Although considerable research achievements have been made to address the plastic crisis using enzymes, their applications are limited due to incomplete degradation and low efficiency. Herein, we report the identification and subsequent engineering of BHETases, which have the potential to improve the efficiency of PET recycling and upcycling. Two BHETases (ChryBHETase and BsEst) are identified from the environment via enzyme mining. Subsequently, mechanism-guided barrier engineering is employed to yield two robust and thermostable ΔBHETases with up to 3.5-fold enhanced kcat/KM than wild-type, followed by atomic resolution understanding. Coupling ΔBHETase into a two-enzyme system overcomes the challenge of heterogeneous product formation and results in up to 7.0-fold improved TPA production than seven state-of-the-art PET hydrolases, under the conditions used here. Finally, we employ a ΔBHETase-joined tandem chemical-enzymatic approach to valorize 21 commercial post-consumed plastics into virgin PET and an example chemical (p-phthaloyl chloride) for achieving the closed-loop PET recycling and open-loop PET upcycling.
Obtaining a robust and applicable enzyme for bioethanol production is a dream for biorefinery engineers. Herein, we describe a general method to evolve an all-round and interpretable enzyme that can be directly employed in the bioethanol industry. By integrating the transferable protein evolution strategy In-SiReP 2.0 (In Silico guided Recombination Process), enzymatic characterization for actual production, and computational molecular understanding, the model cellulase PvCel5A (endoglucanase II Cel5A from Penicillium verruculosum) was successfully evolved to overcome the remaining challenges of low ethanol and temperature tolerance, which primarily limited biomass transformation and bioethanol yield. Remarkably, application of the PvCel5A variants in both first-and secondgeneration bioethanol production processes (i. Conventional corn ethanol fermentation combined with the in situ pretreatment process; ii. cellulosic ethanol fermentation process) resulted in a 5.7-10.1 % increase in the ethanol yield, which was unlikely to be achieved by other optimization techniques.
The safe and efficient stabilization and preservation
of enzymes,
therapeutic proteins, and biomaterials are increasingly important
in biology, medicine, and pharmaceutics. Various protectants have
been explored, but their protective actions on protein structures
remain unclear. Herein, we present an all-around protectant–protein
interaction landscape by investigating the behaviors of Bacillus
subtilis lipase A (BSLA), cellobiohydrolase I from Trichoderma reesei (CBHI), and endoglucanase from Penicillium Verruculosum (EG) in various concentrations
of glycerol. Surprisingly, decreased, neutralization, and activation
effects were observed for three industrial enzymes during the long-term
storage examination, respectively, demonstrating that a universal
condition for protein preservation might not exist. Alignment of the
experimental catalytic activity profiles and computational molecular
dynamics simulation reveals that (1) the overall structure of enzymes
in glycerol remains stable; (2) specific activity reduction mainly
results from three factors: (a) increased structural compactness,
(b) overall water stripping, and (c) competitive inhibition by glycerol
in the substrate binding site; (3) H-bond interactions are the main
driving force that governs the structural dynamics, water stripping,
and glycerol accumulation in glycerol cosolvents. Also, these gained
insights are most likely to be transferred to other polyol additive
systems for rationally stabilizing and preserving biomaterials in
structural biology, biocatalysis, and biotransformation fields.
Phosphorene, a novel member of the two-dimensional nanomaterial family, has been demonstrated a great potential in biomedical applications, such as photothermal therapy, drug delivery and antibacterial. However, phosphorene is unstable...
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