IDENTIFICATION AND ELIMINATION BY SITE-DIRECTED MUTAGENESIS OF THERMOLABILE ASPARTYL BONDS IN ASPERGILLUS-AWAMORI GLUCOAMYLASE

Both native Aspergillus niger glucoamylase and wild-type Aspergillus a wamori 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 domin ant at both pH 3.5 and 4.5, Site-directed mutations Asp126-->Glu and G ly127-->Ala in wild-type enzyme decreased specific activities by simil ar to 60 and 30%, respectively, and increased irreversible thermoinact ivation rates 3- to 4-fold at pH 4.5. Replacement of Asp257 with Glu a nd Asp293 with Glu or Gin decreased specific activities by similar to 20%, but greatly reduced cleavage of the Asp257-Pro258 and Asp293-Gly2 94 bonds. The Asp257-->Glu mutant was produced very slowly and was mor e thermostable than wild-type glucoamylase at pH 4.5 up to 70 degrees C. Replacement of Asp293 with either Glu or Gin significantly raised p rotein production and slightly increased thermostability at pH 3.5 and 4.5, but not at pH 5.5.