A Novel Thermostable Cellulase And The Synergism In Enzymatic Hydrolysis Of Cellulose

Cellulosic biomass is considered to be the most abundant renewable resource and can be converted into fermentable sugars, biofuels, and other materials in an environmentally friendly way. Cellulose is the most abundant organic polymer on Earth; it is a linear homopolymer of glucose units and can theoretically be hydrolyzed to pure glucose. In the conversion of cellulosic biomass, cellulases are regarded as one of the key elements. Especially, thermostable cellulases have some significant advantages in the processes for converting cellulose to sugars, with their intrinsical stability and activities at high temperatures.In this thesis, a novel cellulase gene encoding a thermostable endoglucanase from the thermophilic eubacterium Fervidobacterium nodosum Rt17-B1 was cloned and expressed, which is the first cellulase cloned from the organisms of genus Fervidobacterium and designated as FnCel5A for being a member of glycoside hydrolase family 5, and the enzymatic properties were characterized. The cellulase was overproduced in Escherichia coli with a high protein content and good solubility in water, and could be easily purified. The purified recombinant cellulase shows high hydrolytic activities on carboxylmethyl cellulose, regenerated amorphous cellulose,β-D-glucan from barley and galactomannan, with the optimum temperature of 80–83°C and the optimum pH of 5.0–5.5. Furthermore, this enzyme is highly thermostable and has a half-life of 48 h at 80°C. With such a combination of thermostability and high activities, this cellulase is expected to be useful for hydrolysis of cellulosic and hemicellulosic substrates at high temperatures, and for industrial hydrolysis of cellulosic biomass during long-time processing at the elevated temperatures, particularly in converting biomass into biofuels.Natural cellulose in cellulosic biomass is primarily crystalline cellulose. Its cellulose molecular are long unbranched linear chains linked byβ-1,4-glycosidic bonds, and the chains are precisely arranged in parallel. As a result, they form a compact crystalline structure through strong interchain hydrogen bonding between adjacent chains. These hydrogen-bonding networks make crystalline cellulose insoluble, stable, less accessible, and resistant to degradation. No matter how high cellulases load, only a tiny part of crystalline cellulose is exposed to cellulases, and most cellulose chains have no contact with cellulases. The compactness and resistance of crystalline structures are the bottleneck in the efficient degradation of natural cellulose. For the efficient degradation and bioconversion of cellulosic biomass, it is even more important to efficiently disrupt and convert crystalline regions of cellulose into easily hydrolysable regions than to simply hydrolyze cellulose.Expansin-like proteins such as swollenins have been proven to have disruptive functions on lignocellulose including crystalline cellulose through some non-hydrolytic mechanisms. In this thesis, the swollenin from Trichoderma asperellum was first produced in E. coli. The recombinant protein was then refolded into the bioactive form with simultaneous purification via a novel cellulose-assisted process. In order to characterize the disruptive functions on crystalline cellulose, we introduced a novel, simple and efficient method for the quantitative determination of the non-hydrolytic disruptive activities, via the synergism of the swollenin and the endoglucanase FnCel5A. These methods and processes will be useful for the further study on non-hydrolytic disruptive bioactivities and provide novel approaches for the efficient and economical bioconversion of cellulosic biomass.Carbohydrate-binding modules (CBMs), which disrupt crystalline cellulose via non-hydrolytic mechanisms, are expected to overcome the bottleneck in the efficient degradation of cellulose. However, the lack of convenient methods for quantitative analysis of the disruptive functions of CBMs has hindered further systematic studies and molecular modifications. In this thesis, we established a practical and systematic platform for quantifying and comparing the non-hydrolytic disruptive activities of CBMs via the synergism of CBMs and a catalytic module within designed chimeric cellulase molecules. We also used bioinformatics and computational biology in our investigations. Combined with experiments, these computations lead to a deeper understanding of the relationships and synergisms between CBMs and catalytic modules, and are also helpful in designing and optimizing protein molecules. We constructed a convenient and reliable vector to serve as a cellulase matrix, into which heterologous CBM sequences can be easily inserted. The resulting chimeric cellulases were suitable for studying disruptive functions, and their activities quantitatively reflected the disruptive functions of CBMs on crystalline cellulose. In addition, this cellulase matrix can be used to construct novel chimeric cellulases with high hydrolytic activities on crystalline cellulose.