Improved Adsorption to Starch of a β‐Galactosidase Fusion Protein Containing the Starch‐Binding Domain fromAspergillusGlucoamylase

摘要

AbstractWe have previously shown (Chen et al., 1991) that a β‐galactosidase (β‐gal) fusion protein (BSB133) containing 133 amino acids (aa) from the C‐terminus ofAspergillusglucoamylase (GA) adsorbs strongly to starch compared to β‐gal, due to the presence of the GA starch‐binding domain. We have now made deletions at the N‐terminus of this 133‐aa region to test the minimal size required for starch binding of β‐gal fusion proteins. Three fusion proteins (BSB119, BSB103, and BSB80) were genetically engineered, containing 119, 103, and 80 C‐terminal aa from GA, respectively. The fusion proteins were expressed inEscherichia coliand purified. Purified BSB119 adsorbed to native starch at least 2‐fold more strongly than did BSB133 or fusion proteins with shorter tails. Adsorption isotherms generated over a wide range of initial concentrations indicated a 10‐fold difference in the loading capacity of starch for BSB119 (36.5 mg of protein/g of starch) compared to β‐gal (3.7 mg of protein/g of starch). Adsorption constants calculated from the initial slopes of the isotherms indicated a nearly 30‐fold difference in affinity to starch for BSB119(Kad= 63 mL/g of starch) compared to β‐gal(Kad= 2.3 mL/g of starch). BSB119 in the presence of crude enzyme extracts also bound to starch with a high affinity compared to a β‐gal control. Potential applications of the starch‐binding tail include enzyme immobilization to starch or recovery and purification