Development of a chimeric hemicellulase to enhance the xylose production and thermotolerance

Xylan is an abundant plant cell wall polysaccharide and its reduction to xylose units for subsequent biotechnological applications requires a combination of distinct hemicellulases and auxiliary enzymes, mainly endo-xylanases and ß-xylosidases.  In the present work, a bifunctional chimeric enzyme consisting of catalytic domains from a GH11 (endo-1,4-β-xylanase) and a GH43 (β-xylosidase), both from Bacillus subtilis, was designed taking into account the quaternary arrangement and accessibility to the substrate. The parental enzymes and the resulting chimera were successfully expressed in Escherichia coli, purified and biochemical and biophysically characterized. Interestingly, the substrate cleavage rate was altered by the molecular fusion improving at least 3-fold the xylose production using specific substrates as beechwood xylan and hemicelluloses from pretreated biomass. Moreover, the chimeric enzyme showed higher thermotolerance with a shift of the optimum temperature from 35 to 50ºC for xylosidase activity. Thermal denaturation experiments also showed the improvement of thermal stability with a melting point of ~ 58ºC, a which seems to be related to a gain of stability of the β-xylosidase domain. These results demonstrate the superior functional and stability properties of the chimeric enzyme in comparison to individual parental domains, suggesting the molecular fusion as a promising strategy for enhancing enzyme cocktails aiming at lignocellulose hydrolysis