A thermostable and CBM2-linked GH10 xylanase from Thermobifida fusca for paper bleaching

Abstract

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 <i>Tf</i>Xyl10A, a xylanase from <i>Thermobifida fusca</i>, was greatly induced to transcript by microcrystalline cellulose (MCC) substrate. Biochemical characterization showed that <i>Tf</i>Xyl10A 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 <i>Tf</i>Xyl10A-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 (<i>Tf</i>Xyl10AdC) 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 <i>Tf</i>Xyl10A with the ability to degrade xylan surrounding cellulose. These results indicated that <i>Tf</i>Xyl10A might be an excellent candidate in bio-bleaching processes of paper industry. In addition, the features of active-site architecture of <i>Tf</i>Xyl10A 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 <i>Tf</i>Xyl10A-CD (the catalytic domain of <i>Tf</i>Xyl110A). 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.