| With the global economic development, the fossil resources are consumed quickly. Energy, resources and environment problems have become the major bottleneck restricting human social, economic and ecological sustainable development. Cellulose, the major component of plant cell walls, is the most abundant renewable carbon source on the earth. Making full use of cellulose can ease the environmental crisis caused by excessive use of fossil fuels. However, the effective utilization of cellulose is limited by the high cost of biodegradation.To utilize cellulose effectively, the study focus on building effective cellulases system since cellulases discoveried. It is generally believed that cellulase system mainly composed of glycoside hydrolases, oxidases and auxiliary degradation factors. And the glycoside hydrolases contains cellobiohydrolases (CBH), endoglucanases (EG) and β-glucanases (BG), etc. Compared with ceelobiohydrolases and β-glucanases which are both defined exactly, endoglucanases which often contains several enzymes in one system are not clearly distinguished. Meanwhile, substrate derivatives and analogues for cellulases activity measurement are not able to exactly reflect neither the activity of cellulases nor the roles and functions of each component of the cellulase system. Based on this, the work carried out and the major results are as follows:1. The dynamic expression of glycoside hydrolases was analyzed with secretomics analysis when T. reesei was cultivated on 14 kinds of different carbon sources. It was found that T. reesei could secrete different kinds of cellulases, especially for endoglucanases. This indicated the secretion of variety endoglucanases was due to the adaption of microorganism to diverse physiological conditions. And different endoglucanases were proved to have variety The function adaptions of different endogluanases to variety physiological functions.T. reesei was cultivated on different carbon sources. The reducing sugar, activities of exoglucanases, endoglucanases and xylanases was detected, and the cello-oligosaccharides over time course was analyzed with FACE. The result showed that the induce ability of cello-oligosaccharides with high DP was higher than that with low DP. On different carbon sources. The secreted time and component of endoglucanases was variety when T. reesei grow on different carbon source. This verified that endoglucanases secreted by one microorganism had different action modes. Three natural carbon sources (CSWB, RAC, Xylan) were taken to study the extracellular proteome of T. reesei. The proteome vary considerably. RAC and xylan mainly induced enzyme degraded cellulose and hemicellulose, respectively. CSWB could induced both cellulases and hemicellulases because of its complicate component. Endoglucanases secreted on these three carbon sources were different. This reflected the different physiological functions of endoglucanases. This study was conducive for building effective cellulase system from T. reesei.2. The method for determinating the action modes of cellulases quantitatively with Fluorescence-assisted carbohydrate electrophoresis (FACE). Obvious differences were first found among the action modes of endoglucanases.FACE is a sensitive, simple and relative high-throughput method. In this study, it was used to analyze the activities of cellulases. Concentration changes of oligosaccharides following hydrolysis of a carbohydrate polymer could be quantitatively measured continuously over time using the FACE method. FACE could cover the shortage of the method which measured the cellulase activity with the total mass of reduce sugar at the end point. Based on FACE method, cellobiohydrolase (CBH), endoglucanase (EG) and β-glucanase could be easily differentiated, realizing fast determination of cellulases types and action modes. Moreover, the products profiles of endoglucanases were quantitatively analyzed when amorphous cellulose (RAC) was substrate. This result first conformed the differences among the action modes of endoglucanases. Members from the same GH family (GH5) exhibited various product profiles. This result supplied a direct evidence for explaining why cellulolytic microorganism produced several endoglucanases. It indicated that only based on the sequence consistency to classified glycoside hydrolase was not enough. It could not describe the differences among glycoside hydrolases sufficiently. The analysis of different action modes was helpful for the classification of glycoside hydrolases, especially for endoglucanases.3. The dynamic product profiles of four endoglucanases secreted by Trichoderma reesei were anaylyzed with FACE. Combining the analysis of structure bioinformatics, it was found that endoglucanases with different action modes had a specific molecular structure basis. The structure information of active sites was analyzed.Four endoglucanases of T. reesei (EG Ⅰ, EG Ⅱ, EGⅢ, EG Ⅳ) were expressed heterogenously. Their product profiles of amorphous cellulose (RAC) and cello-oligosaccharides were studied. The results showed that endoglucanases did not simply bind and cut amorphous cellulose chain randomly, but had specific product profiles like CBH. With the comparative analysis, the minimum binding units of endoglucanases were different:EG Ⅰ had strong ability to bind and hydrolysis cellotriose. EG Ⅱ could bind and hydrolysis cellotriose, but the interaction was weaker than EG Ⅰ.EGIII could bind and hydrolysis cellotriose, but it is cellopentose for EGIV.The comparative analysis of three endoglucanases (EG Ⅰ, EGⅡ, EGⅢ) which had crystal structures was carried with structural bioinformatics. The active sites structures and surface potentials of three endoglucanases showed obviously differences. At the same time, the π-H interactions and H-bands between endoglucanase and cello-oligosaccharides were analyzed. The key residues in the interaction between endoglucanases and cello-oligosaccharides were defined. According to the distribution of these residues, the result was consequence with experiment. This study indicated endoglucanases secreted by one microorganism had different action modes and physiological functions.1. The fluorescence spectra method was used to quantitatively measure the kinetics of the cellulase binding to substrate, which supply a feasible method to study the interaction between protein and small molecule ligand. Through this analysis method, it was found that the activity of cellobiohydrolase could be overstated when pNPC was taken as substrate.Cellulases are able to recognize and bind with cellulose specificity. Tryptophan residues in the active sites of glucoside hydrolases play an important role in the process of substrate recognition. The fluorescence emission spectra of tryptophan residues is one wide band with the emission peak at 355nm when they are excited by 295nm exciting light. After tryptophan binding with polysaccharides, the fluorescence is quenched due to the changes of electron trajectory. The emission spectra has a clearly concentration quenching effect. Based on this, fluorescence spectra was used to quantitatively analyze the kinetic process of glucoside hydrolases binding to polysaccharides in this study. The quenching curve of substrate to tryptohpan was fitted by single/multiple sites binding equation. The quantitative relationship between single site specific binding and multiple sites nonspecific binding was clearly described. Through analyzing the single site specific binding, the kinetic params and free energy change were calculated accurately. Meanwhile, the limitation of p-nitrophenol-beta-D-cellobioside (pNPC) as specific substrate of cellobiohydrolase was analyzed. It was found that thee activity of cellobiohydrolase could be estimated with high positive deviance when pNPC was taken as substrate. The kinetic process of glucoside hydrolases binding to substrate could be accurately determined basing on the quantitative analysis of tryptophan fluorescence quenching. It was conducive to further research of the interaction between protein and polysaccharides, and supplied a feasible method to study the interaction between protein and small molecule ligand. |
PDF Full Text Request