Abstract:The polysaccharide-disintegrating carbohydrate-active enzymes (CAZymes) are often found with multimodular architecture, where the catalytic domain is connected to an accessory CBM domain with the help of a flexible linker region. So far, the linker has been understood merely as a flexible spacer between the two domains.
“…However, a small portion of the protein band remained in the precipitant lane, suggesting that the linker unexpectedly retains some substrate binding affinity. Although linkers are generally considered as flexible spacers between two domains, some studies have demonstrated that linkers can contribute to polysaccharide binding ( 36 ), aligning with our observed results. Figure 2 Substrate binding and activity of Mt LPMO9G and its truncated forms .…”
“…However, a small portion of the protein band remained in the precipitant lane, suggesting that the linker unexpectedly retains some substrate binding affinity. Although linkers are generally considered as flexible spacers between two domains, some studies have demonstrated that linkers can contribute to polysaccharide binding ( 36 ), aligning with our observed results. Figure 2 Substrate binding and activity of Mt LPMO9G and its truncated forms .…”
“…Srivastava et al recently investigated the role of the linker region of fungal Bc LPMO9C ( Table 1 ) focusing on its effect on catalytic performance and thermostability. The authors generated three linker truncation variants, where shortening of the linker seemingly resulted in lower cellulose-binding affinity [ 48 ].…”
Lytic polysaccharide monooxygenases (LPMOs) have revolutionized our understanding of how enzymes degrade insoluble polysaccharides. Compared with the substantial knowledge developed on the structure and mode of action of the catalytic LPMO domains, the (multi)modularity of LPMOs has received less attention. The presence of other domains, in particular carbohydrate-binding modules (CBMs), tethered to LPMOs has profound implications for the catalytic performance of the full-length enzymes. In the last few years, studies on LPMO modularity have led to advancements in elucidating how CBMs, other domains, and linker regions influence LPMO structure and function. This mini review summarizes recent literature, with particular focus on comparative truncation studies, to provide an overview of the diversity in LPMO modularity and the functional implications of this diversity.
“…Previous studies have indicated that the linker length and flexibility play an important role in the activities of some cellulases, such as BsCel5A from Bacillus subtilis (Ruiz et al, 2016) and PoCel6A from Penicillium oxalicum (Gao et al, 2015). The linker region promotes enzyme activity and binding efficiency of LPMO towards polymeric substrate (Srivastava et al, 2022). In addition, Sonan et al (2007) have reported that the linker region plays a key role in the cold adaptation of the a cellulase from an Antarctic bacterium.…”
Xylanases have the potential to be used as bio-deinking and bio-bleaching materials and their application will decrease the consumption of the chlorine-based chemicals currently used for this purpose. However, xylanases with specific properties could act effectively, such as having significant thermostability and alkali resistance, etc. In this study, we found that TfXyl10A, a xylanase from Thermobifida fusca, was greatly induced to transcript by microcrystalline cellulose (MCC) substrate. Biochemical characterization showed that TfXyl10A is optimally effective at temperature of 80 °C and pH of 9.0. After removing the carbohydrate-binding module (CBM) and linker regions, the optimum temperature of TfXyl10A-CD was reduced by 10°C (to 70°C), at which the enzyme’s temperature tolerance was also weakened. While truncating only the CBM domain (TfXyl10AdC) had no significant effect on its thermostability. Importantly, polysaccharide-binding experiment showed that the auxiliary domain CBM2 could specifically bind to cellulose substrates, which endowed xylanase TfXyl10A with the ability to degrade xylan surrounding cellulose. These results indicated that TfXyl10A might be an excellent candidate in bio-bleaching processes of paper industry. In addition, the features of active-site architecture of TfXyl10A in GH10 family were further analyzed. By mutating each residue at the -2 and -1 subsites to alanine, the binding force and enzyme activity of mutants were observably decreased. Interestingly, the mutant E51A, locating at the distal -3 subsite, exhibited 90% increase in relative activity compared with wild-type (WT) enzyme TfXyl10A-CD (the catalytic domain of TfXyl110A). This study explored the function of a GH10 xylanase containing a CBM2 domain and the contribution of amino acids in active-site architecture to catalytic activity. The results obtained provide guidance for the rational design of xylanases for industrial applications under high heat and alkali-based operating conditions, such as paper bleaching.
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