Date of Award
Doctor of Philosophy
Food Science and Human Nutrition
Zivko L. Nikolov
We have produced intracellularly 8.8, 3.4, and 3.2 mg/l [beta]-galactosidase and two [beta]-galactosidase fusion proteins containing amino acids 498-616 (BSB119), and amino acids 537-616 (BSB80) of Aspergillus glucoamylase I, respectively. The fusion proteins suffered from extensive proteolytic degradation resulting in a mixture of full-size fusion proteins and [beta]-galactosidase. Granular starch adsorption study of these purified [beta]-galactosidase fusion proteins showed that BSB119 had the strongest interaction with granular starch;To overcome the proteolytic degradation problem associated with the [beta]-galactosidase fusion proteins, we have fused three different lengths of starch-binding fragments encoding amino acids 511-616, 495-616, and 481-616 of glucoamylase I to the C-terminal of the E. coli maltose-binding protein in vectors for both periplasmic and cytoplasmic expression. The fusion proteins produced in E. coli were purified by affinity chromatography. Factor X[subscript] a digestion of the fusion proteins resulted in the release of functional starch-binding fragments that were purified from maltose-binding protein on the basis of differential binding to cross-linked amylose. Immunoblot analysis, size exclusion chromatography, and amino acid analysis confirmed that the correct starch-binding fragments were produced. The first 23 amino acid sequence at the N-terminus of SBD122 corresponds to amino acids 495-517 of Aspergillus glucoamylase I. The C-terminus sequencing of maltose-binding protein possessing SBD122 showed that Arg was the C-terminus amino acid. SBD106, SBD122, and SBD136 bound specifically to [beta]-cyclodextrin with dissociation constant values of 34.3, 43.2, and 51.4 [mu]M, respectively. SBD106 and SBD122 also interacted with maltoheptaose with dissociation constant values of 540 and 570 [mu]M, respectively. The binding of these two ligands to the starch-binding fragments affected Trp and Tyr residues. Amino acids 511-616 of Aspergillus glucoamylase I contains the starch-binding domain;Clones were also constructed for the independent production of the three different-length starch-binding fragments (SBD[subscript]515-616, SBD[subscript]497-616, and SBD[subscript]483-616) in E. coli using the intracellular expression vector pKK233-2. The three starch-binding fragments were produced at very low levels. SBD[subscript]483-616 was expressed at a higher level than either SBD[subscript]515-616 or SBD[subscript]483-616, which were almost undetectable by immunoblotting.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Ann R. Kusnadi
Kusnadi, Ann R., "Characterization of the starch-binding domain of Aspergillus glucoamylase produced in Escherichia coli " (1992). Retrospective Theses and Dissertations. 10127.