Construction?Purification And Functional Analysis Of The Chimera Cellulase EGX-Gluc1C

The energy captured by plants in the form of cellulosic biomass,which is the most abundant carbon source on earth,may provide a renewable and alternative option for transportation fuel.However,the biomass from cellulose is poor of utilization for its molecular complexity and limitation of commercial cellulase.Identification and design of new cellulolytic enzymes with higher catalytic efficiency are a key factor in reducing the production cost of lignocellulosic bioalcohol.We report here construction of bi-functional chimeric proteins based on a novel?-glucosidase Gluc1C and a cullulase EGX.Glu1C was identified from Paenibacillus sp.Strain MTCC 5639 and the 448-amino-acid-long Gluc1C contained a GH superfamily 1 domain and hydrolyzed cellodextrin up to a five-sugar chain length,with highest efficiency toward cellobiose.EGX is reported as a multi-functional cellulase previously purified from the mollusca Ampullaria crossean.The purified EGX from E.coli is a multi-functional enzyme with the activities of exo-beta-1,4-glucanase,endo-beta-1,4-glucanase,and endo-beta-1,4-xylanase.We assumed that the fusion of two cellulases will release more reducing sugars from carboxymethyl cellulose.We therefore constructed one bi-functional chimeric proteins based on EGX and Gluc1C linked by a flexible sequence(G4S)3.A GST-tag is used to separate and purify proteins including EGX?Gluc1C and the chimera.The SDS-PAGE and commasie blue results indicates that there are large part of inclusion body expressed during the induction,then we optimized the condition of expression,like lowing temperature,decreasing the concentration of IPTG.We used the inclusion body of Gluc1C as antigen to inject into the rabbit and obtained the serum.The specificity of the serum was tested by western blot analysis and the result demonstrated that the antibody really works.The recombinant Gluc1C?EGX?EGX-Gluc1C was purified through standard GST purification procedure and the protein concentration is visualized through SDS-PAGE and Coomassie blue staining.The final concentration of three proteins is 0.5 mg/mL.We evaluated the filter paper enzyme activity and P-Glucosidase activity incubating with carboxymethyl cellulose sodium(CMC-Na)of the cullulases.The release of the reducing sugar is determined by spectrophotometry and DNS as color developing reagent.The constructs consisting of EGX-GIuc1C,demonstrated 1.5-and 1.67-fold higher molar specific activities for ?-glucosidase and endoglucanase than Gluc1C and EGX alone,indicating its commercial applicability.The study and application of chimera may improve the potential of EGX-Gluc1C to be a large scale commercial enzyme and significantly enhance the release of reducing sugars from pretreated biomass.