Active Site Architecture And Functional Analysis Of Key Subsites Of Endoglucanases In Trichoderma Reesei

Lignocellulose is an abundant renewable resource on the earth, and efficient conversion of lignocellulose has profound influence on sustainable development and agricultural modernization in our country. Cellulose, the most abundant polysaccharide in nature, constitutes the major component of plant cell walls, the yearly output of cellulose produced by photosynthesis is up to 1011t. Cellulases play an essential role in cellulose degradation, which are classified into different glycoside hydrolase families according to smiliarity of sequences, meanwhile, the space structures and catalytic mechanisms of cellulases in different glycoside hydrolase families show significant difference. The structural bioinformatics platform makes it possible to statistically analysis of cellulase sequences, structures and important amino acids in different glycoside hydrolase families, which provides theoretical foundation for rational design of cellulases. What’s more, it is helpful for us to understand the structure foundation of cellulases in identifying and catalyzing substrates. Based on the above thesis, the relationship of substrate distortion and cellulases classification of cellulases in GH5, GH7, GH12 families has been explored. Furthermore, the structural characteristics, sequence conservation and functions of key amino acids in active site architecture were also analyzed.This work tries to clarify the molecular mechanism of different cellulases in identifying and catalyzing substrates. The major results of the work are as follows: 1. The structural bioinformatics analysis of active site architecture in glycoside hydrolasesAt present, the glycoside hydrolases have been classified into 135 families according to sequence similarity. And specific space structures and catalytic models are characterized in each glycoside hydrolase family. Generally, cellulases have multi-domains, and the active site architectures of catalytic domain play a key role in substrate degradation. Analysis of sequence and structure characteristics in active site architectures is of great significance to study catalytic mechanism of cellulases. This article mainly studied GH5, GH7 and GH12 families, which revealed that cellulases in different families had obvious differences on substrate distortion. The frequency of amino acids in active site architecture of cellulases in the three glycoside hydrolase families had certain preference. The interaction between amino acids and substrates displayed obvious differences, which may be the structural foundation of cellualses in the three glycoside hydrolase families.2. Heterologous expression systems and enzymatic propertiesCellulase,7rCel5A(EGII) was heterologous expressed by E. coli and P. pastoris, respectively. We found that protein properties in two expression systems were similar. E. coli expression system was selected based on two aspects:First, the crystalline structure of cellulase 7rCel5A in PDB database was obtained by E. coli heterologous expression system; Second, the E. coli expression system had advantage in easy operation and short growth cycle. It is more achievable to quickly obtain diverse mutant proteins with the expression system, which could improve the efficiency of the experiment. It paved way for the rational design research of cellulases.3. The functional analysis of key amino acids in active site architecture of TrCel5AThe amino acids around the subsite(-2) of Tr-Cel5A with endoglucanase activity play an important role in recognizing and catalyzing substrates. Mutation experiments were conducted on subsite(-2) of active site architecture in TrCel5A(EGII). Enzyme activity determination experiments demonstrated that different mutations of amino acids resulting in varying degrees of activity change. Amino acids in -2 subsite of TrCe15A combined and catalyzed substrates by hydrophobic interaction. The change in binding force between amino acids and substrates leaded to a significant discrepancy in the proportion of products.4. Functional analysis of important amino acids in active site architecture of TrCe17BGH7 family is the biggest fungal glycoside hydrolase family. Conservative amino acids in active architecture form hydrogen bonds with hydroxyl on C2 and C3 of sugar rings, which play a critical role in binding and catalyzing cellulose. Different amino acids in the same subsite may perform specific functions. The residues Arg108 and Ser318 may have functions in combining substrates and stabilizing the enzyme-substrate transition state, while residue Tyrl46 may play a key role in inducing the substrate distortion. The oligosaccharide species and proportions in the product spectrums of the three mutations changed evidently,which is of great value in commercial application.