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Improved Adsorption to Starch of a β‐Galactosidase Fusion Protein Containing the Starch‐Binding Domain from Aspergillus Glucoamylase
Author(s) -
Chen Luojing,
Ford Clark,
Kusnadi Ann,
Nikolov Zivko L.
Publication year - 1991
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp00009a004
Subject(s) - starch , chemistry , biochemistry , fusion protein , enzyme , escherichia coli , adsorption , carbohydrate binding module , chromatography , glycoside hydrolase , recombinant dna , organic chemistry , gene
We 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 of Aspergillus glucoamylase (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 in Escherichia coli and 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 (K ad = 63 mL/g of starch) compared to β‐gal (K ad = 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 of target proteins from crude extracts.