The extensive application of polyethylene terephthalate (PET) has led to severe environmental pollution. Enzymatic degradation provides a sustainable solution for PET recycling, but the limited hydrolytic activity of current PET hydrolases hinders its practical application. Here, we designed an Energy-Guided Accumulate Mutation Strategy (EGAMS) for the rational design of PET enzyme PHL7. By applying multiple rounds of evolution to enhance the degradation activity and thermostability of PHL7, we achieved the excellent PET degradation enzyme FlashPETase (PHL7 E148K/T158P/S184E/H185Y ) with 238.8% PET degradation activity of PHL7 and 82.93 °C melting temperature. FlashPETase completely degrades PET at 72 °C with an enzyme loading of 1.5 mg enzyme g PET −1 , without any substrate pretreatment or additional processing. As for PET substrates with high crystallinity, FlashPETase still performs well. Furthermore, molecular simulations revealed the structural mechanisms of FlashPETase’s enhanced activity and stability. In summary, the EGAMS protocol and the variants discussed in this work advance the fields of PET degradation and enzyme engineering, offering valuable insights for future research.
Mangrove ecosystems are critical for coastal protection and biodiversity but are increasingly threatened by plastic pollution, particularly polyethylene terephthalate (PET). In this study, a novel marine bacterium, strain SCSIO 80796T, was isolated from PET debris collected from the mangrove in QiAo Island, Zhuhai, China. Using a polyphasic taxonomic approach, including 16S rRNA gene sequencing, genome-based comparisons (average nucleotide identity [ANI] 72.2%, digital DNA-DNA hybridization [dDDH] 19.0%, average amino acid identity [AAI] 70.6%), and phenotypic and chemotaxonomic analyses, the strain was classified as a novel species within the genus Neptunicella. It is proposed as Neptunicella plasticusilytica sp. nov. (type strain SCSIO 80796ᵀ = MCCC 1K08369T = KCTC 92826T). Genomic analysis revealed that strain SCSIO 80796ᵀ encodes a novel PET-degrading enzyme, NmCut, which degrades PET and yields 105–120 µM of degradation products [terephthalic acid (TPA), mono(2-hydroxyethyl) terephthalate (MHET), bis(2-hydroxyethyl) terephthalate (BHET)] within 48 h at 60°C. NmCut exhibits both structural and evolutionary novelty, featuring a unique PET-binding module (PBM) absent in known PETases. PBM is characterized by a long, positively charged α-helix enriched in aromatic residues, forming a distinct substrate-interacting surface with potential as a transferable domain to enhance the efficiency of other plastic-degrading enzymes. This study not only expands the known diversity within Neptunicella but also highlights the potential of marine-derived microbes in addressing plastic pollution through biotechnological applications.
危险品化学品经营许可证（带存储）