| Biomimetic mineralization, which utilizes biological molecules or synthetic analogues to template and catalyze the formation of inorganic oxides under mild conditions, provides a versatile new technology for enzyme immobilization with several inherent advantages. The morphology of the inorganic materials can be controlled by the template self-assembly. The process of LbL self-assembly is conducted in aqueous solution under mild conditions, which can well preserve the enzymes native structures and activities. Both of the biomimetic mineralization and self-assembly process can create benign microenvironment for the immobilized enzyme. This study discusses biomimetic and self-assembly approach for the formation of titania-based materials and their potential application in the preparation of enzyme carriers.The details in this study were summarized as follows:Firstly, protamine was employed for the first time in vitro to induce the formation of a titania nanoparticle from a water-stable titanium precursor, titanium(IV) bis(ammonium lactato) dihydroxide (Ti-BALDH). The resulting titania nanoparticle was extensively characterized and the titania nanoparticle was of amorphous structure. The size of titania nanoparticle can be controlled by the self-assembly of protamine. The catalyzing and templating function of protamine involved in the synthesis of the nanoparticle composite was discussed, and a mechanism was tentatively proposed.Secondly, the biomimetic approach was employed for the entrapment of yeast alcohol dehydrogenase (YADH). Protamine was employed as the template and catalyst for the condensation of Ti-BALDH into titania nanoparticles to encapsulate YADH simultaneously. The mechanism of YADH encapsulation through co-precipitation approach was tentatively proposed. The preliminary investigation showed that encapsulated YADH could retain more than 95% of its initial activity. Compared to free YADH, encapsulated YADH exhibited significantly improved thermal, pH, recycling and storage stability.Thirdly, a novel approach combining biomimetic mineralization with layer-by-layer (LbL) self-assembly was proposed to prepare protamine–titiania hybrid microcapsules. More specifically, these microcapsules were fabricated by alternative deposition of positively charged protamine layers and negatively charged titania layers on the surface of CaCO3 microparticles, followed by dissolution of the CaCO3 microparticles using EDTA. The mechanism involved in the preparation of the microcapsules was clarified. Moreover, these protamine-titania hybrid microcapsules were first employed as the carrier for the immobilization of YADH. The encapsulation efficiency was more than 68% and the encapsulated YADH displayed enhanced recycling stability.Fourthly, a microfactory with Shells-in-Shell structure was prepared for multienzymes immobilization. The Alg-Pro-Ti capsules were loaded with LbL self-assembly microcapsules. The Alg-Pro-Ti capsules worked as structural shell and the LbL microcapsules worked as"workshop". By encapsulating different enzyme-containing LbL microcapsules within Alg-Pro-Ti capsules, an enzymatic'assembly line'could be created to alter the incoming molecules or'manufacture'the product by immobilizing multiple enzymes, the reaction probabilities and efficiencies would be considerably enhanced owing to spatial confinement of the biochemical machinery.Finally, based on biomimetic titanification, protamine and lysozyme were for the first time demonstrated to catalyze the hydrolysis/condensation of the precursor potassium hexafluorozirconate (K2ZrF6) and template the formation of amorphous zirconia particles at room temperature. The catalyzing and templating function of lysozyme involved in the synthesis of zirconia particles was analyzed, and the hydrolysis mechanism of K2ZrF6 was tentatively discussed. In addition, the zirconia was utilized as the carrier for the immobilization of YADH, which displayed enhanced thermal and pH stability, compared to free YADH.
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