| The effective conversion of straw biomass resources to biofuels and biochemical provides a possible route to reduce the environmental pollution caused by burning straw. Currently why straw resources can not yet realize industrialization bottleneck is the high cost of lignocellulolytic enzymes and low catalytic efficiency. The catalytic process of enzymes includes the substrate binding and catalytic bond-breaking two processes. The recognition and binding to substrate is a prerequisite and basis for catalytic process, but also the rate-limiting step. Therefore, the bonding strength is the premise and foundation of high enzyme activity. The binding of enzyme and substrate is a physics course, and it does not involve the breaking of chemical bonds. The formed enzyme-substrate (ES) complex is is transient and unstable, which makes it difficult to determine the binding affinity.The thesis mainly optimized the existing high-throughput binding characterization platform of glycoside hydrolases, and to some extent, through the comparison and analysis of existing binding assay method, improved the binding assay precision and high throughput. We selected three representative cellulase domains, and measured their binding affinity to substrate by various methods, in order to further understand the various cellulase binding domain of the contribution of the direction. Research work carried out in this thesis and the main results are as follows:1. Optimizing the existing binding electrophoresis technology of glycoside hydrolases, its combination with image processing and quantitative analysis laid the foundation for rapid high-throughput characterization of the binding properties of glycoside hydrolases.Based on non-denaturing polyacrylamide gel electrophoresis technology, added a concentration gradient substrate in the polyacrylamide gels while preparation. The binding of enzyme and substrate performed during electrophoresis. We used digital image processing technology to get the relative mobility of protein bands and fit linear with CMC concentration. R2 value of the linear fit above 0.9 indicates the relative mobility of electrophoretic bands with CMC having a negative linear correlation. By comparing the change of relative mobility when CMC concentration increased, we can quickly analyze and compare the different binding affinity of enzymes and substrates. Therefore, optimization in binding electrophoresis of glycoside hydrolases in this thesis can not only qualitative analyze the catalytic glycoside hydrolase activity, and quantitative analysis the activity combined with substrate anti-leaching method*11, also quantitative characterization of binding affinity of non-catalytic glycoside hydrolases to substrate. In this method, it is based on non-denaturing polyacrylamide gel electrophoresis technology, therefore, small amount of sample is needed, and can greatly save enzyme and substrate, and can be used for rapid screening process after glycoside hydrolase engineered, so the technology establishment has important application value and practical significance.2. Determination binding affinity of three existing universal application were systematically analyzed and compared, and for the study to determine the binding force between the protein and carbohydrate provides guidance on the method.In this thesis, we start with ITC-one of the most commonly used and high precision methods, and discuss the advantages and disadvantages of ITC through comparing the binding affinity of the three representative cellulase binding domains and their mutants with the substrate. ITC is now the only access to all parameters in a single experiment, by measuring the heat change in the binding process, it is possible to accurately calculate the binding constant (Ka), enthalpy (ΔH) and entropy (ΔS). The parameters can be determined in the natural state, without fluorescent labeling or immobilization modified. However, ITC needs multiple pre-experiments to explore the most appropriate experimental conditions (for example:the optimal enzyme concentration, the optimum substrate concentration, etc.), and thus demands for large substrate and enzyme, and titration is a time-consuming process, you can only titration an enzyme with a substrate in one time. In order to compensate for the lack ITC method, we have established a binding electrophoresis. Binding electrophoresis is rapid, high-throughput, high sensitivity, and no complicated pre-test, the sample is less needed. Just not as good as the accuracy of ITC, therefore it can be used as a screening tool before ITC.Due to the low binding affinity between the carbohydrate binding module and the substrate, the sensitivity of Binding electrophoresis and ITC method is insufficient to detect it, while fluorescence spectroscopy can do. The method of fluorescence spectroscopy measures binding affinity by detecting the change in fluorescence spectra which is caused by the binding of tryptophan residues in enzyme active site and substrate. This method is more sensitive than ITC, and the operation is simple, less time consuming, need no complicated pre-experiment. But the parameter fluorescence spectroscopy used to characterize the binding affinity is an effective quenching constants, so we cannot obtain thermodynamic parameters like enthalpy, entropy, and it is more suitable for comparing the binding affinity of different enzymes or mutants and substrate.3. We selected three representative domains of cellulase, carried out bioinformatic analysis, and determined the binding affinity of these domains and substrate using three different methods, clarifying that different domains of cellulose play different roles in binding process.Most cellulases are multiple domains, including catalytic domain and a number of catalytically inactive carbohydrate-binding module. They perform different functions in the binding and catalysis of the enzyme and substrate. We selected three representative families:GH12 family, CBM4 family and CBM1 family as the research object, through bioinformatic analysis, constructed phylogenetic trees, and analyzed enzyme species in each family, indicating functional promiscuity of the three families. Through analyzing active architecture and surface potential, we know each family has a different substrate recognition and positioning mechanism; we constructed the sequence profile of active site, and analyzed subsites of binding substrate and the conservative amino acids. From the active architecture, we can rapidly screen for key amino acid residues in binding or catalytic process, in preparation for the follow-up study.We determined the binding affinity of three cellulose domains to substrate. CBM1 binding plane is formed by the three aromatic amino acids and bound to the surface of crystalline cellulose, its binding force is less than CBM4 which has a groove and can bind single cellulosic chain. CBM4 and GH12 family EGⅢ have the same binding type—groove-binding type, while binding force of EGⅢ to CMC is 10 times of CBM4 to CMC. From the sequence profile of active site, we can know within 5A of substrate, hydrogen bonds and π-H interactions between EGⅢ and CMC are all much more than CBM4, in which hydrogen bonds formed between polar amino acids and substrate and π-H interaction formed between aromatic amino acids and substrate.In short, we analyzed and compared three methods to detect binding affinity of protein and carbohydrate, and established a fast, high-throughput method to characterize the binding affinity, which provides a method support and paves the way for elucidating cellulase binding and catalytic mechanism, cellulase rational design and quickly select cellulase excellent mutants. What’s more, we established a method to exactly trace the functional determinants based on the study of the whole family’s active sites and different domains, and the study of binding affinity of different domains provides new ideas for the compound of glycoside hydrolases. |
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