Degree Type

Dissertation

Date of Award

1997

Degree Name

Doctor of Philosophy

Department

Theses & dissertations (Interdisciplinary)

First Advisor

Clark F. Ford

Abstract

Aspergillus awamori glucoamylase (GA) is an enzyme involved in industrial corn starch processing. Genetic and biochemical approaches were used to study the mechanisms governing GA thermostability. Three proline substitution (Xaa → Pro) mutations were constructed that were predicted to increase the enzyme's stability by decreasing its conformational entropy of unfolding. When expressed in Saccharomyces cerevisiae Ser30 → Pro increased, Asp345 → Pro did not alter and Glu408 → Pro greatly decreased GA stability as measured by resistance to irreversible thermoinactivation relative to the wild-type enzyme. The Ser30 → Pro mutation was combined with other previously identified stabilizing mutations to examine whether combining such mutations could cumulatively stabilize the GA. The Ser30 → Pro mutation cumulatively stabilized the enzyme when combined with the Asn20 → Cys/Ala27 → Cys mutations, which create a disulfide bond between positions 20 and 27. Similarly, when Ser30 → Pro was combined with a Gly 137 → Ala mutation the enzyme was cumulatively stabilized. The combined mutant Asn20 → Cys/Ala27 → Cys/Ser30 → Pro/Gly 137 → Ala was the most stable variant constructed and increased the enzyme's activation energy for thermoinactivation by 4.4 kJ/mol at 65°C and its melting temperature (the temperature at which the enzyme was 50% inactivated after 10 minutes) by 3.9°C relative to wild-type GA. None of the combined mutants decreased the enzyme's activity. The Asn20 → Cys/Ala27 → Cys/Ser30 → Pro/Gly 137 → Ala and Ser30 → Pro/Gly 137 → Ala mutants increased resistance to irreversible thermoinactivation in the presence of 1.71 M glucose and outperformed wild-type GA in a high-temperature (65°C) saccharification of DE 10 maltodextrin;Using random mutagenesis, an Asp238 → Asn mutation was identified that increased the expression of recombinant GA by S. cerevisiae when grown at elevated temperatures in both wild-type and Gly396 → Ser genetic backgrounds. This may be due to increased resistance to intra- and/or extra-cellular proteolysis conferred by the Asp238 → Asn amino acid substitution. The screening method used to identify the Asp238 → Asn mutation may be useful to identify other mutations which could increase the expression of other production-deficient GA mutants.

DOI

https://doi.org/10.31274/rtd-180813-13541

Publisher

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

Copyright Owner

Martin John Allen

Language

en

Proquest ID

AAI9814614

File Format

application/pdf

File Size

95 pages

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