Sunday, August 10, 2008
P13

Isolation and characterization of 17 different genes encoding putative endopolygalacturonase genes from Rhizopus oryzae

Jeffrey A. Mertens and Ryan C. Burdick. Fermentation Biotechnology, USDA - ARS NCAUR, 1815 N. University St, Peoria, IL 61604

Polygalacturonase enzymes are a valuable aid in the retting of flax for production of linens and more recently, production of biofuels from citrus wastes.  In a search of the recently sequenced R. oryzae strain 99-880 genome database, 18 putative endopolygalacturonase genes were identified, with 2 genes being identical and only 1 with any similarity to a previously reported Rhizopus endopolygalcturonase gene.  The 17 different genes have from 1 to 3 introns and share 50% to greater than 90% identity at the nucleotide level as well as the deduced protein sequence level.  Only 6 of the 17 cDNA’s could be obtained directly from Rhizopus mycelia cultured on different energy sources in liquid and solid cultures.  Although it is not clear under what conditions or if the remaining 11 genes are expressed, the remaining cDNA’s were obtained by recombinant methods.  The cDNA for each putative endopolygalacturonase was expressed in Pichia pastoris and tested for enzyme activity, with 15 of the 17 genes encoding active enzyme.  Initial characterization demonstrated that some of the enzymes were indeed endopolygalacturonase enzymes with hydrolysis of polygalacturonic acid resulting in the production of multiple products ranging from galacturonic acid to greater than penta-galacturonic acid, while others could be considered exopolygalacturonase enzymes as only galacturonic acid was obtained.  The purpose and/or function of the two inactive enzymes is not clear and work continues to assign potential function to these two putative enzymes as well as a more detailed biochemical understanding of the enzymes demonstrating polygalacturonase activity.      

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