The Research Of Fusing CBM₂ To The N-terminal Of Xylanase

Xylanase has shown specific potentials for application in the feed,paper,textile,and energy industries,et al.Here we isolated a high yield xylanase producing strain Aspergillus niger A-25,which secreted a endo-1,4-?-xylanase Xyn III(X)of GH11 family.The enzyme only contains a catalytic domain,and its optimum temperature(Topt)is 48?.However,this property is not suitable for the high temperature conditions of industrial production.Therefore,it is necessary to reform the thermal stability and catalytic ability of the xylanase.GH10 xylanase XynA(Topt 90?)from the Thermotoga maritima contains a carbohydrate-binding module(CBM)in the C-terminal.The CBM can improve the catalytic ability of enzyme and may have a thermostabilizing effect considering the CBM'origin.In our previous studies,CBM,CBM1(C1)and CBM2(C2)were respectively transplanted into X to create the chimeras X-C,X-C1 and X-C2.The C1 and C2 were obtained by dividing the CBM into two sub-modules.As a result,C2 can significantly improve both the thermostability and catalytic activity of fusion enzyme,but C1 only enhance its catalytic ability.Besides,major relevant research focused on fusing the functional module to the C-terminal,but rarely to the N-terminal,which had ended up with reducing or even losing the catalytic activity.Hence,we designed to fuse C2 to the N-terminal of X with the CBM inherent connection peptide as a linker,in order to advance the thermostability and catalytic activity of Xyn.In this research,we connected C2 to the N-terminal of X and screened four positive transformants.The two recombinant plasmids pET21a-C2-X and a K140R mutant pET20b-C2-X were got by the traditional method of enzyme restriction and ligation;The two recombinant plasmids pET20b-C2-X and the pET20b-C2-X with a four more amino acid residues(DIHM)in the N-terminal of the fusion enzyme was obtained by an inverse PCR-based technique.The recombinant plasmids were transformed into E.coli BL21(DE3)and induced to produce C2-X(pET21a),C2-X(pET20b),C2-XK140R and C2-XDIHM.The properties of the purification enzymes were assayed in parallel with native X.The beech xylan was used to detect optimal pH(pHopt),optimal temperature(Topt),half-life(t1/2).The results showed that:(1)The pHopt of recombinant protein C2-X(pET21a),C2-X(pET20b),C2-XK140R,and C2-XDIHM are 3.6,decreased 0.2 units than the native X(pHopt 3.8),which indicates that the N-terminal fusion of C2 makes enzyme acid resistance improved.(2)The Topt of recombinant protein C2-X(pET21a)and C2-X(pET20b)is 50?,increased 2? than the native X(Topt 48?),it Showed Topt improved after the N-terminal fusion of C2.Whereas the C2-XK140R and C2-XDIHM are equal to the X.This phenomenon may be resulted from the amino acid mutation.(3)The t1/2 of recombinant protein C2-X(pET21a),C2-X(pET20b),C2-XK140R,and C2-XDIHM are 60.62min,50,45min,46.9min and 38.6min.Compared with the native X's 21.04min,the C2 has a positive effect on the thermostability of xylanase.(4)The kinetics were assayed using beech xylan and birch wood xylan.As it is shown,the Kcat of enzyme C2-X(pET21a),C2-X(pET20b),C2-XK140R,C2-XDIHM and X are 1237.64s-1,1167.81s-1,805.65s-1,757.44s-1,280.79s-1 with the beech xylan as the substrate;alternatively,the values of Kcat are 1100.06s-1,889.90s-1,699.67s-1,278.04s-1.The results demonstrate that the N-terminal fusion of C2 enhance the catalytic activity of xylanase.Thus,consistent with this topic originally envisaged,the two fusion enzymes have same properties which two different recombinant plasmids expressed.Fusing C2 to N-terminal of X makes the(C2-X)'s Topt raised 2?,and also significantly improved the thermal stability and catalytic efficiency.At the same time,we explored a novel inverse PCR-based technique to construct the recombinant plasmid,with a high positive rate and with a shorter time than the traditional method.