PDF(Pubmed)
Abstract：
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.
摘要：
具有两种类型的固定化酶的微反应器，表现出优异的正交性能，代表了一种有效的方法来抵消由于缺乏单个酶切割位点而导致的消化效率降低，从而影响蛋白质鉴定。在这项研究中,我们开发了一种亲水性双酶微反应器，其特点是传质迅速，酶活性优异。最初，由于其三维网状孔结构，我们选择KIT-6分子筛作为双IMER的载体。修饰涉及聚乙烯亚胺（PEI）和丙烯酰胺（AM）作为胺供体的共沉积，与多巴胺一起增强材料的亲水性。剩余的氨基和双键官能团促进胰蛋白酶和Glu-C的逐步固定。与基于溶液的消化相比，双IMER上牛血清白蛋白（BSA）和牛血红蛋白（BHb）的消化时间显着减少（1分钟与36h),导致序列覆盖率提高(91.30%vs.BSA为82.7%；90.24%与BHb为89.20%）。此外,双IMER表现出优异的耐用性，29个重复使用周期后保留96.08%的相对活性。蛋白质消化效率的提高可归因于几个因素：（1）KIT-6的大比表面积，实现较高的酶负载能力；(2)其三维网络孔结构，促进更快的传质和物质扩散；（3）胰蛋白酶和Glu-C酶切割位点的正交性；（4）PEI链结构和戊二醛间隔臂引入的空间效应，减少空间障碍，增强酶-底物相互作用；（5）温和稳定的酶固定化。基于KIT-6的双IMER为蛋白质消化提供了一个有前途的技术工具，而PDA/PEI/AM-KIT-6平台具有固定其他蛋白质或活性物质的潜力。
