Submitted to: Current Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 3/9/2007
Publication Date: 5/1/2007
Citation: Mertens, J.A., Skory, C.D. 2007. Isolation and characterization of two genes that encode active glucoamylase without a starch binding domain from Rhizopus oryzae. Current Microbiology. 54:462-466. Interpretive Summary: Starch obtained from agricultural crops is used in food processing, brewing, and the production of fuel ethanol. The starch typically needs to be broken down by enzymes into simple sugars in order to be further utilized. The fungus Rhizopus oryzae produces enzymes called glucoamylase that are very effective in the conversion of starch into pure glucose. Industrial interest in this enzyme has remained strong, despite very little understanding about the genetic control mechanisms regulating glucoamylase production. In this work, we have identified two genes that are responsible for production of previously uncharacterized glucoamylase enzymes from Rhizopus. A new understanding of this particular enzyme helps explain many unresolved questions regarding glucoamylase from this fungus. This discovery further improves the ability to control the efficiency of glucoamylase production and will ultimately decrease the cost of using agricultural crops for the production of high value products.
Technical 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 RA99-880 that has recently been sequenced by the Broad Institute of Harvard and MIT. 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 roughly 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 glucoside 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. No activity was detected in either the growth medium or whole cell extracts when the sequence for putative secretion signal was left intact. However, replacement of this region with the yeast alpha-secretion signal resulted in secretion of active glucoamylase that was able to degrade soluble starch.