Structural Stability Analysis And Functional Design Of The Active-site Architecture In GH11 Family

Industrial biotechnology has become the third wave of modern biotechnology development.Although enzyme preparations have been applied to various industrial fields,the efficiency and stability of enzyme catalysis are still the main bottleneck and restriction for the industrial application of enzymes.Therefore,designing and screening efficient and stable industrial enzyme preparations is a hot topic in the field of industrial biotechnology in recent years.The rapid development of high-throughput sequencing technologies and the drastic reduction in the cost of sequencing have prompted explosive growth of the amount of protein sequences;at the same time,the appearance of protein crystallization and cryo-electron microscopy has increased the structural information of biological macromolecules.Massive data informationpromotes the continuous progress and development of enzyme molecular design strategies.Experimental programs has evolved from high-throughput screening,directed evolution,and semi-rational design to designing enzyme molecules with rational design strategies.In particular,the huge sequence space of enzyme molecules requires the enzyme engineers to analyze the structure and function information of the engineered enzyme molecules,which promotes the development of "structure-based bioinformatics" and the classification of protein sectors.The function and properties of proteins depend on the synergistic effect of the relevant amino acid residues in the protein functional domain.Family analysis based on structural bioinformatics can quickly locate key amino acid residues or combinations in the functional regions of proteins,reducing the search space for the sequences.Hydrolases represented by glycoside hydrolases are important biocatalysts in the industry,which are divided into 152 families in the CAZy database according to the principle of sequence similarity.Different families of glycoside hydrolases employ different topologies and catalytic mechanisms.Through structural bioinformatics analysis,the relationship between sequence,structure and function of different glycoside hydrolase families was analyzed statistically.For proteins with high sequence similarity but functional differences,their "functional plasticity islands"could be also accurately and rapidly located by structural bioinformatics analysis,which provides a target(i.e.,key amino acid residues or residue combinations or local structures that affect function)for protein design,thereby creating "small but smart"library of mutations that increases the success rate of protein design.In this dissertation,the affinity-electrophoresis technology for the high-throughput characterization of glycoside hydrolase was optimized.By using the technology of structural bioinformatics platform,the structures basis influencing thermal stability of the glycoside hydrolases in GH11 family and the common law deciding rapid evolution of catalytic function of theses enzymes were explored.And site-directed mutagenesis and functional analysis of the key sub-site amino acids of the active structure of TlXynA(GH11 family)were performed to further elucidate the mechanism of its recognition of the substrate.The innovative research results obtained in this dissertation are as follows:1.Affinity electrophoresis was used to rapidly display the changes in binding force caused by a single point mutation in the active structure of glycoside hydrolase,providing a high-throughput technique for routine laboratories to rapidly screen mutants with binding force changes.The active-site architecture composed by multiple amino acid residues governed substrate recognition,binding and catalytic process.And the residues in active-site architecture has always been the research hotspot in protein engineering.In this study,the catalytic activity and band mobility of different mutants in the active-site architecture of TrCell2A and TlXynA could be rapidly demonstrated by affinity electrophoresis.Furthennore,the substrate binding affinity can be quantitatively characterized by quantitative regression analysis of the relative mobility of proteins at different substrate concentrations.The parameters Kb measured by affinity electrophoresis were significantly related with those determined by Isothermal Titration Calorimetry and Fluorescence spectroscopy.Therefore.,affinity electrophoresis can be used as routine screening technology in biochemical laboratory to detect the change of series mutants binding affinity in mutant library.2.The structural basis for the thermal stability of the glycoside hydrolase GH11 family was explored,and the thermal stability of the enzyme molecule was enhanced by N-terminal substitutions.Xylanase has been able to efficiently hydrolyze xylan and is widely used in various fields of industry,such as food and feed industry,pulp and paper industry and so on.Thermophilic enzymes have many advantages compared to mesophilic enzymes in industrial applications.For example,they can keep stability and have good compatibility when heat treated,and can be stored for long periods of time.However,phylogenetic analysis of the evolutionary of the GH11 family,it was found that the amino acid differences between the closely related thermophilic and mesophilic enzymes were small,and their sequence identity could reach more than 50%.The use of structure-based bioinformatics analysis of the conservation and specificity of sequences,fragments,or local structures of thermophilic and mesophilic enzymes revealed the thermal sensitivity of the N-terminal and Thumb-loop regions of the enzyme molecules and a higher ratio of polar charged amino acids,especially Arg,of thermophilic enzymes.In order to lock the N-terminus,enhance the rigidity of the local structure and increase the intramolecular polar interactions,the GH11 family xylanases AnXynB(from Aspergillus niger ATCC1015)and TlXynA(from Thermomyces lanuginosus)were studied respectively for mutation experiments and functional analysis.The N-terminus of the thermophilase was introduced into the enzyme molecule AnXyn3 to stabilize the N-terminal structure,thereby increasing the Tm value of the enzyme by 4? but it cannot be overlooked that the mutant enzyme activity was reduced by 36%.3.Based on structural bioinformatics,the structural basis and commonality law of the rapid evolution of the GH11 family glycoside hydrolase catalytic function was explored,and amino acid grafting of AnXyuB active site architecture-3 subsites was explored to increase enzyme activity.The diverse enzyme molecules in nature can adapt their specific functions by adjusting their sequences,local structures,and even the dynamics of proteins.Through the phylogenetic analysis of the entire family,it is possible to find proteins with high sequence similarity but functional differences.Variable amino acids in the active site architecture may regulate the specificity of the catalytic function of the enzymes in the same family.Therefore,under the evolution of the molecules,it is feasible to locate variable amino acid residues that may determine the adaptability of the enzyme molecules to the specific environment,such as high-temperature or alkaline solutions.Based on structural bioinfonnatics analysis,it was found that the amino acids of the-3 subsites in the active site archetecture of glycoside hydrolases in GH11 family are highly variable and may be related to the difference in catalytic function.The evolutionary relationship between the homologous enzymes AnXynB and TlXynA is relatively close,but their functional differences are large,especially the differences in binding ability.Through computational and experimental methods,two amino acid residues of-3 subsite of active site architecture of AnXynB were finely grafted,which improved the binding ability of AnXynB to the substrate,thus obtaining two mutants with high enzyme activity.Among them,the enzyme activity of T43E increased by 20%,and the enzyme activity of S41N/T43E increased by 72%.In addition,the results of catalytic kinetic measurements show the importance of a delicate balance between substrate binding and product releasing.The results of fluorescence-assisted carbohydrate electrophoresis showed that increasing the binding force of the enzymes at the-3 subsite can significantly increase the binding energy of the enzyme molecules to the oligosaccharides,thereby increasing the activity of the enzymes.At the same time,because the mutant S41N/T43E introduced two hydrogen bonding forces in the structure,the thermal stability of the enzyme was also significantly improved.4.Site-directed mutagenesis and functional analysis of key amino acids in the active structure of TlXynA(GH11 family),further elucidating its interaction mechanism with the substrate.A detailed analysis of the active structure of glycoside hydrolases in GH11 family revealed that there is a preference for the frequency of amino acids in the active site architecture.Most of the polar amino acids form hydrogen bonds with 02 and 03 of the sugar rings of the substrate,and aromatic amino acids form CH-?interactions with the sugar rings of the substrate,particularly the-2,+2 and +3 subsites.The amino acid conservation analy-sis of the active center re'vealed that the amino acids at the-2,-1,and +1 subsites are highly conserved,and the-2-+1 subsite is the region with the most interactions.Alanine screening experiments were performed on 23 amino acids in the active site architecture,because the removal of R groups of amino acids may decrease enzyme-substrate binding and catalysis.Enzyme activity assay experiments revealed that mutations of amino acids at the-2 subsite and the +1 subsite resulted in most mutant enzyme activities being less than 5%of the wild-type enzyme activity.This shows that the conserved amino acids at the subsites play a key role in the normal functioning of enzyme activity.The mutation experiments of amino acids at the +2 and +3 subsites show that the main interaction force is the CH-? interaction force.and the size of ? electron have different effect on the enzyme activity.Measuring the binding ability of each amino acid of the active structure to the substrate can effectively classify its function,elucidate its mechanism of action,and provide a theoretical basis for rational design afterwards.5.The influence of flexibility change of the critical loop of the enzyme molecule active architecture of TlXynA on its catalytic function was explored.The dynamics of proteins are closely related to their catalytic functions.and they are involved in the formation of enzyme conformations,effective substrate binding and product release.It has been reported that the dynamic stability of the active center region is an indispensable factor for maintaining the catalytic function of thermophilic enzymes.The analysis of the structure of the GH11 family revealed that there are three loop structures around the active center and affected more by temperature.In the thermophilic enzyme TlXynA,there is a polar amino acid Arg respectively on the outside of Loop2 and Loop3,interacting with R116-D97(Loop2)and R81-D129(Loop3),Compared with the three loop regions of the Mesophilic xylanase AnXynB,it was found that there are more Glys in the Loop structure of the Mesophilic xylanase AnXynB,especially in Loopl and Loop2.Therefore,we used the thermophilic enzyme TlXynA as a research object for mutation research and functional analysis.The results of the kinetic simulations revealed that the mutations reduced the flexibility of the local structure of the enzyme molecules,particularly the A23G and R81Q mutants.The results of enzyme activity assay showed that the decrease of the flexibility of the Loop led to a decrease in the catalytic ability of the enzyme molecule,w-hich mainly affected the binding force between the enzyme and the substrate(increased Km value).This shows that the movement of the Loop structure is necessary for the enzyme molecules to bind the substrate and release product.