Cloning And Expression Of Xanthan Lyases From Two Kinds Of Xanthan-degrading Strains

Xanthan gum is a water soluble extracellular polysaccharide,which is known for its distinctive rheological properties,water solubility,thickening property,emulsifiability and stability.Due to its excellent characteristics,xanthan is applied in many industrial sections,such as food industry,petroleum,cosmetics,medicine,chemical industry,etc.Although xanthan gum has significant advantages over other microbial polysaccharides,the high viscosity and stability of xanthan will make the subsequent petroleum recovery process difficult.Besides,xanthan can cause black rot diseases in plants,but the oligosaccharides degraded from xanthan have very strong antiviral activity.Finally,enzymatic modification of xanthan might result in new types of xanthan gum with new properties.As a result,more and more attentions have been paid to the study of xanthan modification.In this work,we successfully cloned and sequenced the xanthan lyase gene from an excellent food grade xanthan degradation strain XT11 and an oil grade xanthan degradation strain LB37 using degenerate PCR,TAIL-PCR(Thermal Asymmetric Interlaced)and overlap extension PCR(SOE-PCR)techniques.The gene of xanthan lyase gene from XT11 contained an open reading frame consisting of 3417 bp coding for 1138 amino acid residues,including a 50-amino-acid signal sequence,384-amino-acid CBM(carbohydrate-binding modules)sequence;the gene of xanthan lyase from LB 37 consists of 4500 bp.The 78 kDa and 104 kDa mature xanthan lyase without carbonhydrate binding domain could be expressed functionally in Escherichia coli with pET-28 a(+)and pGEX-2T expression vector.A xanthan lyase was purified and characterized.The enzyme is optimally active at 40?.Our work should be valuable to figure out the molecular mechanism of xanthan degradation and the preparation of bioactive oligosaccharides and new type of xanthan gum.