|Title||Downstream processing of polysaccharide degrading enzymes by affinity chromatography|
|Source||Agricultural University. Promotor(en): K. van 't Riet; F.M. Rombouts. - S.l. : Somers - ISBN 9789054850540 - 163|
Laboratory of Genetics
Food Chemistry and Microbiology
Sub-department of Food and Bioprocess Engineering
|Publication type||Dissertation, internally prepared|
|Keyword(s)||fermentatie - voedselbiotechnologie - glycosidasen - chromatografie - enzymen - polysacchariden - affiniteitschromatografie - fermentation - food biotechnology - glycosidases - chromatography - enzymes - polysaccharides - affinity chromatography|
The objective of this study was the development of affinity matrices to isolate and purify a number of polysaccharide degrading enzymes and the application of these adsorbents in the large- scale purification of the enzymes from fermentation broths. Affinity adsorbents were developed for endo-polygalacturonase and α-amylase.
The isolation of two of these enzymes was realized using the specific affinity of the enzymes for the corresponding substrates, viz. pectate and starch. Normally interaction between an enzyme and its substrate is accompanied by hydrolysis of the polymeric substrate, resulting in total biodegradation. By specific modification of the substrate it is possible to obtain adsorbents which are capable of binding the enzyme while being resistant against biodegradation.
Pectate is the natural substrate for endo-polygalacturonase. Alginate, a substrate analogue for pectate, is able to bind endo-polygalacturonase while it is not hydrolyzed by the enzyme. Rigid beads can be obtained by calcium complexation of the alginate. The pH and ionic strength of the incubation medium influence the strength of the interaction between endo-polygalacturonase and alginate beads. Adsorption end desorption can be controlled by these two parameters. In this way the enzyme can be isolated and purified from complex mixtures. The adsorbent can be regenerated at least a hundred times in a continuous process (Chapter 2).
The adsorption of the enzyme to the matrix was subject of further study. By determining relevant mass transport parameters such as adsorption equilibrium parameters, diffusion coefficients and rate parameters it appeared to be possible to describe the adsorption process in mathematical terms. The velocity of adsorption is determined by the diffusion velocity of the enzyme in the beads and not by the reaction kinetics of the complex formation. The velocity of the desorption process is also determined by the diffusion velocity of the enzyme out of the bead (Chapter 3).
The most important substrate for α-amylase is starch. Alpha-amylase is used on a large scale for the enzymic conversion of starch into limit dextrins and other oligosaccharides. By means of a chemical crosslinking procedure of starch an adsorbent is obtained which is capable of binding the enzyme while it is degraded only to a limited extent. The adsorption and desorption characteristics of the interaction between enzyme and adsorbent were studied. It appears that the enzyme has the highest affinity for the adsorbent at the pH where it has its maximum catalytic activity. The interaction is biospecific and this principle allows a very selective isolation of the enzyme. The interaction between enzyme and adsorbent is essentially insensitive to changes in ionic strength of the medium. Desorption can be accomplished by a shift of pH or a raise in temperature of the incubation medium (Chapter 4).
The adsorption characteristics were further evaluated. Continuous use of the adsorbent in an isolation process of α-amylase results in a slow biodegradation of the matrix. This effect is accompanied by an increase of capacity of the adsorbent for the enzyme. It appears that the adsorbent can be used repeatedly for the isolation of the enzyme, the biodegradation is made up for by an improved mass transfer of the enzyme into the matrix combined with the increased capacity. The rate of adsorption is determined by the diffusion rate of the enzyme into the porous gel (Chapter 5).
For the direct application of adsorbents in fermentation broths a number of techniques have been proposed. One of these is the use of a fluid bed column. This imposes a few demands on the density and the diameter of the adsorbent. Particles, suitable for use in fluid bed columns were developed by inclusion of crosslinked starch in alginate particles and by the preparation of an alginate/starch copolymer bead. The adsorption characteristics of these adsorbents are comparable with those of crosslinked starch (Chapter 6).
In conclusion it can be stated that affinity separations for endo-polygalacturonase and α-amylase prove to be a selective process with good potential for a one step purification of these enzymes from a fermentation broth. In addition the procedure of adsorbent preparation offers good opportunities to prepare affinity adsorbents for other hydrolases.
This study was performed in a partnership between the Department of Food Science (Food and Bioprocess Engineering Group and Food Chemistry and Food Microbiology Group) and the Department of Genetics. The project was financed by the Netherlands Technology Foundation (STW).