Differential Proteome Analysis Of The Xanthan-degrading Bacterium Microbacterium Sp. XT11

Xanthan is a highly stable polysaccharide which is to be bio-degraded.We recently isolated an excellent xanthan-degrading strain Microbacterium sp. XT11 which can effectively degrade xanthan to form the bioactive oligosaccharides.At present,xanthan oligosaccharide obtained from the biological method is inhomogenous and the degree of polymerization is dispersive.Because the biodegradation mechanism of xanthan is still unclear,it hinders the indusrial production of xanthan oligosaccharides by biological method.To elucidate the molecular mechanism of microbial degradation of xanthan,in this work,three digestion strategies were applied to demonstrate the complexity of the Microbacterium sp. XT11 proteome in xanthan medium and yeast medium.Then the label free quantitative method was used for screening out the significantly up-regulated proteins induced by xanthan from the proteomes of Microbacterium sp. XT11.Totally 2746 and 2792 proteins were identified from the proteomes of Microbacterium sp. XT11 in xanthan medium and yeast medium individually,which covered 80.6%and 81.9%of the total protein predicted from genome data.Of 545 induced proteins that specifically expressed or up-regulated in xanthan medium,304 protein were annotated for three types of functions（molecular functions,biological processes and cellular components）and distributed into 28 subgroups.Besides,16proteins were found in carbohydrate-active enzymes database and 45 proteins were annotated with transporter activity,which were critical for the degrading of xanthan.Among them,4CAZymes and 2 ABC transporters were consistent with the xanthan degradation gene cluster components predicted from the XT11 genome database.Subsequently,the xanthan oligosaccharide binding protein Xan E（up-regulated to 100-fold）was selected from the XT11differential proteome analysis.It was then heterologous expressed in Escherichia.coli BL21（DE3）and purified.The purification yield was 28.37%.Subsequently,the binding ability of Xan E towards xanthan oligosaccharides was verified by the Surface Plasmon Resonance（SPR）.The results showed that the K_D value of the affinity constant was 5.11×10^-3M,indicating that Xan E has a slight binding ability to xanthan oligosaccharides.The low binding ability is due to the substrate heterogeneity.The result also demonstrated that the substrate binding protein Xan E plays a key role in transporting the xanthan biodegradation product.The results of this study lay the foundation for excavating xanthan degradation related proteins and clarifying the molecular mechanism of xanthan biodegradation.