Cellulosomal expansin: functionality and incorporation into the complex

Additional file 1: Figure S1. Cellulases GH48 and GH9 work in a synergistic manner. The recombinant putative C. clariflavum exoglucanase GH48 and endoglucanase GH9 were used for degradation of PASC (phosphoric acid-swollen cellulose) alone or combined. Reaction tubes were supplemented with 0.5 µM of each enzyme, or 1 µM in total of the two enzymes combined. The duration of the reaction was 3 h, and the level of cellulose degradation was assessed by measuring the amount of released reducing sugars. The combination of the two enzymes resulted in 1.29-fold enhancement of PASC degradation. Synergy was calculated by summation of the released reducing sugars from the degradation by each enzyme alone, and comparing it to the amount of released reducing sugars by the action of the two enzymes together

MOESM2 of Cellulosomal expansin: functionality and incorporation into the complex

Additional file 2: Figure S2. Impact of CclEXL1 is diminished at high cellulosome concentrations. Microcrystalline cellulose (Avicel) degradation was performed using the cellulosome fractions of C. clariflavum, MCCI (A), MCCII (B) and the combination of MCCI and MCCII (C), at a final concentration of 50 µg/mL with the addition of 0.5 µM expansin (full shapes) or without (empty shapes). Samples were taken at 24-h intervals, and the amount of released reducing sugars was assessed. The CclEXL1-mediated enhancement of cellulose hydrolysis that was demonstrated for low cellulosome concentrations (25 µg/mL) was not observed for higher concentrations. Standard deviations are indicated

Colocalization and Disposition of Cellulosomes in Clostridium clariflavum as Revealed by Correlative Superresolution Imaging

Cellulosomes are multienzyme complexes produced by anaerobic, cellulolytic bacteria for highly efficient breakdown of plant cell wall polysaccharides. Clostridium clariflavum is an anaerobic, thermophilic bacterium that produces the largest assembled cellulosome complex in nature to date, comprising three types of scaffoldins: a primary scaffoldin, ScaA; an adaptor scaffoldin, ScaB; and a cell surface anchoring scaffoldin, ScaC. This complex can contain 160 polysaccharide-degrading enzymes. In previous studies, we proposed potential types of cellulosome assemblies in C. clariflavum and demonstrated that these complexes are released into the extracellular medium. In the present study, we explored the disposition of the highly structured, four-tiered cell-anchored cellulosome complex of this bacterium. Four separate, integral cellulosome components were subjected to immunolabeling: ScaA, ScaB, ScaC, and the cellulosome’s most prominent enzyme, GH48. Imaging of the cells by correlating scanning electron microscopy and three-dimensional (3D) superresolution fluorescence microscopy revealed that some of the protuberance-like structures on the cell surface represent cellulosomes and that the components are highly colocalized and organized by a defined hierarchy on the cell surface. The display of the cellulosome on the cell surface was found to differ between cells grown on soluble or insoluble substrates. Cell growth on microcrystalline cellulose and wheat straw exhibited dramatic enhancement in the amount of cellulosomes displayed on the bacterial cell surface

On the distinct binding modes of expansin and carbohydrate-binding module proteins on crystalline and nanofibrous cellulose: implications for cellulose degradation by designer cellulosomes

Transformation of cellulose into monosaccharides can be achieved by hydrolysis of the cellulose chains, carried out by a special group of enzymes known as cellulases. The enzymatic mechanism of cellulases is well described, but the role of non-enzymatic components of the cellulose-degradation machinery is still poorly understood, and difficult to measure using experiments alone. In this study, we use a comprehensive set of atomistic molecular dynamics simulations to probe the molecular details of binding of the family-3a carbohydrate-binding module (CBM3a) and the bacterial expansin protein (EXLX1) to a range of cellulose substrates. Our results suggest that CBM3a behaves in a similar way on both crystalline and amorphous cellulose, whereas binding of the dual-domain expansin protein depends on the substrate crystallinity, and we relate our computed binding modes to the experimentally measured features of CBM and expansin action on cellulose