Degree Type

Dissertation

Date of Award

1992

Degree Name

Doctor of Philosophy

Department

Food Science and Human Nutrition

First Advisor

Zivko L. Nikolov

Abstract

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.

DOI

https://doi.org/10.31274/rtd-180813-11548

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/

Copyright Owner

Ann R. Kusnadi

Language

en

Proquest ID

AAI9311507

File Format

application/pdf

File Size

159 pages

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