Characterization And Crystallization Of Cellulases From Marine Aspergillus Niger

Lignocellulosic biomass is the most abundant renewable resources on the earth.The widespread applications of cellulase have important significances in the use of lignocellulosic biomass.But there were few reports on the efficient expression and structural analysis of cellulases.An Aspergillus niger ZJUBE-1 strain was isolated from the sludge in the East China Sea by our laboratory and the cellulases secreted by it had properties such as salt tolerance,heat resistance and so on.In this paper,the enzymatic characteristics and crystallization of the cellulases were studied.The main results are as follows:(1)Site-directed mutation:the enzymatic active sites and the three-dimensional structures of the cellulases were simulated by homology modeling and site-directed mutation.Three muteins were obtained.(2)Secretion and expression of cellulase components,characterization of enzymatic properties:endoglucanase EGL and cellobiohydrolase CBH were obtained by directional expression.Cellulases with a purity higher than 95%were acquired through fermentation,separation and purification.Then the enzymatic properties of EGL were studied.The results indicated that EGL has special properties of acidophilicity,salt tolerance and heat resistance.(3)Protein crystallization methods were explored,cellulases crystallization conditions were sceened and optimized:hen-egg white,Bacillus and human lysozymes were used as model enzymes.The effects of initial lysozyme concentrations,temperatures,pH values and crystallizing agents on the crystallization of three lysozymes were investigated by sitting drop vapor diffusion.The results showed that the optimal crystallization conditions for hen-egg white and Bacillus lysozymes are as follows:the initial concentration of lysozyme is 40 mg/ml,at 25?,the pH value is 5.5,the crystallizing agents are 0.1 M sodium acetate and 2.0 M sodium formate.The optimal crystallization conditions for human lysozyme were as follows:the initial concentration of lysozyme is 80 mg/ml,at 25?,the pH value is 6.1,the crystallizing agents are 0.1 M bis-tris and 2 M ammonium sulfate.The crystal forms of hen-egg white,Bacillus and human lysozymes were squares,plates and needles,respectively.Screening and optimization of high-purity cellulases' crystallization conditions were conducted by sitting drop vapor diffusion.The results showed that the endoglucanase EGL successfully grow the initial crystals under two crystallization conditions.The first one were 0.2 M triammonium citrate,0.1 M imidazole,20%(w/v)PEG 2K-MME and pH 7.1.And the second one were 18%(v/v)isopropanol,0.1 M disodium citrate,20%(w/v)PEG 4000,pH 6.5.The crystal forms were plates and needles,respectively.This paper focused on the homology modeling,site-directed mutation,fermentation,purification,enzymatic characterization and crystallization.Acquirement of enzymatic active sites,high-purity fermentation broth and crystallization conditions of cellulases in this thesis would be useful to provide ideas for the structural identification of the enzymes and lay the foundation.