Construction Of Endoglucanases Library And Characterization Of Enzymatic Properties

Lignocellulose is the most abundant biopolymer on earth and considered to be an important alternative to non-renewable fossil fuel resources. However, the high cost to convert the recalcitrant cellulosic biomass into sugars is the key bottleneck that requires new biotechnological solutions. To improve the efficiency, cellulases are regarded as one of the key elements. Specially, the attention has been paid to cellulases from extremophiles due to their better performances adapted to the extreme conditions such as high temperatures, low or high pH, and hypersaline concentrations.Cellulases involved in hydrolyzing cellulose include three major types:endoglucanases, exoglucanases and β-glucosidases. Endoglucanases are usually considered to be synergistically active in the first stage of cellulose breakdown. There are abundant cellulose decomposition microbes and complex cellulase system in nature. In this study,55endoglucanase genes from open database such as NCBI, SWISS-PROT were screened to be cloned and expressed in Escherichia coli. The recombinant proteins with endoglucanase activity were identified. The enzymes with better performance were further purified and characterized.Cel5H from Dictyoglomus thermophilum was highly tolerant to high salt concentration although the original organism is not a salt-tolerant bacterium. Meanwhile, Cel5H distinguished it from other salt-tolerant protein as it was independent of high salt concentration. To explore the mechanism, three-dimensional structure of Cel5H was developed by homology modeling and surface electrostatic analysis. It could be inferred that a certain amount of acidic amino acids on the surface guaranteed the enzymatic properties of salt-independant and salt-tolerant.Most cellulases are typically modular enzymes with a catalytic module (CM) and several carbohydrate-binding modules (CBMs). To enhance the performance of endoglucanases,6chimeric enzymes of syncretic CBMs were construted. CBMs from different sources at different locations (N-or C-terminus of the catalytic domain) were investigated and all the CBMs were effective.In addition, the synergistic effect of cellulases was also explored preliminarily. The modified endoglucanase Q9LCC5-CBMRa and CBMRa-Q9LCC5together with exoglucanase CbhA showed remarkable synergy effect.