk shells?with hierarchical structures and superior mechanical properties under ambient temperature and pressure.It has been found that organic matrices especially proteins play fundamental roles in biomineralization,acting as organic templates or nucleation through specific molecular recognition.Tooth enamel is the hardest tissue in vertebrates and composed of 95%hydroxyapatite.Compared to bulk hydroxyapatite mineral,enamel has higher hardness and much better fracture toughness,which make teeth hard to break and resistant to fracture and wear.Amelogenin is the major protein of the developing enamel matrix,and reported as a promoter of mineralization and a modulater of the nanocrystalline hydroxyapatite structure.Amelogenin is digested and exhausted at the enamel maturation stage,it is impossible to extract enough amelogenin from organisms for a series of in-vitro mineralization experiments.In the present work,a recombinant protein his-AmelX based on the amelogenin encoded from X chromosome of murine,was overexpressed and purified in specific one-step heat-treat method,to regulate the mineralization of hydroxyapatite layers at mild temperature.A glutamate tripeptide?EEE?was displayed on the surface of Escherichia coli for synthesizing tin dioxides,which attracted considerable attention due to its various applications in energy and environment fields.First,a recombinant murine amelogenin his-AmelX was constructed and purified using a convenient one-step purification,to provide sufficient protein for the mineralization of hydroxyapatite layers.The mineralization was initiated by enzyme AP,and occurred on L-Cys SAMs.It was demonstrated that his-AmelX was capable of inducing the nucleation and promoting the growth of hydroxyapatite at pH 7.0-7.4.Moreover,with the addition of his-AmelX,a much denser layer of hydroxyapatite was obtained at pH 7.2 in contrast to the sample without protein,attributed to the self-assembly of his-AmelX.The present study not only offers benefits in understanding the natural process of biomineralization,but also opens a feasible way to produce advanced materials with bioprocess inspiration.Second,Amelogenin was divided into two domains and displayed on the outer surface of Escherichia coli cells respectively,by fusing them with an outer membrane protein named ice nucleation protein?INP?.Amelogenin-modified bacteria cells were used in in-vitro mineralization experiments,to further explore the function of each Amelogenin fragment.The results showed that,the C-terminal fragment of Amelogenin played a key role in inducing nucleation and accelerating growth of hydroxyapatite,whereas,the N-terminal fragment would inhibit the nucleation and growth instead.Moreover,the mineral coating on cell outer surface could protect bacteria from ultraviolet radiation efficiently.Finally,we developed a bioprocess-inspired synthesis technique to fabricate the hierarchical SnO₂ nanorods assembled by ultrasmall nanoparticles.Briefly,the bacterial was genetically modified to have its surface displayed with a glutamate tripeptide EEE that can induce the deposition of tin presursors on cell surface.After calcination,the bacterial surface not only provided a confined environment for controlling the monodisperse of particle size,but also acted as the framework for maintaining the hierarchical structure of SnO₂.Carbon coating was achieved by annealing mineralized bacteria in argon atmosphere.The carbon coated SnO₂composite exhibited good lithium storage properties as an anode electrode,which is benefited from ultrasmall particles,carbon coating and mesoporous structure.