Bioadhesion And Biomineralization Inspired Fabrication Of Multienzyme System Construction

In this work, mimicking the multienzyme coexistence approach in mitochondria, organic-inorganic hybrid microcapsules were prepared under mild conditions (room temperature, neutral pH and aqueous solution) through the synergy beyween bioadhesion and biomineralization. Starch was converted into isomaltooligosaccharide(IMOs) byα-amylase,β-amylase, and glucosidase spatially separated immobilized in biomimetic microcapsules. The details in this work were summarized as follows:Firstly, the characteristics ofα-amylase,β-amylase and glucosidase were examined respectively, and appropriate reaction condition of multienzyme catalysis was explored. Considering the reaction rate and enzyme stability, the multienzyme reaction condition was determined as follows: 800KUα-amylase,600Uβ-amylase and 420KU glucosidase were added into every kilogram starch, and the reaciton took place in pH6.0 aqueous solution at 50℃. Under such condition, the yield of IMOs from starch was 39%.Inspired by bioadhesion phenomenon, polydopamine(PDA) microcapsules were prepared through the rapid self-polymerization of dopamine on the surface of CaCO₃(PSS) template under mild condition. The wall thickness of the microcapsules could be tuned by polymerization time and the dopamine concentration in an aqueous solution. The three enzymes were immobilized in the lumen, within the wall and on the out surface of microcapsule under extremely mild conditions, respectively. The yield of IMOs from starch accomplished by the multienzyme system was 53%, the operation stability of the multienzyme system is markly higher than that of free multienzyme system.Inspired by biomineralization and bioadhesion phenomenon, a novel approach of preparation organic-inorganic hybrid material under mild condition was designed. The synthesis of inorganic oxide was induced by biomacromolecule, followed by dopamine self-polymerization on the surface of inorganic oxide. Though this method, protamine-silica-polydopamine (PSi-PDA) microcapsules were prepared for spatially separated multienzyme coimmobilization. Compared with PDA microcapsules and the PSi microcapsules, the mechanical stability of PSi-PDA microcapsules was significantly improved by the synergy of organic layer and inorganic layer. The immobilized multienzyme showed 85% of intial enzyme activity after 8 repeated cycles. The yield of IMOs from starch accomplished by the multienzyme system was 54%.From the phenomenon of metal ions-fortified adhesive system, a novel approach of combining bioadhesion and biomimetic titanification was designed to prepare organic-inorganic hybrid material. Based on this approach, robust protamine-titania-polydopamine (PTi-PDA) hybrid microcapsules were prepared for spatially separated multienzyme coimmobilization. Compared with PSi-PDA microcapsules, the mechanical stability of PTi-PDA microcapsules was further improved due to the complexation between Ti atoms and dopamine. The immobilized multienzyme showed no leakage and no appreciable loss in activity after 20h vigorous stirring. The yield of IMOs from starch accomplished by the multienzyme system was 57%.