Identification of a novel xanthan-binding module of a multi-modular sp. xanthanase.

A new strain of xanthan-degrading bacteria identified as sp. has been isolated from a xanthan thickener for food production. The strain was able to utilize xanthan as the only carbon source and to reduce the viscosity of xanthan-containing medium during cultivation. Comparative analysis of the secretomes of sp. after growth on different media led to the identification of a xanthanase designated as Xan9, which was isolated after recombinant production in . Xan9 could efficiently degrade the β-1,4-glucan backbone of xanthan after previous removal of pyruvylated mannose residues from the ends of the native xanthan side chains by xanthan lyase treatment (XLT-xanthan). Compared with xanthanase from , xanthanase Xan9 had a different module composition at the N- and C-terminal ends. The main putative oligosaccharides released from XLT-xanthan by Xan9 cleavage were tetrasaccharides and octasaccharides. To explore the functions of the N- and C-terminal regions of the enzyme, truncated variants lacking some of the non-catalytic modules (Xan9-C, Xan9-N, Xan9-C-N) were produced. Enzyme assays with the purified deletion derivatives, which all contained the catalytic glycoside hydrolase family 9 (GH9) module, demonstrated substantially reduced specific activity on XLT-xanthan of Xan9-C-N compared with full-length Xan9. The C-terminal module of Xan9 was found to represent a novel carbohydrate-binding module of family CBM66 with binding affinity for XLT-xanthan, as was shown by native affinity polyacrylamide gel electrophoresis in the presence of various polysaccharides. The only previously known binding function of a CBM66 member is exo-type binding to the non-reducing fructose ends of the β-fructan polysaccharides inulin and levan.