Date of Award
Doctor of Philosophy
Zoology and Genetics
Clark F. Ford
To study the molecular basis of Aspergillus awamori glucoamylase (GA) thermostability, eighteen mutants were constructed by site-directed mutagenesis and expressed in Saccharomyces cerevisiae based on thermostability theories. Four lysine residues, K61, K279, K352 and K404, were replaced with arginine, with all but K404 well exposed to the solvent and far away from the enzyme activity site. Mutations K61F/D65E and H254W/E326Q were made to fill a packing void around the inner set of six [alpha]-helices of GA and to displace water molecules inside the void. Five residues (A27, A393, A435, Ser436 and Ser460) were replaced with proline. Two additional disulfide bonds and combined thermostable mutations were engineered at position 20,27 and 72,471, respectively. Finally, The disulfide bond mutant A27C/N20C was combined with two other mutants which have been shown to increase thermostability, G137A (Chen et al., Protein Eng., 9, 499-505) and S436P. The mutants K61R, K352R, K404R, K61F/D65E, A27P, A393P, A435P, S436P, A27C/N20C, A471C/T72C, A27C/N20C/S436P, G137A/S436P and A27C/N20C/G137A had the similar specific activities with the wild-type (WT) GA, while mutants K279R, H254W/E326Q, S460P, A27C and N20C had decreased specific activity. The mutant H254W/E326Q affected the correct folding of GA as demonstrated by circular dichroism (CD) spectrum. Mutants S436P, A27C/N20C, A27C/N20C/G137A, G137A/S436P were more thermostable than WT GA, while mutants K352R and A27C were slightly more thermostable than WT, mutants K279R, K404R, A435P and A472C/T72C had the similar thermostability with WT, and mutants K61R, K61F/D65E, A27P, A393P, S460P and N20C were less stable than WT. Mutants A435P and S436P were more resistant to chemical guanidine hydrochloride (Gdn·HCl) unfolding than WT, whereas mutants A27P, A393P and S460P were more susceptible to chemical unfolding caused by Gdn·HCl than WT GA, as demonstrated by CD spectra. The mutants A27C/N20C and A471C/T72C increased the optimal temperature of catalysis by ~1.5°C over WT GA, while mutant A27C/N20C/G 137A increased it by ~2.0°C. The thermostability of two of the combined mutations, G137A/S436P and A27C/N20C/G137A is shown to be additive.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Li, Yuxing, "Genetic construction and biochemical analysis of thermostability mutants of glucoamylase from Aspergillus awamori " (1996). Retrospective Theses and Dissertations. 11551.