Yi SangKu, Park Junhyeon, Park Jiyoung, Kim Kyung-Jin, Kim Juhyun
School of Life Science, BK21 FOUR KNU Creative BioResearch Group, Kyungpook National University, Daegu 41566, Republic of Korea.
J Microbiol Biotechnol. 2025 Jul 14;35:e2503044. doi: 10.4014/jmb.2503.03044.
The accumulation of plastic waste poses a significant environmental challenge, necessitating the development of efficient plastic-degrading enzymes for bioremediation and recycling. However, traditional enzyme engineering approaches rely on microbial expression systems and are time-consuming and prone to unintended interactions between host cells and recombinant circuits. To address these limitations, a cell-free protein synthesis (CFPS) platform was developed for rapidly screening plastic-degrading enzymes. Using CFPS, cutinase and PET-degrading enzymes (PETases) were successfully synthesized, and their catalytic activities were assessed using polymer-containing agar plates. Clear degradation halos were observed for cutinase and PETase on polycaprolactone (PCL)-containing and bis (2-hydroxyethyl) terephthalate (BHET)-containing plates, respectively. The optimization of CFPS conditions revealed that enzyme synthesis efficacy was higher at room temperature than at 37°C. The screening of PETase variants (C3 N1377, Mipa-P, and C5 N1251), synthesized using the CFPS platform, demonstrated that the catalytic activity of Mipa-P was the highest and surpassed that of IsPETase. This finding was further validated through purified enzyme analysis. Our results establish CFPS as a rapid, scalable, and cell-free alternative platform for screening and optimizing plastic-degrading enzymes, facilitating advancements in enzymatic plastic recycling.
塑料垃圾的积累对环境构成了重大挑战，因此需要开发高效的塑料降解酶用于生物修复和回收利用。然而，传统的酶工程方法依赖于微生物表达系统，既耗时又容易出现宿主细胞与重组回路之间的意外相互作用。为了解决这些局限性，开发了一种无细胞蛋白质合成（CFPS）平台用于快速筛选塑料降解酶。利用CFPS成功合成了角质酶和聚对苯二甲酸乙二醇酯降解酶（PET酶），并使用含聚合物的琼脂平板评估了它们的催化活性。在含聚己内酯（PCL）的平板和含双（2-羟乙基）对苯二甲酸酯（BHET）的平板上，分别观察到角质酶和PET酶产生了清晰的降解晕圈。CFPS条件的优化表明，酶合成效率在室温下高于37°C。使用CFPS平台合成的PET酶变体（C3 N1377、Mipa-P和C5 N1251）的筛选表明，Mipa-P的催化活性最高，超过了IsPETase。这一发现通过纯化酶分析得到了进一步验证。我们的结果确立了CFPS作为一种快速、可扩展的无细胞替代平台，用于筛选和优化塑料降解酶，推动酶促塑料回收利用的进展。
