Isolation and Characterization of Two Genes That Encode Active Glucoamylase Without a Starch Binding Domain from Rhizopus oryzae

Abstract

Glucoamylase obtained from Rhizopus sp. is frequently preferred for certain applications of starch modification or saccharification. The predominant enzyme, which contains a starch-binding domain on the amino terminus, has been previously characterized from several species. Additionally, the cDNA encoding this protein was cloned and found to show 100% identity to a R. oryzae strain 99-880 that has recently been sequenced by the Broad Institute of Harvard and Massachusetts Institute of Technology. Analysis of this genome indicates coding regions for two additional glucoamylase genes, amyC and amyD, lacking a starch-binding domain. The two genes encode proteins that are approximately 50% identical to the catalytic region of the AmyA protein and 67% identical to each other. The predicted AmyC and AmyD proteins contain the highly conserved signature sequences of Family 15 glycoside hydrolases. The two genes appear to be transcriptionally expressed in cultures grown in fermentable and gluconeogenic carbon sources. The predicted 49.7-kD AmyC and 48.8-kD AmyD proteins were expressed in several different ways using Pichia pastoris. When the sequence for putative secretion signal was left intact, glucoamylase activity was detected in the crude cell extracts, but no activity was present in the growth medium. However, replacement of this region with the yeast alpha-secretion signal resulted in secretion of active glucoamylase that was able to degrade soluble starch.