Investigations On The Properties Of Cellulases From Filamentous Fungi And Optimization Of Enzymatic Lignocellulose Hydrolysis Process

The human society has been facing a severe energy crisis accompanying the depletion of traditional fossil fuels, making the search for renewable energy sources a focus of investigations. The lignocellulosic biomass is an excellent candidate energy source because it is abundantly distributed, generally cheap, and mostly from agricultural residues. Producing renewable energy from lignocellulosic biomass not only relieves the world from current energy crisis, but also significantly decreases environmental pollution, in particular the production of greenhouse gases. This environment friendly energy source has received widespread attention. However, two key technical problems still exist:the high cost and low efficiency of lignocellulose degrading enzymes, as well as the large dosage of enzymes required for lignocellulose degradation. This thesis documents the studies on three aspects aiming at improving cellulase efficiency, improvement of ethanol production and lowering ethanol production cost:1) identification of and relief from metal ion induced cellulase-catalyzed cellulose hydrolysis;2) identification of cellulase synergism;3) optimization of the cellulose hydrolysis process during lignocellulosic ethanol production. The results obtained from these studies include:1The impact of metal ions on enzyme-catalyzed cellulose hydrolysis and the removal of metal ion-induced inhibitionMetal ions can largely impact, or even determine the activities of enzymes. During cellulase-catalyzed cellulose degradation, the presence of metal ions is inevitable. Our research investigated this metal ion impact on cellulase-catalyzed cellulose hydrolysis, in particular the impact of Fe3+on reactions catalyzed by cellulases from P. decumbens JUA10-1. We first identified the impact of metal ions on the activities of the cellulases, showing a varied degree of impact for different metal ions, out of which Fe3+leads to the highest level of inhibition. We subsequently investigated the mechanism underlying the inhibition of Fe3+on lignocellulose degradation catalyzed by cellulases from P. decumbens. The inhibitory effects are shown to be present on two different levels:Fe3+inhibits the reaction by reacting on the reducing ends of the substrate and improves the resistance to degradation; Fe3+also inhibits the catalytic abilities of cellulases by competing for enzyme binding with the substrate. Finally, we identified two approaches to abolish the inhibitory effect of Fe3+:the addition of reducing reagent DTT or chelating reagent EDTA.2Synergism between cellulases from Trichoderma reeseil Penicillium decumbens and Aspergillus aculeatusThe P-glucosidase from A. aculeatus ZLF was purified to apparent homogeneity in this study. We further identified the synergistic effects between T. reesei cellulases or A. aculeatus cellulases and β-glucosidases. We found that both A. aculeatus cellulases show identical synergistic effects with cellulases from T. reesei. Our further investigations on the enzymatic activities of cellulase cocktails showed that the improvement of β-glucosidase activities leads to the increase of cellulose hydrolysis, while the filter paperase activity (FPA) of the cellulase cocktails stayed unchanged. The FPA was assayed by using filter paper as the substrate, which has a much simpler structure in comparison with natural substrates and more susceptible to enzymatic degradation. The FPA can therefore not represent the’true’abilities to hydrolyze cellulose for cellulases. For P. decumbens JUA10-1cellulases, although a higher level of β-glucosidase activity is present in the enzyme, supplementation of A. aculeatus ZLF cellulases can still improve the ability of the enzyme cocktails to hydrolyze cellulose. This synergistic effect is only obvious when delignined corncob residues serve as the substrate, while it is not apparent for substrates containing a higher level of lignin such as corncob residues.3Optimization of the cellulose degradation process during lignocellulosic ethanol productionThe low efficiency and high dosage requirement of cellulases have become the biggest problems during lignocellulosic ethanol production. We tried to optimize the cellulose degradation process by changing enzyme loading and substrates, in order to find solutions to these problems and optimize the lignocellulosic ethanol production process. We first used steam exploded Arundo donax Linn or delignined corncob residues as the substrate, changed the enzyme loadings, and carried out saccharification experiments at45℃. We found that both substrates and saccharification performance ceases to improve at an enzyme loading of90mg/g substrate, and the enzymes would be wasted when more enzymes were used. We further characterized the fed batch process and using cellulase cocktails for lignocellulosic ethanol production, and found both improve the production of lignocellulosic ethanol.