Characterization Of An Endo-type Xanthanase MiXen From Microbacterium Sp.XT11

Xanthan gum,a multifunctional soluble polysaccharide,is produced by Xanthomonas campestris.Xanthan oligosaccharide,which is the degradation product of xanthan,has anti-oxidation,antibacterial and anti-tumor bioactivities,so that it has great commercial prospects.At present,the preparation of xanthan oligosaccharides by enzymatic method has many advantages,such as mild conditions,and controllable product structure.However,the degradation mechanism of the xanthan main chain is not particularly clear,and the tertiary structure of xanthan is highly ordered and is not easily degraded by enzymes.These hinder the industrial production process xanthan oligosaccharides.In this word,the xanthanase Mi Xen from xanthan degrading bacteria Microbacterium sp.XT11 was study.Firstly,the sequence and structure of Mi Xen was analyzed.The results indicated that MiXen was a protein of 952 amino acids,which may be a new branch of the GH9 family glycoside hydrolase.It had a multi-module structure including an N-terminal immunoglobulin?Ig?domain,an??/??₆ barrel catalytic center and a C-terminal domain.It has been found that the C-terminal domain of Mi Xen belonged to a new class of carbohydrate binding module?CBM?.Then,the functions of the catalytic domain and the C-terminal domain of MiXen were studied.The heterologous expression and enzymatic properties of MiXen were performed.Its special activity was 15.7±0.98 U/g,the optimum temperature was40^oC,the optimum pH was 8.0 and the optimal ion concentration was 10 mM.The optimal substrate for MiXen is xanthan.Circular dichroism?CD?and enzyme activity results indicated that Mi Xen can effectively and randomly cut the highly ordered xanthan backbone,the disorder fraction of xanthan increased from 0 to 0.425±0.018 after incubation with the enzyme for 12 h.Shear viscosity and gel permeation chromatography analysis showed that MiXen could more effectively degrade xanthan compared with commercial cellulases.Thereafter,Mi Xen was confirmed to be a xanthan endonuclease by viscosity,linear analysis of reducing sugars and gel permeation chromatography.The enzymatic activity of the MiXen mutant without the C-terminal domain and the surface plasmon resonance?SPR?analysis of the C-terminal domain revealed that the C-terminal domain is capable of assisting the specific binding of Mi Xen to highly ordered xanthan.Finally,CD and atomic force microscopy?AFM?analysis,suggested that different modified side chain of xanthan affected the secondary structure of xanthan,and subsequently affected the activity of Mi Xen on xanthan.In this study,we provided a new perspective for revealing the hydrolysis mechanism and enzymatic properties of a novel endoxanthanase,and lay the foundation for the enzymatic preparation of xanthan oligosaccharides.