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United States Department of Agriculture

Agricultural Research Service

Research Project: INDUSTRIALLY ROBUST ENZYMES AND MICROORGANISMS FOR PRODUCTION OF SUGARS AND ETHANOL FROM AGRICULTURAL BIOMASS Title: Isolation and Characterization of a Second Glucoamylase Without a Starch Binding Domain from Rhizopus Oryzae

Authors
item Mertens, Jeffrey
item Skory, Christopher

Submitted to: Enzyme and Microbial Technology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 3, 2006
Publication Date: January 5, 2007
Citation: Mertens, J.A., Skory, C.D. 2007. Isolation and characterization of a second glucoamylase gene without a starch binding domain from Rhizopus oryzae. Enzyme and Microbial Technology. 40:874-880.

Interpretive Summary: Starch derived from agricultural crops is used in many commercial facilities involved in food processing, brewing, and even production of fuel ethanol. However, the starch typically needs to be broken down into simple sugars in order to be further utilized. The fungus Rhizopus oryzae produces enzymes called glucoamylase that accomplish the goal of converting the starch into pure glucose very effectively. Industrial interest in this enzyme has remained strong for many years, yet very little is understood about the genetic control mechanisms regulating the enzyme production. In this work, we have identified a gene that is responsible for production of a previously uncharacterized glucoamylase gene from this organism. A new understanding of this particular enzyme helps to explain many of the unresolved questions regarding glucoamylase from this fungus. This discovery will further improve the ability to control the efficiency of glucoamylase production, which will ultimately decrease the cost of using agricultural residues for production of high value products.

Technical Abstract: Work with Rhizopus oryzae previously suggested that this filamentous fungus only had one glucoamylase. We demonstrate in this study that some R. oryzae strains contain multiple glucoamylase genes with differential regulation. The existence of the two unique, amy genes in R. oryzae NRRL 395, a type I strain, was confirmed by Southern blot analysis, restriction mapping, and sequencing. Sequence analysis of the amy genomic clones reveals extensive homology in the coding region, as well as the flanking ends. A notable difference is the amyB gene does not encode a starch binding domain and also contains a 24 bp insertion in the open reading frame that remains in the cDNA. In addition, isolation of the amyA and amyB cDNA demonstrates that the single common intron is spliced in an identical manner. The deduced amino acid sequence of the amy genes shares 91% homology, excluding the starch binding domain. The putative glucoamylase enzymes contain similar signal sequences, as well as conserved amino acid sequence regions found in family 15 glycoside hydrolases. Transcriptional analyses demonstrate that both amy genes are highly expressed on solid medium with low water activity. However, amyB demonstrates little or no expression in shake flask cultures with various carbon sources. Recombinant expression of AmyB in yeast did not produce detectable protein or glucoamylase activity, even though full length amyB transcript was present.

Last Modified: 7/25/2014
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