Groseclose Thomas M, Kober Erin A, Clark Matilda, Moore Benjamin, Banerjee Shounak, Bemmer Victoria, Beckham Gregg T, Pickford Andrew R, Dale Taraka T, Nguyen Hau B
Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, United States.
BOTTLE Consortium, Golden, Colorado 80401, United States.
ACS Catal. 2024 Sep 17;14(19):14622-14638. doi: 10.1021/acscatal.4c04321. eCollection 2024 Oct 4.
The ability of enzymes to hydrolyze the ubiquitous polyester, poly(ethylene terephthalate) (PET), has enabled the potential for bioindustrial recycling of this waste plastic. To date, many of these PET hydrolases have been engineered for improved catalytic activity and stability, but current screening methods have limitations in screening large libraries, including under high-temperature conditions. Here, we developed a platform that can simultaneously interrogate PET hydrolase libraries of 10-10 variants (per round) for protein solubility, thermostability, and activity via paired, plate-based split green fluorescent protein and model substrate screens. We then applied this platform to improve the performance of a benchmark PET hydrolase, leaf-branch compost cutinase, by directed evolution. Our engineered enzyme exhibited higher catalytic activity relative to the benchmark, LCC-ICCG, on amorphous PET film coupon substrates (∼9.4% crystallinity) in pH-controlled bioreactors at both 65 °C (8.5% higher conversion at 48 h and 38% higher maximum rate, at 2.9% substrate loading) and 68 °C (11.2% higher conversion at 48 h and 43% higher maximum rate, at 16.5% substrate loading), up to 48 h, highlighting the potential of this screening platform to accelerate enzyme development for PET recycling.
酶水解常见聚酯聚对苯二甲酸乙二酯（PET）的能力，为这种废弃塑料的生物工业回收带来了潜力。迄今为止，许多这类PET水解酶已通过工程改造提高了催化活性和稳定性，但目前的筛选方法在筛选大型文库方面存在局限性，包括在高温条件下。在此，我们开发了一个平台，可通过基于平板的成对分裂绿色荧光蛋白和模型底物筛选，同时针对10⁶ - 10¹⁰个变体（每轮）的PET水解酶文库进行蛋白质溶解性、热稳定性和活性的检测。然后，我们应用该平台通过定向进化提高一种基准PET水解酶——叶枝堆肥角质酶的性能。在pH控制的生物反应器中，我们改造后的酶在65℃（在2.9%底物负载下，48小时转化率提高8.5%，最大反应速率提高38%）和68℃（在16.5%底物负载下，48小时转化率提高11.2%，最大反应速率提高43%）下，于无定形PET薄膜试片底物（结晶度约9.4%）上，相对于基准LCC - ICCG表现出更高的催化活性，长达48小时，突出了该筛选平台在加速PET回收酶开发方面的潜力。
