Affinity purification of polysaccharide degrading enzymes with crosslinked substrates

H.J. Rozie

Research output: Thesisinternal PhD, WU


<p>The aim of this work was to find economically favourable, affinity based, purification methods for several polysaccharide splitting bulk enzymes. The framework in which this study is done is described in Chapter 1.<p>Chapter 2 describes the adsorption of endo-polygalacturonase (endoPG) from a commercial enzyme preparation (Rapidase) to calcium alginate beads. Approximately 75% of the various polygalacturonase activities from Rapidase can be adsorbed at pH 4.4 by calcium alginate beads as well as by crosslinked sodium alginate powder. Equilibrium experiments were conducted to determine a parameter (k) that represents the degree of interaction between endoPG and the adsorbent. This parameter can be influenced by a change in pH and ionic strength of the adsorbate. At pH 3.8 the degree of interaction is 20 times larger than at pH 4.2. There is increased adsorption when the ionic strength is lowered, but a small amount of CaCl <sub><font size="-2">2</font></sub> is required to keep the calcium alginate beads stable.<p>Despite the resemblance in structure between L-guluronate blocks and polygalacturonate, a lower k value was found when the alginate, used for the preparation of the beads, contained a larger proportion of guluronic acid residues. There is no evidence that L-guluronic blocks in the alginate chain are responsible for the large affinity of endo-PG to this adsorbent. The influence of the pH and the ionic strength and the lack of endoPG inhibition by sodium alginate are indicative for ionic interactions between endoPG and the alginate chains.<p>Ionic interactions were of no importance in the interaction between ct-amylase and crosslinked starch as is described in the chapters 3 and 4. Crosslinked potato starch was prepared as an affinity adsorbent for bacterial α-amylase. To this end, reaction parameters for crosslinking in an ethanol/water solvent were investigated (Chapter 3). The degree of crosslinking, and consequently the suitability of crosslinked starch as an adsorbent for α-amylase, changed by altering these parameters. An increase in the degree of crosslinking of the adsorbent caused lower affinity for bacterial α-amylase which resulted in an unfavourable decrease in adsorption capacity and a favourable decrease of degradation of the adsorbent by the enzyme.<p>The adsorption and desorption characteristics of two bacterial α-amylases <em>(B.subtilis, B.licheniformis)</em> on crosslinked potato starch are described in Chapter 4. A capacity of about 185 mg <em>(B.subtilis)</em> and 71 mg <em>(B.licheniformis)</em> protein per g adsorbent can be realized. However, at 4 °C a smaller adsorption constant (K <sub><font size="-2">a</font></sub> ) was measured for the enzyme from <em>B.subtilis</em> (0.53 * 10 <sup><font size="-2">5</font></SUP>L/mole) than for the <em>B.licheniformis</em> enzyme (3.8 * 10 <sup><font size="-2">5</font></SUP>L/mole). The K <sub><font size="-2">a</font></sub> decreases with increasing temperature suggesting that association is caused by van der Waals forces. Comparison of the adsorption of the α-amylases to crosslinked starch with the activity of the enzymes on their natural substrate reveals that the velocity constant of the backward reaction of the enzyme-adsorbent complex increases strongly with increasing temperatures <em>(B.subtilis α</em> -amylase, k <sub><font size="-2">2</font></sub> (20 °C)/ k <sub><font size="-2">2</font></sub> (4 °C) ≈30). Desorption can be accomplished by a raise in temperature. Glycerol (20%) is added to the desorption buffer to stabilize the enzymes and protect the adsorbent against enzymic attack. The optimal desorption temperature for the <em>B.subtilis</em> enzyme is 60 °C. For the <em>B.licheniformis</em> enzyme this value is 70 °C or even higher. The adsorption velocity of α-amylases to freshly crosslinked starch is low due to the low accessibility of the adsorbent. This can easily be improved by enzymatic modification. Thus, bacterial α-amylases can be adsorbed and desorbed within short time spans (10 min) in sufficiently high amounts to make such an affinity purification process economically feasible.<p>In order to facilitate the purification of xylanases from <em>Aspergillus niger,</em> an affinity adsorbent has been developed from oat spelts xylan (Chapter 5). A suitable adsorbent was only obtained by crosslinking oat spelts xylan with epichlorohydrin in water but not in ethanol or ethanol water mixtures. After some initial degradation of the adsorbent (approx. 4%), no further biodegradation was measured with a reused adsorbent. Up to 60% of the xylanase activity from an <em>Aspergillus niger</em> enzyme mixture (50 mU/ml) was adsorbed at pH 4 The degree of adsorption to crosslinked xylan of four fractions of this preparation, previously separated by DEAE-Biogel A chromatography, varied between 40 and 90%.<p>Adsorption was strongly dependent on pH and ionic strength and desorption was easily accomplished by an increase in ionic strength. In addition to xylanases, polygalacturonases also adsorbed to the matrix probably due to the D-glucuronic acid moieties in xylan. No significant adsorption of β-D-xylosidase, α-L-arabinofuranosidase, β-D-galactosidase, β-(1,4)- galactanase, β-(1-3/6)-D-galactanase or cellulase activities was found.<p>The binding behaviour of four commercial fungal enzyme preparations on crosslinked xylan is presented in Chapter 6. The xylanase activity in Pectinol Al (Röhm GmbH, Darmstadt, Germany) was efficiently purified with crosslinked xylan. The specific endo-xylanase activity increased from 5.5 U/mg up to 160 U/mg. Two proteins were found with SDS-PAGE in purified Pectinol (29 and 51 kD) whereas a K <sub><font size="-2">m</font></sub> of 1.1 mg/ml was measured. Equilibrium adsorption studies revealed a rather low capacity for the Pectinol endo- xylanase (1.5 mg xylanase/g adsorbent). The calculated K <sub><font size="-2">a</font></sub> was 4 * 10 <sup><font size="-2">6</font></SUP>L/mole.<p>Some endo-xylanases were also adsorbed by cation exchange material. However, from crosslinked xylan chromatography and additional FPLC studies it appeared that the adsorption properties of crosslinked xylan were not only due to the cation-binding properties of this adsorbent.<p>Chapter 7 is an evaluation of the foregoing chapters. The adsorption properties of three kinds of economically important polysaccharide splitting enzymes are studied in this work. A cheap substrate analog and crosslinked substrates were used as adsorbents. The magnitude of the capacities of calcium alginate and crosslinked starch towards endo-polygalacturonases and α-amylases, respectively, is such that commercial applications can be considered. Only laboratory applications are foreseen for crosslinked xylan as affinity adsorbent for specific endo-xylanases since the capacity of this adsorbent is rather low.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Rombouts, F.M., Promotor, External person
Award date11 Dec 1992
Place of PublicationS.l.
Print ISBNs9789054850533
Publication statusPublished - 1992


  • fermentation
  • food biotechnology
  • glycosidases
  • adsorption
  • adsorbents


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