Dynamic Behavior, Scission Mechanism And Reaction Kinetics During Enzymatic Hydrolysis Of Polysaccharides

In this thesis, enzymatic hydrolysis of celluloses with different crystallinities and Konjac Glucomannan (KGM) were systematically investigated by a combination of chromatographic and spectroscopic analysis. On the basis of enzyme mechanism and polysaccharides structure, the reaction behavior of celluloses and KGM during enzymatic hydrolysis was extensively studied. The main aspects and conclusions were displayed as follows.1. The multilevel structural nature, scission mechanism and reaction kineitcs during enzymatic hydrolysis of three kinds of celluloses with different crystallinities were studied and compared.(1) For Avicel, microcrystalline cellulose, a rapid hydrolysis rate occurred at the beginning of hydrolysis and then decreased for the time went up, with a sugar conversion of 59.04% after 72h. The molecular weight distribution (MWD) of original Avicel showed a single peak at MW=7.72×104, with polydispersity of 2.43. During enzymatic hydrolysis, no significant change in MWD of Avicel was observed, indicating"one by one"mechanism of cellobiohydrolase (CBH) for crystalline cellulose. The hydrodynamic radius (Rh) of Avicel decreased from 95.2 to 50.5nm, and there were no significant changes in chemical structure and crystalline pattern of Avicel during enzymatic hydrolysis.(2) For Whatman filter paper, cellulose involved both crystalline and amorphous region, the rate of hydrolysis was faster than that of Avicel. After 144h, the sugar conversion was 87.86%. The MWD of original filter paper exhibited a single peak centered over MW=1.06×106, which firstly became broader shifting to lower MW and then became narrower shifting to higher MW, and eventually returned the original position during enzymatic hydrolysis. It was attributed to the synergistic effect of"zipper"mechanism of endoglucanase (EG) and"one by one"mechanism of CBH with respect to amorphous and crystalline region of filter paper.(3) Two kinds of ionic liquids (ILs) 1-butyl-3-methylimidazolium chloride ([BMIM]Cl) and 1-allyl-3-methylimidazolium chloride ([AMIM]Cl) were synthesized and used to pretreat Avicel and Whatman filter paper to obtain amorphous celluloses. At the early stage of enzymatic hydrolysis, the MWD of IL-treated celluloses became broader and rapidly shifted to lower MW, suggesting the"zipper"mechanism of EG for amorphous celluloses. At the later reaction stage, MWD of IL-pretreated celluloses became narrower which did not change as time went up, indicating that there was still crystalline region with very low content in IL-pretreated celluloses. The sugar conversion of IL-celluloses was nearly 100% after 24h hydrolysis, which was attributed to the significant decrease of hydrogen bonding and crystalline degree of celluloses after IL-pretreatment.(4) The adsorption of enzyme to Avicel and Whatman filter paper followed Langmuir isotherm, however, the enzyme adsorption of IL-treated cellulose did not obey Langmuir isotherm. The adsorption models were 1ΓA = 0.04097 + 0.00589 [ E]f ,Aand 1ΓW = 0.02309 + 0.00477 [ E]f ,Was well as the kinetic models were 1 rA = 22.68ΓA+ 1.202 and 1 rW = 17.01ΓW + 0.6777 with respect to Avicel and Whatman filter paper. The reaction rate of filter paper was faster than that of Avicel. Besides, the reaction rate of IL-treated cellulose was much faster than that of the original cellulose.2. MWD evolution, enzyme scission mechanism, and reaction kinetics during enzymatic hydrolysis of KGM was studied.(1) During the enzymatic hydrolysis, the number- and weight-average molecular weight of KGM decreased rapidly with the MWD becoming broader firstly and then becoming narrower; the polydispersity index of KGM became larger at first and then became smaller, which did not obey the random scission mechanism; the enzyme mode ofβ-mannanase on KGM followed the mechanism of multiple scission associated with central scission.(2) On the experimental condition of fixed enzyme loading, based on the evolution of molecular weight and Michaelis-Menton equation, the kinetic models of KGM were established to simulate the enzymatic hydrolysis. For the initial stage of depolymerization, the kinetic model was M w 0 3 M w 3= 1+ M n 0k ct; for the late stage of depolymerization, the kinetic model was 1 M w = 1M w 0 + kM w0 2 m 2w0 ? t and degradation kinetics followed zero-order. It was demonstrated that the two models were in good agreement with the experimental results.(3) The diffusion analysis of KGM solution during enzymatic hydrolysis indicated the interactive movement restriction of molecules with the similar size. At the beginning of enzymatic hydrolysis, KGM molecules were in large-size, which would not hinder the enzyme molecules, resulting in a rapid reaction rate. As the hydrolysis continued, the size of KGM molecules decreased, which restricted the diffusion movement of enzyme molecules, slowing the reaction rate. On the experimental condition of fixed amount ratio of enzyme to substrate, a diffusion-reaction kinetic model: 01/ M t= 1/ M 0+ 17.175t ? 17.398e ?wt was established to demonstrate the influence of diffusion effect on the hydrolysis reaction.