Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Building 68-370A, Cambridge, MA, USA.
Department of Biological Engineering, Massachusetts Institute of Technology, 500 Technology Square, NE47-140, Cambridge, MA, USA.
Sci Rep. 2021 Jan 13;11(1):928. doi: 10.1038/s41598-020-79031-5.
Increased interest in poly(ethylene terephthalate) (PET)-degrading enzymes (PETases) have generated efforts to find mutants with improved catalytic activity and thermostability. Here, we present a simple and fast method to determine relative enzyme kinetics through bulk absorbance measurements of released products over time. A thermostable variant of PETase from Ideonella sakaiensis was engineered (R280A S121E D186H N233C S282C) with a denaturation temperature of 69.4 ± 0.3 °C. This was used to assess the method's ability to determine relative enzyme kinetics across variants and reveal structure-function relationships. Measurements at 24 and 72 h at 400 nM of enzyme suggest that the mutations improved catalytic rates 5- to 7-fold. On the contrary, kinetic analyses of the thermostable variant and wild-type reveal different reaction trajectories despite similar maximum catalytic rates, resulting in higher product accumulation from the thermostable variant over time. The results of the assay support the necessity for kinetic measurements to determine relationships between sequence and function for IsPETase and other PET hydrolases.
人们对聚对苯二甲酸乙二醇酯（PET）降解酶（PETases）越来越感兴趣，这促使人们努力寻找具有更高催化活性和热稳定性的突变体。在这里，我们提出了一种简单而快速的方法，通过随时间释放产物的体相吸光度测量来确定相对酶动力学。我们对来自解淀粉芽孢杆菌的耐热型 PETase 进行了工程改造（R280A S121E D186H N233C S282C），其变性温度为 69.4±0.3°C。该变体用于评估该方法在变体间测定相对酶动力学的能力，并揭示结构-功能关系。在 400nM 酶浓度下 24 小时和 72 小时的测量表明，这些突变使催化速率提高了 5-7 倍。然而，对耐热变体和野生型的动力学分析表明，尽管最大催化速率相似，但反应轨迹不同，导致耐热变体随时间产生更高的产物积累。该测定结果支持了在 IsPETase 和其他 PET 水解酶中，需要进行动力学测量以确定序列与功能之间关系的必要性。
