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This means that, despite the removal of polysaccharides during saccharification, diffusion of probes was not improved since they became hindered by changes in lignin conformation, whose relative amount increased over time. Probes’ diffusion was mainly affected by probes size and pretreatments but only slightly by saccharification time. In addition to chemical composition and porosity analyses, the diffusion of polyethylene glycol probes of different sizes was measured at three different time points during the saccharification. To address this issue, poplar fragments were submitted to hot water and ionic liquid pretreatments selected for their contrasted effects on both the structure and composition of lignocellulose. Although many chemical features are considered as detrimental to saccharification, enzymes’ dynamics within the cell walls remains poorly explored and understood. Improving lignocellulolytic enzymes’ diffusion and accessibility to their substrate in the plant cell walls is recognised as a critical issue for optimising saccharification. This growing interest may eventually lead to applications involving SBSs in industrial and biomedical settings as SBSs provide an interesting way to modulate enzymatic behavior without the need to alter the often highly sensitive active site of the enzyme. Despite a relatively long history, it is only in recent years that SBSs have been studied in great detail as researchers have developed strategies for identifying and characterising these sites, using techniques that measure their binding properties as well as looking at the influence on enzymatic activity of altering these sites through mutagenesis. Although SBSs share many roles with CBMs they may not simply be an alternative to CBMs, but rather complementary as SBSs and CBMs frequently co-occur in enzymes. The roles attributed to SBSs are not limited to targeting the enzyme to the substrate, but also include a variety of others such as guiding the substrate into the active site, altering enzyme specificity and acting as an allosteric site. SBSs have been identified in enzymes from a wide variety of families, though almost half are found in the α-amylase family GH13. Such a site is termed a surface (or secondary) binding site (SBS). In some cases an alternative to possessing a CBM is for the enzyme to bind to the substrate at a site on the catalytic domain, but away from the active site. However, the presence of CBMs is not universal and is in fact rare among some families of enzymes.
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The results clearly demonstrate that both substrate hydrolysis and substrate targeting are key factors for XBS mobility.Ĭarbohydrate active enzymes, particularly those that are active on polysaccharides, are often found associated with carbohydrate binding modules (CBMs), which can play several roles in supporting enzyme function, such as localizing the enzyme to the substrate. On WU-AX, in contrast, the N54W-N141Q mutant displayed a lower mobility than the wild-type enzyme, while the G56A-T183A-W185A mutant showed higher mobility. On OSX, the two modified enzymes both showed higher mobility than the wild-type enzyme. Furthermore, the importance of substrate binding to a secondary xylan binding site (SBS) for enzyme mobility was studied by testing two mutants with a modified SBS (N54W-N141Q and G56A-T183A-W185A) that showed different behavior on the tested substrates. For the inactive mutant, however, substrate binding but no fluorescence recovery was observed on WU-AX, while very slow recovery was observed on OSX. For the wild-type enzyme, substrate binding and a complete recovery of fluorescence after photobleaching was observed on both substrates. To assess the importance of substrate hydrolysis, FRAP of a catalytically inactive mutant was compared to that of the wild-type enzyme. Fluorescence recovery after photobleaching (FRAP) experiments using different Bacillus subtilis xylanase A (XBS) mutants were conducted on water-unextractable wheat flour arabinoxylan (WU-AX) and insoluble oat spelt xylan (OSX). In this study, the relationship between their substrate binding affinity and hydrolysis, on the one hand, and their movement over natural substrates, on the other hand, was investigated. Therefore, they are important for biomass breakdown and they are also often added in various biotechnological applications. Xylanases (EC 3.2.1.8) are enzymes that can hydrolyze the xylan backbone internally.