Ahsan Afrin, Wagner Dominique, Varaljay Vanessa A, Roman Victor, Kelley-Loughnane Nancy, Reuel Nigel F
Department of Chemical and Biological Engineering, Iowa State University, Ames, IA, USA.
Materials and Manufacturing Directorate, Air Force Research Laboratory, Wright-Patterson AFB, OH, USA.
Synth Biol (Oxf). 2024 Feb 13;9(1):ysae005. doi: 10.1093/synbio/ysae005. eCollection 2024.
Cell-free expression (CFE) has shown recent utility in prototyping enzymes for discovery efforts. In this work, CFE is demonstrated as an effective tool to screen putative polyester polyurethane degrading enzyme sequences sourced from metagenomic analysis of biofilms prospected on aircraft and vehicles. An automated fluid handler with a controlled temperature block is used to assemble the numerous 30 µL CFE reactions to provide more consistent results over human assembly. In sum, 13 putative hydrolase enzymes from the biofilm organisms as well as a previously verified, polyester-degrading cutinase were expressed using in-house extract and minimal linear templates. The enzymes were then tested for esterase activity directly in extract using nitrophenyl conjugated substrates, showing highest sensitivity to shorter substrates (4-nitrophenyl hexanoate and 4-nNitrophenyl valerate). This screen identified 10 enzymes with statistically significant activities against these substrates; however, all were lower in measured relative activity, on a CFE volume basis, to the established cutinase control. This approach portends the use of CFE and reporter probes to rapidly prototype, screen and design for synthetic polymer degrading enzymes from environmental consortia. Graphical Abstract.
无细胞表达（CFE）最近已在用于发现工作的酶原型设计中显示出效用。在这项工作中，CFE被证明是一种有效的工具，可用于筛选从飞机和车辆上采集的生物膜宏基因组分析中获得的假定聚酯聚氨酯降解酶序列。使用带有控温模块的自动液体处理器来组装众多30µL的CFE反应，以提供比人工组装更一致的结果。总之，使用内部提取物和最小线性模板表达了来自生物膜生物体的13种假定水解酶以及一种先前已验证的聚酯降解角质酶。然后使用硝基苯基共轭底物直接在提取物中测试这些酶的酯酶活性，结果表明它们对较短的底物（4-硝基苯基己酸酯和4-硝基苯基戊酸酯）具有最高的敏感性。该筛选鉴定出10种对这些底物具有统计学显著活性的酶；然而，以CFE体积为基础，所有这些酶的实测相对活性均低于既定的角质酶对照。这种方法预示着使用CFE和报告探针从环境群落中快速进行合成聚合物降解酶的原型设计、筛选和设计。图形摘要。
