尽管已经做出了许多努力来修饰聚对苯二甲酸乙二醇酯(PET)水解酶以提高PET降解效率,这些酶在接近环境温度下的催化性能仍然是一个挑战。在这里,开发了一种多酶级联系统(PT-EC),并通过组装三种成熟的PETase进行了验证,PETaseEHA,快速PETase,和Z1-PETase,分别,与羧酸酯酶TfCa一起,和使用支架蛋白的疏水结合模块CBM3a。由此产生的PT-ECEHA,PT-ECFPE,PT-ECZPE均表现出优异的PET降解功效。值得注意的是,与PETaseEHA相比,PT-ECEHA的产品释放量增加了16.5倍,PT-ECZPE产生的产品数量最高。随后,PT-EC显示在大肠杆菌的表面,分别,并研究了它们对三种PET类型的降解效率。与表面显示的PETaseEHA相比,显示的PT-ECEHA在PET膜的降解效率上提高了20倍。值得注意的是,在一个星期的降解期间,观察到显示的PT-ECZPE的产品释放几乎线性增加,7天后达到11.56±0.64mM。使用基于对接的虚拟筛选策略进化的TfCaI69W/L281Y显示PET降解的产物释放进一步增加2.5倍。总的来说,PT-EC的这些优点证明了多酶级联系统用于PET生物循环的潜力。
Although many efforts have been devoted to the modification of polyethylene terephthalate (PET) hydrolases for improving the efficiency of PET degradation, the catalytic performance of these enzymes at near-ambient temperatures remains a challenge. Herein, a multi-enzyme cascade system (PT-EC) was developed and validated by assembling three well-developed PETases, PETaseEHA, Fast-PETase, and Z1-PETase, respectively, together with carboxylesterase TfCa, and hydrophobic binding module CBM3a using scaffold proteins. The resulting PT-ECEHA, PT-ECFPE, PT-ECZPE all demonstrated outstanding PET degradation efficacy. Notably, PT-ECEHA exhibited a 16.5-fold increase in product release compared to PETaseEHA, and PT-ECZPE yielded the highest amount of product. Subsequently, PT-ECs were displayed on the surface of Escherichia coli, respectively, and their degradation efficiency toward three PET types was investigated. The displayed PT-ECEHA exhibited a 20-fold increase in degradation efficiency with PET film compared to the surface-displayed PETaseEHA. Remarkably, an almost linear increase in product release was observed for the displayed PT-ECZPE over a one-week degradation period, reaching 11.56 ± 0.64 mM after 7 days. TfCaI69W/L281Y evolved using a docking-based virtual screening strategy showed a further 2.5-fold increase in the product release of PET degradation. Collectively, these advantages of PT-EC demonstrated the potential of a multi-enzyme cascade system for PET bio-cycling.