Improved catalytic efficiency of chimeric xylanase 10B from Thermotoga petrophila RKU1 and its synergy with cellulases

TpXyl10B is a glycoside hydrolase family 10 xylanase of hyperthermophile Thermotoga petrophila RKU-1. This enzyme is of considerable importance due to its thermostability. However, in its native state, this enzyme does not possess any carbohydrate-binding module (CBM) for efficient binding to plant biomass. In this study CBM6 from Clostridium thermocellum was attached to the N- and C-termini of TpXyl10B, thereby producing the variants TpXyl10B-B6C and TpXyl10B-CB6, respectively. TpXyl10B-B6C showed 5–7 folds increased activity on Beechwood xylan and the different types of plant biomass as compared to that from the catalytic domain only. However, the activity of TpXyl10B-CB6 decreased 0.6–0.8 folds on Beechwood xylan and plant biomass compared to the catalytic domain. We explained these results through molecular modeling, which showed that binding residues of CBM6's cleft B, which were previously reported to show no contribution towards binding due to steric hindrance from a loop region, were exposed in a favorable position in TpXyl10B-B6C such that they efficiently bound the substrate. In contrast, these binding residues of CBM6 in TpXyl10B-CB6 were exposed opposite to the catalytic residues; thus, binding to the substrate resulted in decreased exposure of catalytic residues to the substrate. CD spectroscopy and thermostability assays showed that TpXyl10B-B6C was highly thermostable, having a melting point > 90 °C, which is relatively higher than that of the other variant, TpXyl10B-CB6. In addition, this xylanase variant showed synergism with cellulases for the hydrolysis of plant biomass. Therefore, TpXyl10B-B6C, an engineered xylanase in this study, can be a valuable candidate for industrial applications. •Engineering of xylanase 10B of Thermotoga petrophila by fusion of CBM6 from Clostridium thermocellum.•TpXyl10B-B6C showed up to 7-fold enhanced activity on Beechwood xylan as compared to that of TpXyl10B catalytic domain only.•Molecular modelling showed that novel ligand-enzyme interactions in CBM6 contributed to enhanced activity in TpXy110-B6C.