Submitted to: Applied Biochemistry and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 10, 2010
Publication Date: June 13, 2010
Citation: Mertens, J.A., Braker, J.D., Jordan, D.B. 2010. Catalytic properties of two Rhizopus oryzae 99-880 glucoamylase enzymes without starch binding domains expressed in Pichia pastoris. Applied Biochemistry and Biotechnology. 162(8):2197-2213. 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 and remaining questions with regard to catalytic details of glucoamylase conversion of starch to glucose. In this work, we have characterized the catalytic properties of two 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 understanding of catalysis as it relates to this enzyme class and will ultimately aid in decreasing the cost of using agricultural crops for the production of high value products.
Technical Abstract: Catalytic properties of the two glucoamylases, AmyC and AmyD, without starch binding domains from Rhizopus oryzae strain 99-880 were heterologously expressed and purified to homogeneity. AmyC and AmyD demonstrate pH optima of 5.5 and 6.0, respectively, nearly 1 unit higher than most fungal glucoamylases. Optimal initial activities are at 50 deg C, and enzyme inactivation occurs at 60 deg C (AmyC) and 50 deg C (AmyD) following 30 min pre-incubation. AmyC has similar kcat and Km for oligosaccharides as other Rhizopus and Aspergillus glucoamylases; however, the enzyme has a 2-fold lower Km**maltose. AmyD has a 3-fold higher Km and lower kcat for maltooligosaccharides than AmyC and other glucoamylases. kcat/Km values of AmyC acting on DP2 through DP6 define 5 subsites, and those of AmyD define 6 or more subsites. AmyC (but not AmyD) exhibits substrate inhibition. Substrate inhibition increases with increasing length of the substrate. Data from pre-steady state binding of AmyC to maltose and maltotriose and pre-steady state to steady-state catalytic turnover experiments of AmyC acting on maltotriose were used to interrogate models of substrate inhibition. In the preferred model, AmyC accumulates an enzyme-maltose-maltotriose dead-end complex in the steady state.