Okal Evelyn F, Romero Philip A, Heinzelman Pete
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA.
Department of Biomedical Engineering, Duke University, Durham, NC, USA.
J Biol Eng. 2025 Feb 3;19(1):12. doi: 10.1186/s13036-025-00482-3.
Angiotensin-Converting Enzyme 2 (ACE2) is a crucial peptidase in human peptide hormone signaling, catalyzing the conversion of Angiotensin-II to Angiotensin-(1-7), which activates the Mas receptor and elicits vasodilation, increased blood flow, reduced inflammation, and decreased pathological tissue remodeling. This study leverages protein engineering to enhance ACE2's therapeutic potential for treating conditions such as respiratory viral infections, acute respiratory distress syndrome, and diabetes. Surrogate substrates used in traditional high-throughput screening methods for peptidases often fail to accurately mimic native substrates, leading to less effective enzyme variants. Here, we developed an ultra-high-throughput droplet microfluidic platform to screen peptidases on native peptide substrates. Our assay detects substrate cleavage via free amino acid release, providing a precise measurement of biologically relevant peptidase activity.
Using this new platform, we screened a large library of ACE2 variants, identifying position 187 as a hotspot for enhancing enzyme activity. Further focused screening revealed the K187T variant, which exhibited a fourfold increase in catalytic efficiency (k/K) over wild-type ACE2.
This work demonstrates the potential of droplet microfluidics for therapeutic peptidase engineering, offering a robust and accessible method to optimize enzyme properties for clinical applications.
血管紧张素转换酶2（ACE2）是人类肽激素信号传导中的一种关键肽酶，催化血管紧张素II转化为血管紧张素 - （1 - 7），后者激活Mas受体并引发血管舒张、增加血流量、减轻炎症以及减少病理性组织重塑。本研究利用蛋白质工程来增强ACE2在治疗诸如呼吸道病毒感染、急性呼吸窘迫综合征和糖尿病等病症方面的治疗潜力。传统的肽酶高通量筛选方法中使用的替代底物往往无法准确模拟天然底物，导致产生效果较差的酶变体。在此，我们开发了一种超高通量液滴微流控平台，用于在天然肽底物上筛选肽酶。我们的检测方法通过游离氨基酸释放来检测底物切割，从而精确测量生物学相关的肽酶活性。
使用这个新平台，我们筛选了一个大型的ACE2变体文库，确定第187位是增强酶活性的热点。进一步的重点筛选发现了K187T变体，其催化效率（k/K）比野生型ACE2提高了四倍。
这项工作证明了液滴微流控技术在治疗性肽酶工程中的潜力，为优化酶特性以用于临床应用提供了一种强大且易于使用的方法。
