The Research Of Obtaining The Activity Of Xylanase By Introducing Thumb Into CBM9₂

The carbohydrate binding module?CBM?has independent folding conformation,which promotes the combination of enzyme and substrate,but does not have catalytic activity.Through structure comparison,I found that the structure of CBM9₂?C2?from T.maritima Xyl10 A is very similar to that of A.niger Xyn III.But the necessary structural element-the Thumb is absent from C2.Thumb is the unique structure of GH11 family xylanase,which is highly conservative and has a length of 11 Aa.It plays an important role in the combination and hydrolysis of enzyme and substrate.In this study,on the basis of the mutation?D169E,D177E?of the analogous active center,I expect to transform C2 into a thermostable xylanase with highly catalytic activity by introducing the Thumb?N118-F128?of X into C2 between K136 and T137.Considering that Thumb is from the medium-temperature enzyme and may not suit for high-temperature C2,I carry out site-directed mutations and expect to further improve the stability of the mutant enzyme.The selection of the mutation sites which are E119 Q T123E S126 T is based the on the Thumb sequence of high-temperature xylanase.The results are as follows:?1?Construction of recombinant plasmid: Using reverse PCR method.Taking p ET20b-C2 as the template,mutate the analogous active center to obtain the mutations: p ET20b-C7?D177E,constructed by predecessor?,p ET20b-C9?D169E?,p ET20b-C79?D169E/D177E?.Insert Thumb respectively: p ET20b-CT,p ET20b-C7 T,p ET20b-C9 T,p ET21a-C79 T.Site-directed mutate in Thumb: p ET21a-C7T-9,p ET21a-C7T-3,p ET21a-C7T-6,p ET21a-C7T-93,p ET21a-C7T-96,p ET21a-C7T-36,p ET21a-C7T-936,totally 14 recombinant plasmids.?2?Measure the activity of pure protein after concentrated: None of the 15 crude proteins include C2 are active.But after purified by Ni²⁺ chromatography and concentrated 100 times,the degradation activity of BWX is measured in the mutanted enzyme C7 T.?3?Optimal pH(pH_opt): The p Hopt of C7 T is 4.2,which is 0.4 units higher than the wild-type X(pH_opt 3.8).The relative residual enzyme activity is still remained more than 60% in the range of p H3.6-4.8.?4?Optimum temperature(T_opt): The Topt of C7 T is 48 ?,which is 2 ? higher than X(T_opt 46 ?).The relative residual enzyme activity is 62 % at 40 ? and 69 % at 51 ?.?5?Half the deactivation time(t_1/2): The t1/2 of C7 T under 50 ? is 11.0 min,which is 8.8 min shortened of X(t_1/2 19.8 min).?6?Enzyme kinetics parameters:1)K_m value: 1.2 mg·ml^-1,lower than X(3.6 mg·ml^-1),show that the affinity of C7 T and substrate is 3 times higher than that of the wild type X.2)K_cat value: 1.4 s^-1,lower than X(600 s^-1),show that the catalytic activity of C7 T is decreased.In conclusion,in this study,based on the mutation of the analogous active center and introducing the Thumb structure into C2,I successfully transform C2 into a xylanase with catalytic activity.Compared with wild type xylanase X,the substrate affinity of C2 increased by 3 times,and the optimum reaction temperature increased by 2 ?.This strategy,based on rational design and structural analysis,provides an entirely new way for enzymatic modification.