The microreactor with two types of immobilized enzymes, exhibiting excellent orthogonal performance, represents an effective approach to counteract the reduced digestion efficiency resulting from the absence of a single enzyme cleavage site, thereby impacting protein identification. In this study, we developed a hydrophilic dual-enzyme microreactor characterized by rapid mass transfer and superior enzymatic activity. Initially, we selected KIT-6 molecular sieve as the carrier for the dual-IMER due to its three-dimensional network pore structure. Modification involved co-deposition of polyethyleneimine (PEI) and acrylamide (AM) as amine donors, along with dopamine to enhance material hydrophilicity. Remaining amino and double bond functional groups facilitated stepwise immobilization of trypsin and Glu-C. Digestion times for bovine serum albumin (BSA) and bovine hemoglobin (BHb) on the dual-IMER were significantly reduced compared to solution-based digestion (1 min vs. 36 h), resulting in improved sequence coverage (91.30% vs. 82.7% for BSA; 90.24% vs. 89.20% for BHb). Additionally, the dual-IMER demonstrated excellent durability, retaining 96.08% relative activity after 29 reuse cycles. Enhanced protein digestion efficiency can be attributed to several factors: (1) KIT-6\'s large specific surface area, enabling higher enzyme loading capacity; (2) Its three-dimensional network pore structure, facilitating faster mass transfer and substance diffusion; (3) Orthogonality of trypsin and Glu-C enzyme cleavage sites; (4) The spatial effect introduced by the chain structure of PEI and glutaraldehyde\'s spacing arm, reducing spatial hindrance and enhancing enzyme-substrate interactions; (5) Mild and stable enzyme immobilization. The KIT-6-based dual-IMER offers a promising technical tool for protein digestion, while the PDA/PEI/AM-KIT-6 platform holds potential for immobilizing other proteins or active substances.

To solve the problems of separating dual enzymes from the carriers of dual-enzyme immobilized micro-systems and greatly increase the carriers\' recycling times, photothermal-responsive micro-systems of IR780-doped cobalt ferrite nanoparticles@poly(ethylene glycol) microgels (CFNPs-IR780@MGs) are prepared. A novel two-step recycling strategy is proposed based on the CFNPs-IR780@MGs. First, the dual enzymes and the carriers are separated from the reaction system as a whole via magnetic separation. Second, the dual enzymes and the carriers are separated through photothermal-responsive dual-enzyme release so that the carriers can be reused. Results show that CFNPs-IR780@MGs is 281.4 ± 9.6 nm with a shell of 58.2 nm, and the low critical solution temperature is 42 °C, and the photothermal conversion efficiency increases from 14.04% to 58.41% by doping 1.6% of IR780 into the CFNPs-IR780 clusters. The dual-enzyme immobilized micro-systems and the carriers are recycled 12 and 72 times, respectively, and the enzyme activity remains above 70%. The micro-systems can realize whole recycling of the dual enzymes and carriers and further recycling of the carriers, thus providing a simple and convenient recycling method for dual-enzyme immobilized micro-systems. The findings reveal the micro-systems\' important application potential in biological detection and industrial production.

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.