Identification and elimination by site-directed mutagenesis of thermolabile aspartyl bonds in Aspergillus awamori glucoamylase.

Both native Aspergillus niger glucoamylase and wild-type Aspergillus awamori glucoamylase expressed in Saccharomyces cerevisiae, which have identical primary structures, undergo hydrolysis at aspartyl bonds at low pH values and elevated temperatures. In native A.niger enzyme the Asp126-Gly127 bond was preferentially cleaved at pH 3.5, while at pH 4.5 cleavage of the Asp257-Pro258 and Asp293-Gly294 bonds was dominant. In wild-type A.awamori glucoamylase, cleavage of the latter was dominant at both pH 3.5 and 4.5. Site-directed mutations Asp126-->Glu and Gly127-->Ala in wild-type enzyme decreased specific activities by approximately 60 and 30%, respectively, and increased irreversible thermoinactivation rates 3- to 4-fold at pH 4.5. Replacement of Asp257 with Glu and Asp293 with Glu or Gln decreased specific activities by approximately 20%, but greatly reduced cleavage of the Asp257-Pro258 and Asp293-Gly294 bonds. The Asp257-->Glu mutant was produced very slowly and was more thermostable than wild-type glucoamylase at pH 4.5 up to 70 degrees C. Replacement of Asp293 with either Glu or Gln significantly raised protein production and slightly increased thermostability at pH 3.5 and 4.5, but not at pH 5.5.