| The effective conversion of lignocellulose to ethanol and fuel is quite promising to solve the energy crisis in the future Thus, multifunctional glycoside hydrolases have earned themselves very important spots in the cellulosic biomass refinery The thesis mainly optimized the existing functional characterization platform of glycoside hydrolases for its further high-throughput detection and clarified catalytic mechanism of the functional promiscuity of GH12family Its clarification is not only of great significance for further increasing the accuracy of automatic functional prediction, but also layed the foundation for better understanding the structural and functional features of glycoside hydrolases. Active-site architecture of promiscuity can thus be rationally designed in the future.Based on this, works were carried out and the main results are as follows;1.Optimizing the native electrophoresis of glycoside hydrolases,its combination with digital image processing techniques lay the foundation for high-throughput demonstration of the intrinsic properties of carbohydrate-active enzymes.Native polyacrylamide gel was applied in the display of the enzymatic kinetics of glycoside hydrolases, including xylanase, β-glycosidase, endoglucanase and cellobiohydrolase, in the gel. Bands’ gray values were quantitatively extracted, and it showed that linear relations existed between bands’ gray values and the protein concentration or relative enzyme activities The pH or temperature profile of each component in multienzyme systems were comprehensively displayed by native electrophoresis combined with digital image processing techniques and obvious cooperation of the component was further proved in the polymer degradation. What’s more, the binding affinity of glycoside hydrolase can then be detected easily by comparing the relative migration distance before and after the addition of substrate in the separation gel. Therefore, optimization in native electrophoresis of glycoside hydrolases in this thesis will surely aid the high-throughput and comprehensive detection of the diversity as well as their intrinsic properties in nature. The screening speed of protein engineering will also be accelerated effectively meanwhile. Thus, it has great application values and practical significances.2. Tracing specificity determinants of glycoside hydrolase family12and active-site architecture based on this has converted the function rationally.The evolutionary history as well as the structural and functional features of GH12family have been collected and systematic analyzed using a phylogenomic approach. The specificity determinants of GH12family were exactly traced by constructing the phylogenetic tree and a local alignment of the active-site structure and its compositing residues. Active-site architectures of the two members in GH12family from Trichoderma reesei QM9414(EGⅢ) and Aspergillns niger CBS120.49(EglA) were carried out. It showed that the two carboxylates EI16and E200in EGIII, which were absolutely conservative in GH12family, catalyzed the hydrolysis of glycosidic bond, while the whole families’optimal pH activity depended on the N95site, which formed the only hydrogen bond with the nucleophilic E200. Meanwhile, the aromatic-carbohydrate ring π-stacking between W22and the glucose at-2site was proved to play an important role in the substrate recognizing and subsequent binding process, it can even be the government for the functional promiscuity. The N95D mutant of EGⅢ has shifted its optimal pH towards the acidic direction by0.6pH units and the catalytic efficiency of W22Y mutant on pNPC were enhanced by338%(for EGⅢ) and468%(for EglA) respectively. Meanwhile, it also showed that mutant W22Y has increased its binding capacity with various cellulosic substrates, possessing some processive properties for some extent. The results of fluorescence assisted analysis by carbohydrate gel electrophoresis (FACE) proved that lots of cellotetrose existed in the hydrolysis product of the mutant W22H on PASC, providing a rich resource of cellulase inducer. Besides, it also showed that there was an exquisite balance between the substrate binding and the product release. Rational design of the binding determinants can bring about effective changes in the enzymatic function, while enzymatic properties can be best engineered by making strategic amino acid substitutions at positions outside of the "core"’ active sites It provides important guidelines for further improvement of the glycoside hydrolases.3. Clarifying the functional promiscuity mechanism of GH12familyStructural mechanism for the functional promiscuity of GH12family lies on the partial recognition of function determining residues to the mutual motif of different binding substrate. The result of X-ray polycrystalline powder diffraction (XRD) on the degradated cellulose CF11showed that, it can break the well-organized microfibril structure to some extent, with the crystallinity degree decreasing Meanwhile, the diameter of the outmost layer microfibrilis was much bigger than that of the inner in the section based on the statistics through atomic force microscope (ATM) Its potential ability likewise "Expansin" was further proved by the unique binding pattern on microfibril. The clarification will surely lay the foundation for accurately predicting the potential function(s) of a given sequence or structure, which also provides ratioanl guides for the reconstituting cellulase cocktailIn a word, a method to exactly trace the (multi-) functional determinants was established based on the study of the whole family’ active sites. Architecture on the active groups can not only pave the way for clarifying its catalytic mechanism, which further improves the accuracy of automatic functional prediction, but also provide rational guides for its protein engineering Meanwhile, establishment of the high-throughput functional proteomics lays the foundation for its diversity demonstration, and the screening rate will also be accelerated effectively... |
PDF Full Text Request