We report a dual-pathway decarbonization strategy for high-impact polystyrene (HIPS) that integrates renewable bio-rubbers and marine biomass fillers to reduce reliance on fossil-derived components. Poly(butadiene-co-myrcene) copolymers with 20–50 wt% myrcene were synthesized via neodymium-catalyzed coordination polymerization, achieving high cis-1,4 stereoregularity and molecular weights suitable for impact modification. These bio-rubbers were incorporated in situ during styrene polymerization to produce Bio-HIPS with tunable morphology, transitioning from a salami to a core–shell structure as the myrcene content increased. Concurrently, Caribbean Sargassum biomass was chemically treated to remove non-cellulosic components and used as a 20 wt% bio-filler in both commercial and bio-HIPS matrices. Comprehensive characterization revealed that treated Sargassum enhanced matrix-filler adhesion, improving mechanical properties and maintaining processability. Bio-HIPS composites exhibited increased stiffness, preserved damping capacity, and elevated glass transition temperatures compared to commercial counterparts. This work demonstrates a scalable, sustainable approach to producing high-performance, partially bio-sourced HIPS, valorizing marine waste and advancing circular materials design.

We present a systematic study of thermoplastic polypropylene (PP) composites reinforced with wood fibers (WF) derived from Norway spruce industrial residues (FibraQ) as scalable, sustainable alternatives to conventional polymers. The wood fibers retain a characteristic softwood monosaccharide profile and display robust morphological integrity and uniform dispersion across loadings from 20 to 50 wt%. Mechanical characterization demonstrates a linear increase in tensile modulus and strength with increasing WF content, counterbalanced by reduced ductility and impact toughness due to increasing fiber network density. Thermal analyses confirm enhanced stability and elevated Vicat softening temperatures upon WF addition. Importantly, these composites exhibit outstanding closed-loop mechanical recyclability: after three industrially relevant processing cycles, PPWF retains >90% of initial stiffness and >94% tensile strength, significantly outperforming neat PP and previously reported biocomposite systems. Our study provides the first direct quantitative comparison of recyclability and structural retention for industrially relevant PPWF composites. These advances offer a pathway for integrating renewable residues into high-performance, durable, and circular materials platforms beyond the capabilities of conventional polymers.

Carbohydrate-binding modules (CBMs) are non-catalytic proteins found appended to carbohydrate-active enzymes. Soil and marine bacteria secrete such enzymes to scavenge nutrition, and they often use CBMs to improve reaction rates and retention of released sugars. Here we present a structural and functional analysis of the recently established CBM family 92. All proteins analysed bind preferentially to β−1,6-glucans. This contrasts with the diversity of predicted substrates among the enzymes attached to CBM92 domains. We present crystal structures for two proteins, and confirm by mutagenesis that tryptophan residues permit ligand binding at three distinct functional binding sites on each protein. Multivalent CBM families are uncommon, so the establishment and structural characterisation of CBM92 enriches the classification database and will facilitate functional prediction in future projects. We propose that CBM92 proteins may cross-link polysaccharides in nature, and might have use in novel strategies for enzyme immobilisation.

In nature, complex carbohydrates are rarely found as pure isolated polysaccharides. Instead, bacteria in competitive environments are presented with glycans embedded in heterogeneous matrices such as plant or microbial cell walls. Members of the Bacteroidota phylum thrive in such ecosystems because they are efficient at extracting nutrients from complex substrates, secreting consortia of synergistic enzymes to release metabolizable sugars. Carbohydrate-binding modules (CBMs) are used to target enzymes to substrates, enhancing reaction rate and product release. Additionally, genome organizational tools like polysaccharide utilization loci (PULs) ensure that the appropriate set of enzymes is produced when needed. In this study, we show that the soil bacterium Chitinophaga pinensis uses a PUL and several CBMs to coordinate the activities of enzymes targeting two distinct polysaccharides found in fungal cell walls. We describe the enzymatic activities and carbohydrate-binding behaviors of components of the fungal cell wall utilization locus (FCWUL), which uses multiple chitinases and one β-1,3-glucanase to hydrolyze two different substrates. Unusually, one of the chitinases is appended to a β-glucan-binding CBM, implying targeting to a bulk cell wall substrate rather than to the specific polysaccharide being hydrolyzed. Based on our characterization of the PUL’s outer membrane sensor protein, we suggest that the FCWUL is activated by β-1,3-glucans, even though most of its enzymes are chitin-degrading. Our data showcase the complexity of polysaccharide deconstruction in nature and highlight an elegant solution for how multiple different glycans can be accessed using one enzymatic cascade.

In biomass-processing industries there is a need for enzymes that can withstand high temperatures. Extensive research efforts have been dedicated to finding new thermostable enzymes as well as developing new means of stabilising existing enzymes. The attachment of a stable non-catalytic domain to an enzyme can, in some instances, protect a biocatalyst from thermal denaturation. Carbohydrate-binding modules (CBMs) are non-catalytic domains typically found appended to biomass-degrading or modifying enzymes, such as glycoside hydrolases (GHs). Most often, CBMs interact with the same polysaccharide as their enzyme partners, leading to an enhanced reaction rate via the promotion of enzyme-substrate interactions. Contradictory to this general concept, we show an example of a chitin-degrading enzyme from GH family 18 that is appended to two CBM domains from family 92, both of which bind preferentially to the non-substrate polysaccharide β-1,6-glucan. During chitin hydrolysis, the CBMs do not contribute to enzyme-substrate interactions but instead confer a 10–15 °C increase in enzyme thermal stability. We propose that CBM92 domains may have a natural enzyme stabilisation role in some cases, which may be relevant to enzyme design for high-temperature applications in biorefinery.

Use of the 3,5-dinitrosalicylic acid reagent allows the simple, rapid quantification of reducing sugars. The method can be used for analysis of biological samples or in characterization of enzyme reactions, as new reducing ends are generated when a polysaccharide substrate undergoes hydrolytic cleavage. Presented here is an application of the method in measuring the kinetics of a glycoside hydrolase reaction, including the optimization of the DNSA reagent, and the production of a standard curve of absorbance versus sugar concentration.

Use of the 3,5-dinitrosalicylic acid reagent allows the simple, rapid quantification of reducing sugars. The method can be used for analysis of biological samples or in characterization of enzyme reactions, as new reducing ends are generated when a polysaccharide substrate undergoes hydrolytic cleavage. Presented here is an application of the method in measuring the kinetics of a glycoside hydrolase reaction, including the optimization of the DNSA reagent, and the production of a standard curve of absorbance versus sugar concentration.

Glucose 6-phosphate is the phosphorylated form of glucose and is used as a reagent in enzymatic assays. Current production occurs via a multi-step chemical synthesis. In this study we established a fully enzymatic route for the synthesis of glucose 6-phosphate from cellulose. As the enzymatic phosphorylation requires ATP as phosphoryl donor, the use of a cofactor regeneration system is required. We evaluated Escherichia coli glucokinase and Saccharomyces cerevisiae hexokinase (HK) for the phosphorylation reaction and Pseudomonas aeruginosa polyphosphate kinase 2 (PPK2) for ATP regeneration. All three enzymes were characterized in terms of temperature and pH optimum and the effects of substrates and products concentrations on enzymatic activities. After optimization of the conditions, we achieved a 85% conversion of glucose into glucose 6-phosphate using the HK/PPK2 activities within a 24 h reaction resulting in 12.56 g/l of glucose 6-phosphate. Finally, we demonstrated the glucose 6-phosphate formation from microcrystalline cellulose in a one-pot reaction comprising Aspergillus niger cellulase for glucose release and HK/PPK2 activities. We achieved a 77% conversion of released glucose into glucose 6-phosphate, however at the expense of a lower glucose 6-phosphate yield of 1.17 g/l. Overall, our study shows an alternative approach for synthesis of glucose 6-phosphate that can be used to valorize biomass derived cellulose.

The use of enzymes in aqueous solutions of ionic liquids (ILs) could be useful for the enzymatic treatment of lignocellulose. Hydrophilic ILs that dissolve lignocellulose are harmful to enzymes. The toleration limits and enzyme-friendly superbase IL combinations were investigated for the hyperthermophilic Thermopolyspora flexuosa GH10 xylanase (endo-1,4-β-xylanase EC 3.2.1.8) TfXYN10A and Dictyoglomus thermophilum GH11 xylanase DtXYN11B. TfXYN10A was more tolerant than DtXYN11B to acetate or propionate-based ILs. However, when the anion of the ILs was bigger (guaiacolate), GH11 xylanase showed higher tolerance to ILs. 1-Ethyl-3-methylimidazolium acetate ([EMIM]OAc), followed by 1,1,3,3-tetramethylguanidine acetate ([TMGH]OAc), were the most enzyme-friendly ILs for TfXYN10A and [TMGH]+-based ILs were tolerated best by DtXYN11B. Double-ring cations and a large size anion were associated with the strongest enzyme inhibition. Competitive inhibition appears to be a general factor in the reduction of enzyme activity. However, with guaiacolate ILs, the denaturation of proteins may also contribute to the reduction in enzyme activity. Molecular docking with IL cations and anions indicated that the binding mode and shape of the active site affect competitive inhibition, and the co-binding of cations and anions to separate active site positions caused the strongest enzyme inhibition.

Thermopolyspora flexuosa GH11 xylanase (XYN11A) shows optimal activity at pH 6-7 and 75-80 °C. We studied how mutation to aspartic acid (N46D and V48D) in the vicinity of the catalytic acid/base affects the pH activity of highly thermophilic GH11 xylanase. Both mutations shifted the pH activity profile toward acidic pH. In general, the Km values were lower at pH 4-5 than at pH 6, and in line with this, the rate of hydrolysis of xylotetraose was slightly faster at pH 4 than at pH 6. The N46D mutation and also lower pH in XYN11A increased the hydrolysis of xylotriose. The Km value increased remarkably (from 2.5 to 11.6 mg/mL) because of V48D, which indicates the weakening of binding affinity of the substrate to the active site. Xylotetraose functioned well as a substrate for other enzymes, but with lowered reaction rate for V48D. Both N46D and V48D increased the enzyme inactivation by ionic liquid [emim]OAc. In conclusion, the pH activity profile could be shifted to acidic pH due to an effect from two different directions, but the tightly packed GH11 active site can cause steric problems for the mutations.