The Research Of Enhancement Thermostability Of Aspergillus Niger Xylanase? By C-Terminal Fusion Of CBM9₂ Structural Units

Xylanase is widely used in industrial production,but wild-type xylanase can not meet the conditions required in industrial production,so the transformation of xylanase is very necessary,where the focus is on the xylanase The catalytic activity and thermal stability are modified.The carbohydrate-binding structural module CBM has an independent folded conformation and does not have catalytic activity,and can promote the binding of the enzyme to the substrate.The thermostability of xylanase can be effectively improved by fusing the thermophile-derived CBM with a xylanase.In the early stage of the research group,CBM9₂?C2?derived from Thermotoga maritima was divided into four structural units?U1,U2,U3,U4?by bioinformatics analysis and fused to the Xyn III?X?of Aspergillus niger xylanase.At the N-terminus,fusion enzymes with improved thermal stability were obtained,but a large number of inclusion bodies appeared during the expression process,presumably because the resolution of a complete structure was not conducive to the correct folding of the protein.Therefore,it is expected that the C2 structural units will be fused at the C-terminus of X to obtain soluble expression.The recombinases X-U1,X-U2,X-U3 and X-U4 containing a single structural element,and the recombinases X-U12,X-U123,X-U234 and X-U34 combined with a plurality of structural elements were separately constructed.By comparing the enzymatic properties,the function of each structural element of C2 was analyzed,and it was hoped that a xylanase with a simplified structure and improved thermal stability could be obtained.At the same time,it was also desired to obtain a functional module with significant improvement in enzymatic properties.The experimental results are as follows:?1?Construction of recombinant plasmid: Recombinant plasmids pET21a-X-U1,pET21a-X-U2,pET21a-X-U3 and pET21a-X-U4 containing a single structural element;recombinant plasmid pET21a-X containing multiple structural elements-U12,pET21a-X-U123,pET21a-X-U234 and pET21a-X-U34.The recombinant plasmid was transformed into E.coli BL21?DE3?,and transformants were selected for screening.After sequencing was correct,the recombinant enzyme was induced by IPTG.?2?Optimum pH(pH_opt): pH_opt of pure enzyme X-U1 and crude enzyme X-U3,X-U4 and X-U34 was 3.8,which was consistent with wild-type X;pH_opt of crude enzyme X-U2,X-U12,X-U123 was 4.2,which was 0.4 units higher than X.Among them,X-U1 can maintain more than 75% of enzyme activity from pH 2.6 to pH 5.0.It is speculated that the structural units U1 and U2 are related to the pH adaptability of the enzyme molecules.?3?Optimal temperature(T_opt): T_opt of crude enzyme X-U1,X-U3 and X-U123 are all 46 ?,which is consistent with X;T_opt of crude enzyme X-U2,X-U4,X-U12 is50?,4 ? higher than X.Among them,X-U4 has a wide temperature adaptability and can maintain more than 89% of enzyme activity between 34? and 54?.It is assumed that the structural units U2 and U4 are related to the T_opt of the enzyme molecule.?4?Stability(t_1/2): The semi-inactivation time t_1/2 of the crude enzyme X-U1,X-U2,X-U3,and X-U4 at 50? was: 231 min,30 min,and 500 min.And 340 min,while the t_1/2 of X is: 19.7 min.The t_1/2 of the crude enzyme X-U12 and X-U123 at50 ? were 23.9 min and 10 min,respectively.It is speculated that the structural elements U1,U3 and U4 are related to the stability of the enzyme molecules.?5?Kinetics determination: When using beech xylan as substrate,the Km value of X-U1 was 6.853,and the K_cat value was 146.7.The affinity and catalytic ability for the substrate were lower than that of the wild type X.Thus,C2 is composed of different structural units,in which structural units U1 and U2 are related to the pH adaptability of the enzyme molecules;structural units U2 and U4 are related to the T_opt of the enzyme molecules;structural units U1,U3 and U4 are related the stability of the enzyme molecules.Through the analysis of the functions of C2 structural units,new ideas are provided for the rational design of enzyme molecules.