Abstract
•Variants of xynlanase Z of Clostridium thermocellum in combination with CBM6 and CBM22 were expressed in E. coli.•The construct with CBM22 showed 5-fold higher activity as compared to the one containing CBM6.•Location of the binding site inside a tunnel like structure of the CBM6 construct seemed to restrict the substrate binding.•The construct with CBM22 produced a more open structure, facilitating substrate binding and release of the product.
Xylanase Z of Clostridium thermocellum exists as a complex in the cellulosome with N-terminus feruloyl esterase, a carbohydrate binding module (CBM6) and a dockerin domain. To study the role of the binding modules on the activity of XynZ, different variants with the CBM6 attached to the catalytic domain at its C-terminal (XynZ-CB) and N-terminal (XynZ-BC), and the CBM22 attached at N-terminus (XynZ-B′C) were expressed in Escherichia coli at levels around 30% of the total cell proteins. The activities of XynZ-BC, XynZ-CB and XynZ-B′C were 4200, 4180 and 20,700UμM−1 against birchwood xylan, respectively. Substrate binding studies showed that in case of XynZ-BC and XynZ-CB the substrate birchwood xylan remaining unbound were 51 and 52%, respectively, whereas in the case of XynZ-B′C the substrate remaining unbound was 39% under the assay conditions used. The molecular docking studies showed that the binding site of CBM22 in XynZ-B′C is more exposed and thus available for substrate binding as compared to the tunnel shape binding pocket produced in XynZ-BC and thus hindering the substrate binding. The substrate binding data for the two constructs are in agreement with this explanation.