Enhanced xyloglucan-specific endo-β-1,4-glucanase efficiency in an engineered CBM44-XegA chimera

Xyloglucan-specific endo-β-1,4-glucanase (Xeg, EC 3.2.1.151) exhibits high catalytic specificity for b-1,4 linkages of xyloglucan, a branched polysaccharide abundant in dicot primary cell walls and also present in many monocot species. Although  glycosyl hydrolase family 12 (GH12) Xegs are widely distributed in prokaryotes and eukaryotes, they are not associated with carbohydrate-binding modules (CBMs). Here we have investigated the effect of gene fusion of the xyloglucan-specific CBM44 on the structure and function of a GH12 Xeg from Aspergillus niveus (XegA). After expression and purification, the resulting protein chimera presented enhanced catalytic properties and conferred superior thermal stability relative to the parental XegA. Furthermore, an increased k_cat (chimera, 177.03 s^-1, XegA, 144.31 s^-1) and reduced K_M (chimera, 1.30 mg/mL, XegA, 1.50 mg/mL) resulted in a 1.3-fold increase in catalytic efficiency of the chimera over the parental XegA. Although both parental and chimeric enzymes presented catalytic optima at pH 5.5 and 60^oC, the thermostabilitiy of the chimera at 60^oC was greater than the parental XegA. Moreover, the crystallographic structure of XegA together with SAXS (small-angle X-ray scattering) and molecular dynamics simulations revealed that the spatial arrangement of the domains in the chimeric enzyme resulted in the formation of an extended binding cleft that may underly the improved kinetic properties of the CBM44-XegA chimera.