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.

One strategy to decrease both the consumption of crude oil and environmental damage is through the production of bioethanol from biomass. Cellulolytic enzyme stability and enzymatic hydrolysis play important roles in the bioethanol process. However, the gradually increased ethanol concentration often reduces enzyme activity and leads to inactivation, thereby limiting the final ethanol yield. Herein, we employed an optimized Two-Gene Recombination Process (2GenReP) approach to evolve the exemplary cellulase CBHI for practical bioethanol fermentation. Two all-round CBHI variants (named as R2 and R4) were obtained with simultaneously improved ethanol resistance, organic solvent inhibitor tolerance, and enzymolysis stability in simultaneous saccharification and fermentation (SSF). Notably, CBHI R4 had a 7.0-to 34.5-fold enhanced catalytic efficiency (k cat /K M ) in the presence/absence of ethanol. Employing the evolved CBHI R2 and R4 in the 1G bioethanol process resulted in up to 10.27% (6.7 g/L) improved ethanol yield (ethanol concentration) than non-cellulase, which was far more beyond than other optimization strategies. Besides bioenergy fields, this transferable protein engineering routine holds the potential to generate all-round enzymes that meet the requirement in biotransformation and bioenergy fields.

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.

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