| Cellulosic biomass from plants is the most abundant renewable resource on the earth. With the running out of nonrenewable fossil resource, more and more attention has been paid to the use of cellulosic biomass these years. However, people still do not understand cellulases well. And it is still unable to obtain general rules. So using bioinformatics analysis the relationship among the sequences, structures and function of enzyme molecules will not only help elucidating its catalytic mechanism, but aslo help looking for general rules. Based on structural bioinformatic analysis, the design of the glycoside hydrolase GH12family and the key amino acids functional analysis have been carried out in this work with the purpose of preliminarily elucidating its binding mechanism and catalytic progress. The major results of this thesis are as follows:1. Active-site sequence profile of GH12family and the analysis of the key amino acids.The memebers that have functional annotation in GH12family are distributed in Bacteria, Archaea. and Eukaryota. From the NJ tree of these proteins we find that there are far evolutionary relationships between them. The topological structure is-jelly roll, with a catalytic groove in its ventral side that can accommodate six glucose residues carrying the strong negative charge. Moreover, two Glu catalytic sites are located in the central point of the catalytic groove. Structure alignments of proteins in GH12family displays that little spatial differences exist in these structures. Then we bulit the active-site sequence profile of the GH12family by structural alignments and tested its validity. The method, which has family specificity, can reduce the intensity of mutation greatly which postion the functional residues and spatial combination fast in this profile. Based on the conservative degree of the amino acids in the catalytic pore and the spatial information of superimposed structures, we choose10key amino acids within the profile contained25amino acids. Then we conjecture that the sites (N95, D99, M11.8, E116and E200) are related to the catalysis while the sites (N20, W22) are related to the substrate binding.2. Heterologous expression of GH12family and enzymatic propertiesCellulase EGⅢand EglA of GH12family were heterologous expressed by Pichia pastoris and E. Coli respectively. We found that cellulase EGIIIwas not induced in E. Coli with extremely low content in periplasmic space. While the molecular weight protein cellulase EglA induced in E. Coli is bigger than theoretical value and has a10%lower in activity, with partial content forming inclusion body. To the contrast, the two proteins were expressed in Pichia pastoris, whose endoglucanase is similar to protein reduced in primary strains. Experiments determinated that the optimum pH of EglA is3.9while EGIIIis5.0. Their optium temperature are all50℃. Therefore, Pichia pastoris expression system is more suitable for GH12family cellulases, laying a technological platform for subsequent directed mutation research.3. The alanine screening of key amino acids of GH12family active site and property analysisRepresented by EGⅢ in GH12family enzymes, some key amino acid sites N20, Y60, D99, M118, P129alanine screening, found that mutations had a loss in cellulase activity. So key amino acids in active site play a decisive role in the enzyme function. And different damage degrees illustrate that different amino acids in active-site play different functions. The key amino acids are located in the inner side of the groove which interact with one side of glucose residue, result in endoglucanase and xyloglucanase. With the similar catalytic properties in different substrates, it demonstrates that the spacical combination of key amino acids is the common structural foundation of recognizing different substrates.4. The functional analysis of the key animo acids in GH12family active-siteWith analysis of amino acid residues N20, Y60, D99enzyme activity decreased more obviously through alanine screening. The strictly conserved D99hydrogen bonds to the nucleophile E116. Mutated into D99A, D99E and D99N, analysis shows that all are lost in the related hydrolytic activity, and the catalytic mechanism of GH12 family is a carboxylic acid trio. When Y60is mutated to A, the activity has a sharp decrease, while Y60is mutated to F, the activity is consistent with the wild-type enzyme. With third-structure study speculats that it does not participate in the formation of the PI-H, which plays an important role in maintaining the correct conformation and trajectory of the substrate. N20, a residue interacting with Glc(-2), playing a important role in substrate binding. When mutated to D endocellulase activity loses48%, it shows that hydrogen bond binding force weaken. While N20is mutated to A, the product spectrum changes without monosaccharides, and enhance oligosaccharide content by530%, which implied that function evloves fast from EC3.2.1.74to EC3.2.1.4and it has a commercial value. |
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