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

A new strain of xanthan-degrading bacteria identified as Cohnella 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 xanthancontaining medium during cultivation. Comparative analysis of the secretomes of Cohnella sp. after growth on different media led to the identification of a xanthanase designated as CspXan9, which was isolated after recombinant production in Escherichia coli. CspXan9 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 (XLTxanthan). Compared with xanthanase from Paenibacillus nanensis, xanthanase CspXan9 had a different module composition at the N- and C-terminal ends. The main putative oligosaccharides released from XLT-xanthan by CspXan9 cleavage were tetrasaccharides and octasaccharides. To explore the functions of the Nand C-terminal regions of the enzyme, truncated variants lacking some of the non-catalytic modules (CspXan9-C, CspXan9-N, CspXan9-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 CspXan9-C-N compared with fulllength CspXan9. The C-terminal module of CspXan9 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. [ABSTRACT FROM AUTHOR]