Location: Bioenergy ResearchTitle: Kinetic properties of two Rhizopus exo-polygalacturonase enzymes hydrolyzing galacturonic acid oligomers using isothermal titration calorimetry) Author
Submitted to: Applied Biochemistry and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/16/2013
Publication Date: 8/1/2013
Citation: Mertens, J.A. 2013. Kinetic properties of two Rhizopus exo-polygalacturonase enzymes hydrolyzing galacturonic acid oligomers using isothermal titration calorimetry. Applied Biochemistry and Biotechnology. 170(8):2009-2020. Interpretive Summary: Future fuel and specialty chemical needs will require the use of agricultural biomass. To be used efficiently and effectively, agricultural biomass needs to be broken down by chemical pre-treatments and enzymes into simple sugars. Rhizopus oryzae, a filamentous fungus, make enzymes called polygalacturonases that are successful in the conversion of pectin, a component of biomass, into simple sugars. Interest in polygalacturonases is strong due to its use in clarifying fruit juices, textile production, and role in damage to economically important crops. In this work the affinity and activity towards complex sugars of two exo-polygalacturonases was uncovered. This work aids in a greater understanding of how these enzymes breakdown complex sugars and paves the way for engineering and development of more effective enzymes.
Technical Abstract: The kinetic characteristics of two Rhizopus oryzae exo-polygalacturonases acting on galacturonic acid oligomers (GalpA) were determined using isothermal titration calorimetry (ITC). RPG15 hydrolyzing (GalpA)2 demonstrated a Km of 55 uM and kcat of 10.3 s^-1^ while RPG16 was shown to have greater affinity for (GalpA)2 with a Km of 16 uM, but lesser catalytic activity with a kcat of 3.9 s^-1^. Both enzymes were inhibited by the product, galacturonic acid, with appKi values of 886 uM and 501 uM for RPG15 and RPG16, respectively. RPG15 exhibited greater affinity for (GalpA)3 with a Km of 9.2 uM and a similar kcat at 10.7 s^-1^ relative to (GalpA)2. Catalytic constants for RPG16 hydrolyzing (GalpA)3 could not be determined, however, single injection ITC assays suggest a distinct preference and catalytic rate for (GalpA)3 relative to (GalpA)2. Thermodynamic parameters of a series of galacturonic acid oligomers binding to RPG15 were determined and exhibited some distinct differences from RPG16 binding thermodynamics, providing potential clues to the differing kinetic characteristics of the two exo-polygalacturonase enzymes.