Claire Dumon

Cellulosomes are multi‐enzyme assemblies whose catalytic efficiency depends on the spatial organization of their components. However, their pronounced conformational flexibility has precluded quantitative characterization of inter‐enzyme distances and overall topology. Here, we present a methodological framework to tailor and analyze the architecture of constrained multi‐enzyme complexes composed of two endoglucanases At Cel8A and At Cel9R and one xylanase At Xyn11A by fixing enzyme positions using the Jo‐In scaffold. This approach enables generation of defined assemblies structurally characterized by small‐angle X‐ray scattering (SAXS) and/or atomic force microscopy (AFM). SAXS analysis of the two‐glucanase complexes reveals distinct scattering profiles corresponding to different degrees of compaction depending on the enzyme spatial arrangement. Complementary AFM imaging of bifunctional assemblies supports SAXS derived models at the single‐particle level, reinforcing the robustness of the proposed workflow. The three‐enzyme assemblies' SAXS measurements distinguish different constructions while showing relatively homogeneous radii of gyration (53 ± 2 Å) and maximum dimensions of 180 to 200 Å. Atomistic modeling using two independent approaches, DADIMODO and BILBO‐MD, converges toward consistent average spatial organizations with constrained interdomain distance ranges, and consistent quantitative parameters validating the structural models. Altogether, the results establish a quantitative framework for designing tailored multi‐enzyme assemblies. Jo‐In scaffold provides a versatile tool for structural analysis of modular, dynamic protein complexes, advancing our understanding of structure–function relationship in multi‐enzyme systems.

Microscale thermophoresis (MST) is an emerging technology for studying a broad range of biomolecular interactions with a high sensitivity. The affinity constant can be obtained for a wide range of molecules within minutes based on reactions in microliters. Here we describe the application of MST in quantifying protein–carbohydrate interactions. A CBM3a and a CBM4 are titrated with insoluble substrate (cellulose nanocrystal) and soluble oligosaccharide (xylohexaose), respectively. Key words Microscale thermophoresis (MST) Binding studies Fluorescence quenching Fluorescent label K_d (dissociation constant)

Valorizing plant cell wall, marine and algal polysaccharides is of utmost importance for the development of the circular bioeconomy. This is because polysaccharides are by far the most abundant organic molecules found in nature with complex chemical structures that require a large set of enzymes for their degradation. Microorganisms produce polysaccharide-specific enzymes that act in synergy when performing hydrolysis. Although discovered since decades enzyme synergy is still poorly understood at the molecular level and thus it is difficult to harness and optimize. In the last few years, more attention has been given to improve and characterize enzyme synergy for polysaccharide valorization. In this review, we summarize literature to provide an overview of the different type of synergy involving carbohydrate modifying enzymes and the recent advances in the field exemplified by plant cell-wall degradation.

This paper aims to investigate the effect of extrusion at high solid loading on corn stover (CS) properties and its enzymatic hydrolysis. This biomass was extruded under different screw speeds and different solid loadings and the impact of these parameters on the physicochemical properties was evaluated. It was found that lignocellulosic components were not significantly affected by the pretreatment, while the surface area increased with solid loading and rotation speed. Different enzyme cocktails were used for the enzymatic hydrolysis of extruded and untreated CS. Overall, mild twin‐extrusion enhanced the enzymatic hydrolysis of CS through an increase in glucose and xylose yields by 134–212% and 214–294%, respectively, when using Trichoderma longibrachiatum cellulase. The highest sugar content was obtained from CS extruded under 400 g total solids per liter and 200 rpm. The energy efficiency of the pretreatment was also assessed and was found to be maximal at 400 g total solids per liter and 200 rpm. © 2022 Society of Chemical Industry and John Wiley & Sons, Ltd.

With the growing need for renewable sources of energy, the interest for enzymes capable of biomass degradation has been increasing. In this paper, we consider two different xylanases from the GH-11 family: the particularly active GH-11 xylanase from Neocallimastix patriciarum, NpXyn11A, and the hyper-thermostable mutant of the environmentally isolated GH-11 xylanase, EvXyn11^TS. Our aim is to identify the molecular determinants underlying the enhanced capacities of these two enzymes to ultimately graft the abilities of one on the other. Molecular dynamics simulations of the respective free-enzymes and enzyme–xylohexaose complexes were carried out at temperatures of 300, 340, and 500 K. An in-depth analysis of these MD simulations showed how differences in dynamics influence the activity and stability of these two enzymes and allowed us to study and understand in greater depth the molecular and structural basis of these two systems. In light of the results presented in this paper, the thumb region and the larger substrate binding cleft of NpXyn11A seem to play a major role on the activity of this enzyme. Its lower thermal stability may instead be caused by the higher flexibility of certain regions located further from the active site. Regions such as the N-ter, the loops located in the fingers region, the palm loop, and the helix loop seem to be less stable than in the hyper-thermostable EvXyn11^TS. By identifying molecular regions that are critical for the stability of these enzymes, this study allowed us to identify promising targets for engineering GH-11 xylanases. Eventually, we identify NpXyn11A as the ideal host for grafting the thermostabilizing traits of EvXyn11^TS.

Xylan is the major hemicellulosic polysaccharide in cereals and contributes to the recalcitrance of the plant cell wall toward degradation. Bacteroidetes , one of the main phyla in rumen and human gut microbiota, have been shown to encode polysaccharide utilization loci dedicated to the degradation of xylan. Here, we present the biochemical characterization of a xylanase encoded by a bacteroidetes strain isolated from the termite gut metagenome.

Ionic liquids (ILs) are organic salts molten at room temperature that can be used for a wide variety of applications. Many ILs, such as 1-ethyl-3-methylimidazolium acetate ([C₂C₁Im][OAc]), have been shown to remove a significant fraction of the complex biopolymer lignin from biomass during pretreatment. Valorizing lignin via biological pathways (e.g., enzymes) holds promise but is limited by the low biocompatibility of many ILs used for pretreatment. The discovery of thermostable enzymes and the application of enzyme engineering techniques have yielded biocatalysts capable of withstanding high concentrations of ILs. Converting lignin from a waste product to value-added chemicals is vital to the success of future cellulosic biorefineries. To that end, we screened the activity of the lignolytic enzyme laccase from a hyperthermophilic bacterium (Thermus thermophilus) in aqueous [C₂C₁Im][OAc]. Despite the thermophilicity (T_opt > 90°C) of this laccase, significant activity loss (>50%) was observed in only 2% (w/v) [C₂C₁Im][OAc]. Kinetics studies show that the IL can bind to the free enzyme and the enzyme-substrate complex. Docking simulations suggest that the cation favors binding to a region close to the active site. We then used a rational design strategy to improve the activity of the laccase in [C₂C₁Im][OAc]. A total of 8 single amino acid mutations were made; however, there were no significant improvements in the activity of the mutants in [C₂C₁Im][OAc] compared to the wild type. The results of this study shed light on the complex nature of enzyme-IL interactions and the challenges faced when designing a biological lignin valorization strategy.

Synergism between enzymes is of crucial importance in cell metabolism. This synergism occurs often through a spatial organisation favouring proximity and substrate channelling. In this context, we developed a strategy for evaluating the impact of the geometry between two enzymes involved in nature in the recycling of the carbon derived from plant cell wall polymers. By using an innovative covalent association process using two protein fragments, Jo and In, we produced two bi-modular chimeric complexes connecting a xylanase and a xylosidase, involved in the deconstruction of xylose-based plant cell wall polymer. We first show that the intrinsic activity of the individual enzymes was preserved. Small Angle X-rays Scattering (SAXS) analysis of the complexes highlighted two different spatial organisations in solution, affecting both the distance between the enzymes (53 Å and 28 Å) and the distance between the catalytic pockets (94 Å and 75 Å). Reducing sugar and HPAEC-PAD analysis revealed different behaviour regarding the hydrolysis of Beechwood xylan. After 24 h of hydrolysis, one complex was able to release a higher amount of reducing sugar compare to the free enzymes (i.e., 15,640 and 14,549 µM of equivalent xylose, respectively). However, more interestingly, the two complexes were able to release variable percentages of xylooligosaccharides compared to the free enzymes. The structure of the complexes revealed some putative steric hindrance, which impacted both enzymatic efficiency and the product profile. This report shows that controlling the spatial geometry between two enzymes would help to better investigate synergism effect within complex multi-enzymatic machinery and control the final product.