Characterization of a novel endo-type xanthanase Mi Xen from xanthan-degrading Microbacterium sp. XT11.

Under general aqueous conditions, xanthan appears in an ordered conformation, which makes its backbone largely resistant to degradation by known cellulases. Therefore, the xanthan degradation mechanism is still unclear because of the lack of an efficient hydrolase. Herein, we report the catalytic properties of Mi Xen, a xanthan-degrading enzyme identified from the genus Microbacterium Mi Xen is a 952-amino-acid protein that is unique to strain XT11. Both the sequence and structural features suggested that MiXen probably belongs to a new branch of GH9 family and has a multi-modular structure, in which a catalytic (α/α)6 barrel is flanked by an N-terminal Ig-like domain and by a C-terminal domain that has very few homologues in sequence databases and functions as a carbohydrate-binding module (CBM). Based on circular dichroism, shear-dependent viscosity, reducing sugar and gel permeation chromatography analysis, we demonstrated that recombinant Mi Xen could efficiently and randomly cleave glucosidic bonds within the highly ordered xanthan substrate. Mi Xen mutant free of the C-terminal CBM domain partially lost its xanthan-hydrolyzing ability because of decreased affinity towards xanthan, indicating the CBM domain assisted Mi Xen in hydrolyzing highly ordered xanthan via recognizing and binding to the substrate. Furthermore, side chain substituents and terminal mannosyl residue significantly influenced the activity of Mi Xen via the formation of more or less barriers to enzymolysis, respectively. Overall, the results of this study provide insight into the hydrolysis mechanism and enzymatic properties of a novel endo-type xanthanase that will benefit future applications. IMPORTANCE This work characterized a novel endo-type xanthanase Mi Xen and elucidated that C-terminal carbohydrate-binding module of Mi Xen could drastically enhance the hydrolysis activity of the enzyme towards highly ordered xanthan. Both the sequence and structural analysis demonstrated that the catalytic domain and carbohydrate-binding module of Mi Xen belong to the novel branch of the GH9 family and CBMs, respectively. This xanthan 'cleaver' can help further reveal the enzymolysis mechanism of xanthan and provide an efficient tool for the production of molecular modified xanthan with new physicochemical and physiological functions.