Cellulase Active Architecture And Functional Analysis Of Key Subsites In TrCel12A

Lignocellulose, composed of cellulose, hemicelluloses, and lignin, is the most abundant renewable biomass on earth. Degradation and conversion of lignocellulosic biomass is attracting attention because of its potential for the development of sustainable and environment-friendly energy. Given the natural recalcitrance of lignocellulose to enzymatic hydrolysis,the highcost of lignocellulolytic enzymes is still a major bottleneck in lignocellulosic biofuel production. The key material for biofuel production is cellulose,which is one of the main components of the plant cell wall. Cellulases is one of most importent components to synergistically and codependently depolymerize cellulase. All cellulases are grouped into glycoside hydrolase (GH) families based on sequence and 3D structure similarity.And cellulases often show function promiscuity in the same family or differernt families.Better understanding the relationship between funtions and its stuctures would be a great help to analysis its promiscuity mechanism and also its fundamentals structure of cellulase to identify the substrate.Based on structural bioinformatics method,this thesis try to find out fundamentals structure of different promiscuity cellulase families. And key amino acids mutation have also been carried out in this work with the purpose of elucidating its binding and recongnise mechanism.The main results of this work are as follows:(1) Analysising active site architecture of different promiscuity cellulases families30% sequence similarity make glycoside Hydrolases family having similar structural characteristics. Given the function of cellulases are just part of the catalytic domain,identifying the active architecture based on sequence and structural character is of great significance.In this paper,we found that amino acid residues relative frequency in Active site architecture are different in main cellulases families.And binding site amino acids and conservative position mode veried in different GH families,this to a certain extent, reveals the fundermental structure of promiscuity in GH families.(2)Mutation of key amino acids in the active architecture of TrCel12A and its promiscuity analysisTrCel12A is an functional promiscuity enzyme,which has CMCases and xyloglucanase,it can also degrade different polymerization degree of soluble substrate. we selected two aromatic residues, W22 and W7, with different degrees of conservation at the subsites of -2 and -4, and constructed the structurally-guided mutations of W22Y and W7Y. Mutational analysis confirmed that tyrosine exhibits maximal subsite-binding energy among the aromatics when additional hydrogen bonds could be formed and that it was the good balance between substrate binding and product release that guaranteed a higher catalytic efficiency. Increased binding affinity at the -4 subsite (W7Y mutant) resulted in a marked increase of the intermediate and enhanced reactivity of endoglucanase by 138%; while tighter binding at the -2 subsite (W22Y) made the enzyme bind and hydrolyze oligosaccharides more easily, leading a delayed release of product and a decreased catalytic efficiency. Clarification of the aromatic-carbohydrate roles in mediating substrate binding and catalysis has paved the way for a more rational design of glycoside hydrolase.(3)Analysis of key amino acids in determining multifunctions in TrCel12ATrCel12A and HsCel12A enzyme show a high sequence similarity according to sequence alignment. Only 14 amino acid differences, but different functions occur. These residues appear as crucial for promiscuity. Mutated into T16I, S63V and A66N, analysis showed that all are decreased in the related xyloglucan activity.This reveal that further sequence classification have certain guiding significance to determine the substrate specificity.(4) The key amino acid and the substrate interaction analysis of TrCel12AThe highly conserved residue Asn at the position 20 in subsite (-2) of TrCel12A is likely to play a crucial role through the presence of possible hydrogen bonds with the 02 of glucose. When mutated to D, CMCase activity loses 48%,but no change has seen in enzymatic RAC product profile. Kinetic parameters show that Km increases, Kcat also increases, but Kcat/Km decline. Thermodynamic constants indicate the binding force weakened compared to WT.Contribution in the -2 subsite between Asp and Asn can be showen through N20 mutations.