Core Insights - The article discusses a significant advancement in enzyme engineering achieved through collaboration between YuanTian Bio and Tianjin University, focusing on a new strategy called Rationality-informed Protein Evolution (RIPE) that enhances the catalytic activity of a PET hydrolase enzyme by 6.91 times, setting a new record in enzyme performance [2][3][43]. Group 1: Research Background - Polyethylene terephthalate (PET) waste poses severe environmental challenges, and while enzymatic degradation offers advantages over traditional chemical methods, the inherent catalytic activity of PET hydrolases has been insufficient for industrial needs [3][6]. - The RIPE strategy was developed to improve the efficiency of the EVOLVEpro framework, which integrates protein language models and machine learning for protein evolution, addressing the weak correlation between evolutionary information and enzyme activity [3][6][7]. Group 2: RIPE Strategy Overview - The RIPE strategy consists of a two-stage process that begins with rationally designed mutants as an initial dataset for EVOLVEpro, enhancing the robustness of the evolutionary path [3][7]. - The optimal variant IYLL (F208I/H183Y/Q142L/S27L) was developed, showing 6.91 times higher hydrolytic activity against amorphous PET and 2.58 times higher against post-consumer PET compared to the wild-type enzyme [3][7]. Group 3: Experimental Methods - The study involved cloning the BhrPETase gene into a vector, constructing various mutants through PCR, and purifying the proteins using Ni-NTA affinity chromatography [8][9]. - The hydrolytic activity was measured under optimized conditions, and the kinetic parameters were analyzed using the Michaelis-Menten equation [18][20]. Group 4: Enzyme Properties and Performance - The optimal reaction temperature for IYLL was found to be 72°C, with a pH stability range of 7.5-10.0, demonstrating excellent performance in various conditions [28][30]. - IYLL exhibited significantly improved kinetic parameters, including a kcat of 131.73 nmol g⁻¹ s⁻¹, which is 3.81 times higher than the wild-type [30][31]. Group 5: Comparative Analysis - IYLL's degradation performance was superior to ten other reported high-activity PET hydrolases, achieving complete degradation of both amorphous and post-consumer PET within 12 hours under optimized conditions [34][36]. - The variant maintained high product tolerance and stability, outperforming wild-type and other variants in various experimental setups [34][36]. Group 6: Molecular Mechanism - The enhanced activity of IYLL is attributed to a combination of structural stabilization, increased substrate affinity, and reduced catalytic energy barriers, as demonstrated through molecular dynamics simulations [39][41][43]. - The study highlights the synergistic effects of the four mutations in optimizing the enzyme's catalytic microenvironment and reaction pathway, leading to a significant performance boost [39][41].