Study On PH-Dependent Activity Of GH11 Family Xylanase Based On High Resolution X-ray Crystallography

Xylan is the main component of hemicellulose in nature.With the implementation of the green development strategy,hemicellulose has received much attention as a renewable biomass resource.Xylanase is widely used in industry(feed and papermaking),there are different requirements for xylanase in different industries.For example,In feed industry,it is needed for xylanase to have enzyme activity at low pH.In papermaking industry,it is also needed for xylanase to have enzyme activity in strong alkaline environment due to bleaching and dyeing.Therefore,the study of pH-dependent activity mechanism of xylanase has a profound impact on meeting the requirements of industrial industry.Because of the characteristics of low molecular weight,high specificity,high enzyme activity and wide pH distribution,GH11 family can be served as the best object to study the enzymatic properties and applications of xylanase.In this study,Xyn? secreted and expressed by Trichoderma reesei was investigated.The relationship between pH and enzyme activity shows a typical bell curve,and the scope of optimal pH is 5.0-5.5.Based on the principle and method of protein structure,protein crystals grown from scratch under different pH conditions were obtained to explored the mechanism of Xyn? enzyme activity reduction.So far,all members of the GH11 family have surprisingly similar three-dimensional structure and catalytic site geometry.In particular,they all contain two glutamates which are necessary for catalysis and a complex hydrogen bond network which is composed of highly conserved nei'ghboring residues.One glutamic acid residue plays a nucleophilic role in the double displacement mechanism,and the other glutamic acid residue plays an acid-base catalyst role in the double displacement mechanism.In the future,the amino acid sequence of xylanase from GH11 family with different optimal pH was compared,and a significant correlation was found.In neutral and alkaline xylanase,the amino acid is asparagine near the acid-base catalyst glutamic acid;in acid xylanase,the amino acid near the acid-base catalyst glutamic acid is aspartic acid.In Xyn?,the amino acid near 177 glutamate is 44 asparagine N44.Through site-specific mutation,44 aspartic acid N was mutated into aspartic acid D or glutamic acid;177 glutamic acid E was mutated into glutamine Q or asparagine N,which made the ability of 177 glutamic acid E to get protons and lose protons disappeared.Study on the relationship between the 44 amino acid and pH dependent activity.The main results as follows:1.Using protein crystallography technology,Xyn? crystals were obtained under different pH crystallization conditions.The structure of these crystals was analyzed and compared to analyze the effect of pH on the enzyme activity of Xyn?.By comparing the calculation and comparison of the B-factors of the crystal structures of pH 5.0,pH 5.5,pH 6.5 and pH 8.0,it was found that the B-factors of the crystal structures obtained at pH 6.5 and pH.8.0 are lower than the B-factors of the crystal structures of pH 5.0 and pH 5.5,it is speculated that the decrease in protein flexibility is one of the reasons for the decrease in Xyn? enzyme activity.By comparing and analyzing the Ca Difference of the pH 5.5 crystal structure and the pH 8.0 crystal structure,it was found that the main change of the C? Difference in the two crystal structures is located in the "finger area" of Xyn?.It is speculated that the change in the "finger area" is due to the decrease of Xyn? enzyme activity one of the reasons.The catalytic area of Xyn? is located in the crack area.By comparing the size of the crack surface area in the crystal structure of pH 5.5 and pH 8.0,it can be known that the crack surface area is the largest in the crystal structure of pH 5.5.Therefore,the size of the crack pocket may be one of the reasons for the decreased activity of Xyn II enzyme.2.Using protein engineering site-directed mutagenesis techniques,the crystal structures of N44D,N44E/E177Q,N44E/E177N and the crystal structures of N44Dligand complex,N44E/E177Q-ligand complex,N44E/E177N-ligand complex were obtained.The crystal structure was analyzed and analyzed,and it was found that after N44 was mutated to E44 or D44,although it had the potential to be a proton donor,it could not play a role because it was far away from the cleavage site.The pKa of E177 in the crystal structure of N44D-pH 4.5and pH 4.0(4XQD)was calculated through the H++website;the results show that the pKa of E177 in the crystal structure of N44D-pH 4.5 is 8.4,but the pKa of E177 in the crystal structure of pH 4.0(4XQD)s 6.9.Therefore,D44 can change the microscopic pKa of E177,so that the optimal pH of N44D is 4.5-5.0,which is reduced by 0.5 relative to the optimal pH of wild-type 5.0-5.5.