| Bioethanol from lignocellulosic materials as the substitute of petrol is being studied in many countries. The process of bioethanol production contains four main steps: pretreatment, enzyme hydrolysis of cellulose, fermentation, ethanol distillation. The most important step is pretreatment of lignocellulosic materials. The purpose of pretreatment is to destruct physical and chemcial structures of the biomass which hinder enzyme hydrolysis, to destroy the lignin protection to cellulose, which boost the cellulose reacting sufficiently with cellulase. However, the residual lignin in pretreated cellulosic materials can adsorp the cellulase which is a complex system, and inhibit enzymatic hydrolysis of cellulose in solid residues from pretreatment process. Up to now, the characteristics and mechanism of adsorption of cellulase onto lignin is not clear. Properties and structure of lignin were also changed by different pretreatment process, which affect adsorption of cellulase. In view of the above reasons, the influence of the pretreatments on adsorption of cellulase enzymes onto lignin is researched in the thesis.Dilute acid pretreatment, alkali pretreatment and sulfite pretreatment were used in this paper, and chemical compositions of corn stover and lignin properties before and after pretreatment were firstly studied. It was found that hemicellulose degradation mainly occured during dilute acid pretreatment, and lignin content in solid residues decreased after alkali pretreatment and bisulfite pretreatment. At the same time, lignin structures also be changed after pretreatment, for example, dilute acid pretreatment makes lignin condensation, and alkali and sulfites pretreatment led to lignin degradation.In addition, adsorption experiments between cellulase from liquor fermentation of Penicillium A10-T and lignins showed that lignin extracted from preteated corn stover with dilute acid adsorps more cellulase enzymes compared to lignin from corn stover preteated by alkali and bisulfite pretreatment, and lignin from untreated corn stover has weak adsorption ability on cellulase. we also found that, for all the lignin above, the adsorbed proteins are mostly proteins with molecular weight of 25 to 70 kDa. As far as lignin samples used in the thesis is concerned, after adsorption experiments, the reduction of activity of CBH enzyme is the biggest, then EG, but changes on enzymes activities of Xyn and BGL are different. Lignin from dilute acid pretreated corn stover has the strongest influence on enzymes activities, then lignin from sulfite pretreated corn stover, and effect of lignin from untreated corn stover is the weakest.Combined with two-dimensional electrophoresis of crude enzyme and partial purification cellulase, we found that the adsorption characterization of cellulase onto different lignins is different. The lignin extracted from unpretreated corn straw mainly adsorbs xylanase and EG The lignin extracted from the dilute acid pretreated and alkali pretreated corn straw mainly adsorb CBH, xylanase and EG. In addition to CBH, EG, and Xyn three enzymes, the lignin extracted from bisulfite pretreated corn stover strong adsorbs amylase too. The study about adsorption of cellulase onto different types of lignin could help to look for some ways to decrease negative influence of residual lignin in pretreated corn stover on enzymatic hydrolysis with cellulase. |
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