| In the early stage of the research group,the GH11 family of glucanase Cel12 B was incorporated into the Thumb structure of the GH11 family enzyme,and the mutant enzyme 17 QT was obtained,which retained the thermostability of the GH12 family of enzymes and resulted in the hydrolysis of xylan substrate.The content of sugar,xylotriose and xylotetraose increased,but the affinity of 17 QT for the two substrates of CMC(carboxymethyl cellulose sodium salt)and BWX(beech xylan)decreased and the catalytic ability of the enzyme decreased.Because the constructed 17 QT Thumb structure is derived from Aspergillus medium a with low temperature,17 QT has good stability at 100°C,and Thumb structure may not be suitable for the function high temperature conditions.In this study,the screening and comparison of the GH11 family Thumb structure and amino acid site-directed mutagenesis of the Thumb structure were carried out to explore the key amino acid sites for the degradation of xylan substrates by Thumb.A single point mutation and multiple point mutations were made to the key amino acid sites identified in the Thumb of 17 QT,and the dextranase affinity to the xylan substrate and the hydrolysis ability were improved.From the alignment of the Thumb structure amino acid sequence of the thermophilic enzyme,based on the highly conserved nature of the Thumb structure sequence,the three mutation sites on the Thumb structure and the mutation site on the junction with the Thumb were determined.A single amino acid single point mutation was performed on the Thumb structure of GH11 family xylanase 23A2,and four mutants E119 Q,T123E,S126 T,and T129 D were constructed.In glucanase 17 QT,single mutation and multiple point mutations were performed on the three mutant sites of Thumb structure.Seven mutants E177 Q,T181E,S184 T,E177Q-T181 E,T181E-S184 T,E177Q-S184 T and three point mutations were constructed.The results of the enzymatic properties of the Thumb structural mutants were as follows:Xylanase Thumb Structural Mutant Pure Enzymes Detection(1)The optimal p H values of the mutants E119 Q,T123E,S126 T,and T129 D were 4.2,4.0,4.0,and 4.4,and they were neutral to acidic with respect to the optimum p H 3.8 of 23A2.(2)In the optimum temperature detection,the optimum temperature of 23A2 is 50?,and the optimum temperatures of the mutants E119 Q,T123E,and T129 D are all 50?.The mutations at these three points have no significant effect on the optimum temperature of the enzyme.The optimum temperature of S126 T is 45?,and the optimal temperature is reduced,indicating that the mutation has an effect on the temperature adaptability of the enzyme.(3)At 50? the semi-inactivation time of 23A2,E119 Q,T123E,S126 T and T129 D was 23.1 min,15.4 min,6.3 min,7.0 min and 9.3 min respectively.Half-life detection showed that the enzyme half-life shortened after point mutation..(4)In the enzymatic kinetic assay,the fastest reaction rate was T129 D,and Vmax was 10.3 ?mol·L-1·s-1,which was 1.48 times that of 23A2.Comparing with Km 3.4 of 23A2,the mutants E119 Q,T123E,and T129 D all showed increased substrate affinity.The Km values of the mutants E119 Q,T123E,and T129 D were 3.0,2.9,and 3.3,respectively.The highest substrate affinity was T123 E,which was 1.167 times that of 23A2.Prove that mutations in these sites in Thumb can increase the affinity for xylans substrate.Detection of Crude Enzymes in Dextranase Thumb Structural Mutant(1)The optimum p H and temperature of the seven mutants were the same as those of 17 QT crude enzyme.The optimum p H was 6.0 and the optimal reaction temperature was 100 ?.The semi-inactivation time of crude enzyme was measured at 100 ?.Semi-inactivation time of E177 Q,T181E,S184 T,E177Q-T181 E,T181E-S184 T,E177Q-S184 T,and three-point mutations was 58 min,54 min,33 min,214 min,81 min,82 min,and 150 min,the half-lost time of the 17 QT crude enzyme assay at 100°C was 65 min.It can be seen that the mutant and the 17 QT were better than the semi-inactivation time of the crude enzyme.The p H of the original glucanase was retained and the thermal stability was good.Moreover,after the two-point and three-point mutation,the half-inactivation time of the crude enzyme was longer than 17 QT,and showing better thermal stability.(2)In the comparison of the activity of CMC and BWX substrates with mutants,the substrate activity of single point mutants was similar to that of 17 QT,and the activity of crude enzyme detection activity of CMC OD550 in two-and three-point total mutants was significantly reduced.OD550 decreased from above 0.4 to below 0.1,from BWX above single point mutations,to below BWX.In the two-point and three-point mutants,the activity of CMC substrate was significantly decreased,the relative value of BWX/CMC activity was increased by 5 to 7 times,and the activity of BWX substrate was enhanced.(3)In the relative activity of the mutant crude enzyme,the single-point mutant showed the highest specific activity was T181 E,and the activity of E177Q-T181 E and T181E-S184 T mutation did not increase significantly in the double-point mutant.The specific activity of the T181E-S184 T mutant decreased,and the three-point mutation was the highest in all relative livelihoods.It can be seen that the single-point mutant showed similar properties to 17 QT in the activity,and the multi-point combination was mutated,and the affinity for degradation of the xylan substrate was improved with good thermal stability of the 17 QT.Among the three amino acid positions of the Thumb structure,three sites play a key role in the degradation of xylan substrates,and their combined mutations are more conducive to exerting the Thumb structure to improve the affinity and hydrolysis of xylan substrates.Ability,there is a certain additive gain ability between the three amino acid mutation sites in different positions,the mutant has changed the hydrolysis ability of CMC and BWX after performing multi-point combination mutation,which is more conducive to improve the affinity degradation of xylan substrate..In this study,by mutating key amino acids in the Thumb structure of the GH11 family xylanase-specific element,dextranase gains affinity for xylan substrates and increases the ability to hydrolyze,which is specific for glucanase substrates.The transformation provides a new direction and also provides a new method for the improvement of enzymes. |
PDF Full Text Request