A dual enzyme-containing microreactor for consecutive digestion based on hydrophilic ZIF-90 with size-selective sheltering

Abstract Recently, new developments of multiple-enzyme catalysis in enzyme scaffold designs have garnered much attention for their important applications. The reactions catalyzed by multiple protease which couldn’t co-exist in solution would greatly facilitate the bottom up strategy for proteome analysis. In this study, a dual-enzyme microreactor with two proteases was successfully constructed for consecutive digestion under mild reaction conditions in aqueous solution based on hydrophilic ZIF-90 with size-selective sheltering, where chymotrypsin was encapsulated into the ZIF-90 framework through a biomimetic mineralization procedure and trypsin was then covalently adsorbed on the outer surface of ZIF-90. With extraordinarily uniform size and high protein loading capacity, the microreactor exhibited enhanced stability (including thermostability, pH stability and storage stability) and better digestion performance compared to in-solution digestion. Thus, the work presents a novel and general strategy for the design of high-performance biomimetic reactors for multienzymatic catalysis by altering the type of enzyme and further develops the great potential of biomacromolecules in catalysis application. Moreover, we also demonstrate that although the biomacromolecules such as proteins couldn’t directly tune crystallinity and morphology of ZIF-90, they can perform as a stabilizer for anchoring metal ion to prevent crystal fast transformation of ZIF-90 during synthetic process.