Hoekstra Matthew, Smith Myron L
Institute of Biochemistry, Faculty of Science, Main Campus, Carleton University, Ottawa, ON K1S 5B6, Canada.
Department of Biology, Faculty of Science, Main Campus, Carleton University, Ottawa, ON K1S 5B6, Canada.
Polymers (Basel). 2023 May 24;15(11):2439. doi: 10.3390/polym15112439.
We present a spectrophotometric-based assay to identify enzymes that degrade commercially available bioplastics. Bioplastics comprise aliphatic polyesters with hydrolysis-susceptible ester bonds and are proposed as a replacement for petroleum-based plastics that accumulate in the environment. Unfortunately, many bioplastics can also persist in environments including seawater and waste centers. Our assay involves an overnight incubation of candidate enzyme(s) with plastic, followed by A610 spectrophotometry using 96-well plates to quantify both a reduction in residual plastic and the liberation of degradation by-products. We use the assay to show that Proteinase K and PLA depolymerase, two enzymes that were previously shown to degrade pure polylactic acid plastic, promote a 20-30% breakdown of commercial bioplastic during overnight incubation. We validate our assay and confirm the degradation potential of these enzymes with commercial bioplastic using established mass-loss and scanning electron microscopy methods. We show how the assay can be used to optimize parameters (temperature, co-factors, etc.) to enhance the enzyme-mediated degradation of bioplastics. The assay endpoint products can be coupled with nuclear magnetic resonance (NMR) or other analytical methods to infer the mode of enzymatic activity. Overall, the screening capacity of the spectrophotometric-based assay was demonstrated to be an accurate method to identify bioplastic-degrading enzymes.
我们提出了一种基于分光光度法的检测方法，用于鉴定能够降解市售生物塑料的酶。生物塑料由具有易水解酯键的脂肪族聚酯组成，被提议作为在环境中积累的石油基塑料的替代品。不幸的是，许多生物塑料在包括海水和垃圾处理中心在内的环境中也会持续存在。我们的检测方法包括将候选酶与塑料进行过夜孵育，然后使用96孔板通过A610分光光度法来量化残留塑料的减少以及降解副产物的释放。我们使用该检测方法表明，蛋白酶K和聚乳酸解聚酶这两种先前已被证明能降解纯聚乳酸塑料的酶，在过夜孵育期间可使商业生物塑料分解20 - 30%。我们使用既定的质量损失和扫描电子显微镜方法验证了我们的检测方法，并确认了这些酶对商业生物塑料的降解潜力。我们展示了该检测方法如何用于优化参数（温度、辅助因子等）以增强酶介导的生物塑料降解。该检测方法的终点产物可与核磁共振（NMR）或其他分析方法相结合，以推断酶促活性的模式。总体而言，基于分光光度法的检测方法的筛选能力被证明是一种鉴定生物塑料降解酶的准确方法。
