W. Somers

Crosslinked potato starch as an affinity adsorbent for bacterial α-amylase

Abstract 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. 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 in the degradation of the adsorbent by the enzyme. 1 g of a suitable adsorbent for bacterial α-amylase, prepared with an epichlorohydrin/glucose monomer ratio of 0·65 (starch concentration 150 mg/ml, ethanol/water ratio 2·0, sodium hydroxide/epichlorohydrin ratio 1·0), can adsorb 9·8 mg of an α-amylase from B. licheniformis at 4°C in 20 h. The equilibrium constant between bound and unbound α-amylase is dependent on the temperature. An effective desorption was possible by a shift to higher temperatures. Degradation values smaller than 0·1% were measured after an incubation of 1 h at 70°C in a desorption buffer with 20% glycerol. It was concluded that coulombic interactions and hydrogen bonds are of no or little importance in enzyme adsorption. Van der Waals forces, which are responsible for the large temperature effect, are the main forces in the interaction between α-amylase and crosslinked starch.

Developments in downstream processing of (poly)saccharide converting enzymes.

Recently novel techniques have been developed, based on liquid-liquid extractions and affinity interactions. Although they show considerable potential for the purification of several industrial enzymes they have not been widely introduced into existing processes yet. The introduction and applications of these techniques in the field of polysaccharidases are discussed

On the interaction of α-amylase with crosslinked starch: Evaluation of process conditions

Abstract The interaction of α-amylase with crosslinked starch is described. The adsorption characteristics are influenced especially by pH and temperature. Adsorption preferentially takes place at 4°C. The adsorption behavior corresponds with the catalytic activity of the enzymes studied. α-Amylase of Bacillus licheniformis , which has a broad pH optimum, adsorbs over a larger range (pH 5.0–9.0) than the α-amylase from Bacillus subtilis (pH 5.0–7.0). Capacities and Langmuir constants were determined in the relevant pH range. At pH values of 9.0–11.0 the catalytic activity and the adsorption levels drop, but the enzyme activity is not irreversibly lost. These conditions are used to recover the enzyme from the matrix. The crosslinked starch matrix is a competitive inhibitor for the enzyme in the enzyme assay. The K i was determined to be 6–8 g ml −1 for the inhibition of B. licheniformis α-amylase. The affinity for soluble starch appears to be approximately 30 times higher than for the matrix. As a result, limit dextrin solutions can be used as competitive eluents for the recovery of the enzyme from the adsorbent. A temperature shift from 4°C to 70°C can be used to recover the enzyme from the adsorbent, although this makes the matrix susceptible to biodegradation and enzyme activity is lost. The latter effect can be reduced by adding Ca 2+ to the system. Sodium chloride and glycerol have an influence on the interaction between α-amylase and the adsorbent. V max of the enzyme and the adsorption levels of α-amylase decrease among others as the water activity of the system is lowered. The matrix adsorbs a variety of α-amylases from bacterial and mammalian origin.

Crosslinked xylan as an affinity adsorbent for endo-xylanases

Abstract In order to facilitate the purification of xylanases from Aspergillus niger, an affinity adsorbent has been developed from oat spelts xylan. 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 (approximately 4%), no significant biodegradation was measured with a reused adsorbent. Up to 60% of the xylanase activity from an Aspergillus niger enzyme mixture (50 mU/ml) was adsorbed at pH 4 (50 m m sodium acetate buffer). 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%. 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 were also adsorbed to the matrix. 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.