DEVELOPMENT OF AQUEOUS SIZE EXCLUSION CHROMATOGRAPHY FOR THE STUDY OF SUBSTRATE STRUCTURE AND SUSCEPTIBILITY IN ENZYMATIC STARCH HYDROLYSIS
Abstract
A physical model of starch structure during hydrolysis is presented. The model states that various crystalline states of the constituent starch molecules are present throughout the course of degradation. These crystalline states are associated with the natural state of the strach granule as well as with recrystallized states caused by retrogradation. In order to properly describe enzymatic starch liquefaction, these crystalline states must be accounted for. An appropriate means of determining the macromolecular size and molecular size distribution of starch during hydrolysis, aqueous size exclusion chromatography, is developed. The method employs a strong alkaline mobile phase and porous gel chromatographic supports which are stable to the basic solutions. The separation range of molecular sizes can be extended by coupling size exclusion chromatographic columns in a manner that will maintain linearity in molecular size separation with elution volume. Although separation efficiency is mass transfer limited, efficiency of separation can be increased by controlling eluent flow rate and gel particle packing size and packing size distribution. Although the size exclusion chromatographic system cannot be calibrated directly using well characterized starches due to their non-availability, secondary calibrants can be used for this purpose. Commercially available polyelectrolytes (sodium polystyrene sulfonates) and polysaccharides (dextrans) are appropriate calibrants for aqueous size exclusion chromatography, provided the basic phenomena governing separation are understood. Molecular size in solution, as measured by hydrodynamic volume, controls separation by size exclusion. Excluded volume effects are incorporated into the calculation of the molecular hydrodynamic volume. The electrostatic interactions present in polyelectrolytes are modeled as an excluded volume effect. When the hydrodynamic volume of the two secondary standards is determined using these concepts, a common calibration curve results for moderate to high ionic strength alkaline solutions. Other effects dominate for basic solutions of low ionic strength and for eluents with different co-solutes. Monitoring enzymatic starch depolymerization by aqueous size exclusion chromatography shows that molecular size distribution, reaction extent and reaction rate strongly depends upon the crystalline state of the substrate and the activity of the enzyme. The susceptibility of starch to hydrolysis by (alpha)-amylase is closely related to the crystalline states of the substrate. Starch crystals are present both in the natural granule structure and in retrograded starch; retrogradation lowers reaction extent. Due to thermal deactivation, the highest overall conversion achieved for a given amount of enzyme is obtained at temperatures below the upper limits of the gelatinization range of starch. The initial rate of the reaction is greatest at temperatures above the gelatinization range. The rate and extent of depolymerization is increased by increasing the enzyme concentration within the reactor or by the addition of certain stabilizing agents.
Degree
Ph.D.
Subject Area
Chemical engineering
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