The biocatalytic degradation of poly(ethylene terephthalate) (PET) through enzymatic methods has garnered considerable attention due to its environmentally friendly and non-polluting nature, as well as its high specificity. While previous efforts in enhancing IsPETase performance have focused on amino acid substitutions in protein engineering, we introduced an amino acid insertion strategy in this work. By inserting a negatively charged acidic amino acid, Glu, at the right-angle bend of IsPETase, the binding capability between the enzyme\'s active pocket and PET was improved. The resulted mutant IsPETase9394insE exhibited enhanced hydrolytic activity towards PET at various temperatures ranging from 30 to 45 ℃ compared with the wild-type IsPETase. Notably, a 10.04-fold increase was observed at 45 ℃. To further enhance PET hydrolysis, different carbohydrate-binding modules (CBMs) were incorporated at the C-terminus of IsPETase9394insE. Among these, the fusion of CBM from Verrucosispora sioxanthis exhibited the highest enhancement, resulting in a 1.82-fold increase in PET hydrolytic activity at 37 ℃ compared with the IsPETase9394insE. Finally, the engineered variant was successfully employed for the degradation of polyester filter cloth, demonstrating its promising hydrolytic capacity. In conclusion, this research presents an alternative enzyme engineering strategy for modifying PETases and enriches the pool of potential candidates for industrial PET degradation.

The important role of Carbohydrate-binding module (CBM) in the cellulases catalytic activity has been widely studied. CBM3 showed highest affinity for cellulose substrate with 84.69 % adsorption rate among CBM1, CBM2, CBM3, and CBM4 in this study. How CBM affect the catalytic properties of GH5 endoglucanase III from Trichoderma viride (TvEG3) was systematically explored from two perspectives: the deletion of its own CBM(TvEG3dc) and the replacement of high substrate affinity CBM3 (TvEG3dcCBM3). Compared with TvEG3, TvEG3dc lost its binding ability on Avicel and filter paper, but its catalytic activity did not change significantly. The binding ability and catalytic activity of TvEG3dcCBM3 to Avicel increased 348.3 % and 372.51 % than that of TvEG3, respectively. The binding ability and catalytic activity of TvEG3dcCBM3 to filter paper decreased 51.7 % and 33.33 % than that of TvEG3, respectively. Further structural analysis of TvEG3, TvEG3dc, and TvEG3dcCBM3 revealed no changes in the positions and secondary structures of the key amino acids. These results demonstrated that its own CBM1 of TvEG3 did not affect its catalytic activity center, so it had no effect on its catalytic activity. But CBM3 changed the adsorption affinity for different substrates, which resulted in a change in the catalytic activity of the substrate.

Alginate is a commercially important polysaccharide widely distributed in brown algae. Carbohydrate-binding modules (CBMs), a class of commonly used polysaccharide-binding proteins, have greatly facilitated the investigations of polysaccharides. Few alginate-binding CBMs have been hitherto reported and structurally characterized. Herein, an unknown domain from a potential PL6 family alginate lyase in the marine bacterium Vibrio breoganii was discovered and recombinantly expressed. The obtained protein, designated VbCBM106, displayed the favorable specificity to alginate. The unique sequence and well-defined function of VbCBM106 reveal a new CBM family (CBM106). Moreover, the structure of VbCBM106 was determined at a 1.5 Å resolution by the X-ray crystallography, which shows a typical β-sandwich fold comprised of two antiparallel β-sheets. Site-directed mutagenesis assays confirmed that positively charged polar residues are crucial for the ligand binding of VbCBM106. The discovery of VbCBM106 enriches the toolbox of alginate-binding proteins, and the elucidation of critical residues would guide the future practical applications of VbCBM106.

Enzyme-driven recycling of PET has now become a fully developed industrial process. With the right pre-treatment, PET can be completely depolymerized within workable timeframes. This has been realized due to extensive research conducted over the past decade, resulting in a large set of engineered PET hydrolases. Among various engineering strategies to enhance PET hydrolases, fusion with binding domains has been used to tune affinity and boost activity of the enzymes. While fusion enzymes have demonstrated higher activity in many cases, these results are primarily observed under conditions that would not be economically viable at scale. Furthermore, the wide variation in PET substrates, conditions, and combinations of PET hydrolases and binding domains complicates direct comparisons. Here, we present a self-consistent and thorough analysis of two leading PET hydrolases, LCCICCG and PHL7. Both enzymes were evaluated both without and with a substrate-binding domain across a range of industrially relevant PET substrates. We demonstrate that the presence of a substrate-binding module does not significantly affect the affinity of LCCICCG and PHL7 for PET. However, significant differences exist in how the fusion enzymes act on different PET substrates and solid substrate loading, ranging from a 3-fold increase in activity to a 6-fold decrease. These findings could inform the tailoring of enzyme choice to different industrial scenarios.

Broad-acting antiviral strategies to prevent respiratory tract infections are urgently required. Emerging or re-emerging viral diseases caused by new or genetic variants of viruses such as influenza viruses (IFVs), respiratory syncytial viruses (RSVs), human rhinoviruses (HRVs), parainfluenza viruses (PIVs) or coronaviruses (CoVs), pose a severe threat to human health, particularly in the very young or old, or in those with pre-existing respiratory conditions such as asthma or chronic obstructive pulmonary disease (COPD). Although vaccines remain a key component in controlling and preventing viral infections, they are unable to provide broad-spectrum protection against recurring seasonal infections or newly emerging threats. HEX17 (aka Neumifil), is a first-in-class protein-based antiviral prophylactic for respiratory viral infections. HEX17 consists of a hexavalent carbohydrate-binding module (CBM) with high affinity to sialic acids, which are typically present on terminating branches of glycans on viral cellular receptors. This allows HEX17 to block virus engagement of host receptors and inhibit infection of a wide range of viral pathogens and their variants with reduced risk of antiviral resistance. As described herein, HEX17 has demonstrated broad-spectrum efficacy against respiratory viral pathogens including IFV, RSV, CoV and HRV in multiple in vivo and in vitro studies. In addition, HEX17 can be easily administered via an intranasal spray and is currently undergoing clinical trials.

The widespread utilization of polyethylene terephthalate (PET) has caused a variety of environmental and health problems. Compared with traditional thermomechanical or chemical PET cycling, the biodegradation of PET may offer a more feasible solution. Though the PETase from Ideonalla sakaiensis (IsPETase) displays interesting PET degrading performance under mild conditions; the relatively low thermal stability of IsPETase limits its practical application. In this study, enzyme-catalysed PET degradation was investigated with the promising IsPETase mutant HotPETase (HP). On this basis, a carbohydrate-binding module from Bacillus anthracis (BaCBM) was fused to the C-terminus of HP to construct the PETase mutant (HLCB) for increased PET degradation. Furthermore, to effectively improve PET accessibility and PET-degrading activity, the truncated outer membrane hybrid protein (FadL) was used to expose PETase and BaCBM on the surface of E. coli (BL21with) to develop regenerable whole-cell biocatalysts (D-HLCB). Results showed that, among the tested small-molecular weight ester compounds (p-nitrophenyl phosphate (pNPP), p-Nitrophenyl acetate (pNPA), 4-Nitrophenyl butyrate (pNPB)), PETase displayed the highest hydrolysing activity against pNPP. HP displayed the highest catalytic activity (1.94 μM(p-NP)/min) at 50 °C and increased longevity at 40 °C. The fused BaCBM could clearly improve the catalytic performance of PETase by increasing the optimal reaction temperature and improving the thermostability. When HLCB was used for PET degradation, the yield of monomeric products (255.7 μM) was ∼25.5 % greater than that obtained after 50 h of HP-catalysed PET degradation. Moreover, the highest yield of monomeric products from the D-HLCB-mediated system reached 1.03 mM. The whole-cell catalyst D-HLCB displayed good reusability and stability and could maintain more than 54.6 % of its initial activity for nine cycles. Finally, molecular docking simulations were utilized to investigate the binding mechanism and the reaction mechanism of HLCB, which may provide theoretical evidence to further increase the PET-degrading activities of PETases through rational design. The proposed strategy and developed variants show potential for achieving complete biodegradation of PET under mild conditions.

Chondroitin sulfate (CS) is the predominant glycosaminoglycan within the human body and is widely applied in various industries. Carbohydrate-binding modules (CBMs) possessing the capacity for carbohydrate recognition are verified to be important tools for polysaccharide investigation. Only one CS-specific CBM, PhCBM100, has hitherto been characterized. In the present study, two CBM96 domains present in the same putative PL8_3 chondroitin AC lyase were discovered and recombinantly expressed. The results of microtiter plate assays and affinity gel electrophoresis assays showed that the two corresponding proteins, DmCBM96-1 and DmCBM96-2, bind specifically to CSs. The crystal structure of DmCBM96-1 was determined at a 2.20 Å resolution. It adopts a β-sandwich fold comprising two antiparallel β-sheets, showing structural similarities to TM6-N4, which is the founding member of the CBM96 family. Site mutagenesis analysis revealed that the residues of Arg27, Lys45, Tyr51, Arg53, and Arg157 are critical for CS binding. The characterization of the two CBM96 proteins demonstrates the diverse ligand specificity of the CBM96 family and provides promising tools for CS investigation.

Chondroitinases play important roles in structural and functional studies of chondroitin sulfates. Carbohydrate-binding module (CBM) is generally considered as an accessory module in carbohydrate-active enzymes, which promotes the association of the appended enzyme with the substrate and potentiates the catalytic activity. However, the role of natural CBM in chondroitinases has not been investigated. Herein, a novel chondroitinase ChABC29So containing an unknown domain with a predicted β-sandwich fold was discovered from Segatella oris. Recombinant ChABC29So showed enzyme activity towards chondroitin sulfates and hyaluronic acid and acted in a random endo-acting manner. The unknown domain exhibited a chondroitin sulfate-binding capacity and was identified as a CBM. Biochemical characterization of ChABC29So and the CBM-truncated enzyme revealed that the CBM enhances the catalytic activity, thermostability, and disaccharide proportion in the final enzymatic products of ChABC29So. These findings demonstrate the role of the natural CBM in a chondroitinase and will guide future modification of chondroitinases.

Extensive decoration of backbones is a major factor resulting in resistance of enzymatic conversion in hemicellulose and other branched polysaccharides. Employing debranching enzymes is the main strategy to overcome this kind of recalcitrance at present. A carbohydrate-binding module (CBM) is a contiguous amino acid sequence that can promote the binding of enzymes to various carbohydrates, thereby facilitating enzymatic hydrolysis. According to previous studies, CBMs can be classified into four types based on their preference in ligand type, where Type III and IV CBMs prefer to branched polysaccharides than the linear and thus are able to specifically enhance the hydrolysis of substrates containing side chains. With a role in dominating the hydrolysis of branched substrates, Type III and IV CBMs could represent a non-catalytic approach in overcoming side-chain recalcitrance.

Porphyran is a favorable functional polysaccharide widely distributed in Porphyra. It displays a linear structure majorly constituted by alternating 1,4-linked α-l-galactopyranose-6-sulfate (L6S) and 1,3-linked β-d-galactopyranose (G) units. Carbohydrate-binding modules (CBMs) are desired tools for the investigation and application of polysaccharides, including in situ visualization, on site and specific assay, and functionalization of biomaterials. However, only one porphyran-binding CBM has been hitherto reported, and its structural knowledge is lacking. Herein, a novel CBM16 family domain from a marine bacterium Aquimarina sp. BL5 was discovered and expressed. The recombinant protein AmCBM16 exhibited the desired specificity for porphyran. Bio-layer interferometry assay revealed that the protein binds to porphyran tetrasaccharide (L6S-G)2 with an association constant of 1.3 × 103 M-1. The structure of AmCBM16 was resolved by the X-ray crystallography, which displays a β-sandwich fold with two antiparallel β-sheets constituted by 10 β-strands. Site-directed mutagenesis analysis demonstrated that the residues Gly-30, Trp-31, Lys-88, Lys-123, Phe-125, and Phe-127 play dominant roles in AmCBM16 binding. This study provides the first structural insights into porphyran-binding CBM.