Time-dependent Nature In Enzymatic Hydrolysis Of Konjac Glucomannan

During the past decades, considerable interests have developed in enzymatic hydrolysis of polysaccharides, because of its great industrial applications ranging from bioenergy refining, oil/gas exploration, to drug release and food additives. In this thesis, based on the analysis of characteristics of several polymer enzymatic depolymerization, enzymatic degradation of konjac glucomanan (KGM) byβ-mannanase was systematically investigated by using gel permeation chromatography (GPC) and dynamic light scattering (DLS), including changes in number- and weight-average molecular weights, molecular weight distribution (MWD), the diffusion behaviors of enzyme and substrates, and bond scission manner of enzyme. The main aspects and conclusions were displayed as follows:Analysis in typical kinetics of depolymerization: General reaction kinetic equation told number-average or weight-average molecular weight ( M n, M w) had linear relationship with time, no matter which number the order was. The slop of line between surface reaction rate ks and concentration C had direct relationship with the reaction order. In the mathematic zero-order kinetic, 1/ M nand M wboth were linear functions of time, polydispersity index (PI) had a parabolic distribution during the reaction. MM kinetic showed M w3 and M n had the similar reaction kinetics, also gave the solution of PI for the case of last reaction period.Time-dependent nature in enzymatic hydrolysis of KGM: The changes in molecular weight of hydrolysis at given times were studied by GPC. The high was the concentration of KGM, the fast was the decrease of molecular weight in the hydrolysis with constant ratio of enzyme to substrate, which showed that the reaction was related to the diffusion of enzyme; MWD gradually fragmented into double parts, in the macromolecular part, molecular weight had little change while whose mass is decreasing, indicating the fragmentation of multi-scission probability. The analytical solution for the kinetics showed at the beginning the reaction which was dependent on the net structure of system, was a surface reaction; in the middle time, the change of molecular explained a zero-order reaction; in the latter period, the diffusion of enzyme had significant effect on the hydrolysis.Reaction-diffusion kinetics for enzymatic degradation of KGM: integrating the empirical reaction kinetics and diffusion characteristics, a reaction-diffusion kinetic model ( ) was deduced from a series of chromatographic results. The degradation rate was shown to be greatly affected by the diffusion of enzyme as well as the concentration of substrate. This equation of reaction kinetic presented the importance of diffusion, and the experimental data can be well predicted at the semi dilute concentrations (0.1%~0.5%) of KGM.Diffusion behavior of KGM during enzymatic hydrolysis: The diffusion of KGM during the whole reaction term obtained by DLS showed it's an unsteady period in the earlier time, major mover consisted of the biggest part and the smallest part of molecules, and biggest molecules prevented the middle ones'movement, but not small ones'. As big ones were not enough to cover the whole surface, the molecules with medium size were the most active, meantime kept the smaller ones down. All above suggested system structure was mostly dependent on macromolecules.Analysis in bond scission: The results from computer simulation showed that the initial molecular distribution had significant effect on the variation of polymer undergoing degradation:Narrow peak was degraded into a lower one. In three modes (random, Gaussian, central), the central scission mode had the fastest change in MWD, which also had remarkable behavior of two peaks during the enzymatic degradation of polymer with narrow MWD. To certain extent, there were some similar between the experimental data of enzymatic hydrolysis of KGM with the simulated data based on central scission, such as double peaks trend. The differences from them further verified that multi-scission and central scission combined to work in enzymatic degradation of KGM.