Degree Type

Dissertation

Date of Award

1996

Degree Name

Doctor of Philosophy

Department

Chemical and Biological Engineering

First Advisor

Peter J. Reilly

Abstract

Glucoamylase (GA) is an industrial enzyme involved in the production of glucose and fructose syrups from starch. Exhibiting a large spectrum of selectivities, GAs are able to cleave glucose from the nonreducing ends of [alpha]-(1,4) glycosidic bonds of maltooligosaccharide chains and the [alpha]-(1,6) bonds initiating their branches. The high temperatures and glucose concentrations occurring in saccharification lead to the formation of condensation products, reducing the overall glucose yield. Modified versions of Aspergillus GA that better satisfy industrial requirements are desirable. To achieve this, a structure-based multisequence alignment of the primary sequences of the catalytic domains, linkers, and starch-binding domains of GAs from filamentous fungal, yeast, eubacterial, and archaeal origin has been made to correlate structure to GA stability and selectivity. Evolutionary interpretation of the alignment has elucidated the improvements undergone naturally by GA to improve its catalytic properties. To understand the molecular interactions between GA and its substrates, a protocol was formulated to combine the conformational characterization of substrates using MM3(92) and the Monte Carlo-based docking software AutoDock that allows the interaction of flexible ligands with proteins. This method was applied to study GA active-site interactions with (1) different inhibitors and monosaccharide substrates, yielding very good correspondence with results obtained by X-ray crystallography and so verifying the validity of the approach; (2) several isomaltose analogues, giving a structural basis for some unexplained kinetic properties; and (3) ten glucopyranosyl-based disaccharides, identifying the different requirements that substrates need to satisfy for GA hydrolysis to occur, and revealing that flexibility of the second subsite explains GA properties and preferences. The combined method constitutes a successful approach to study protein-carbohydrate interactions. Finally, an extensive table of GA kinetic and inhibition properties of different natural and genetically modified GAs has been compiled. An assessment of A. niger GA properties reveals that at higher temperatures larger amounts of condensation products are expected.

DOI

https://doi.org/10.31274/rtd-180813-10546

Publisher

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

Copyright Owner

Pedro Maldonado Coutinho

Language

en

Proquest ID

AAI9712549

File Format

application/pdf

File Size

233 pages

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