Catalytic properties of two Rhizopus oryzae 99-880 glucoamylase enzymes without starch binding domains expressed in Pichia pastoris.
Catalytic properties of two glucoamylases, AmyC and AmyD, without starch binding domains from Rhizopus oryzae strain 99-880 are determined using heterologously expressed enzyme purified to homogeneity. AmyC and AmyD demonstrate pH optima of 5.5 and 6.0, respectively, nearly one unit higher than the Rhizopus AmyA glucoamylase enzyme. Optimal initial activities are at 60 and 50 °C for AmyC and AmyD, respectively. Inactivation of both enzymes occurs at 50 °C following 30 min pre-incubation. The two enzymes demonstrate substantially slower catalytic rates toward soluble starch relative to AmyA. AmyC has similar k(cat) and K(m) for oligosaccharides to other Rhizopus and Aspergillus glucoamylases; however, the enzyme has a 2-fold lower K(m) (maltose) . AmyD has a 3-fold higher K(m) and lower k(cat) for maltooligosaccharides than AmyC and other glucoamylases. AmyC (but not AmyD) exhibits substrate inhibition. K(i) for substrate inhibition decreases with increasing length of the oligosaccharides. 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.
Mertens
Jeffrey A
JA
Bioenergy Research Unit, National Center for Agricultural Utilization Research, USDA Agricultural Research Service, 1815 N. University St., Peoria, IL 61604, USA. jeffrey.mertens@ars.usda.gov
Braker
Jay D
JD
Jordan
Douglas B
DB
D055162:Biocatalysis; D004795:Enzyme Stability; D015870:Gene Expression; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010843:Pichia / Q000235:genetics*; D017434:Protein Structure, Tertiary; D012233:Rhizopus / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013213:Starch / Q000378:metabolism*; D013696:Temperature; D014312:Trisaccharides / Q000378:metabolism
D014312:Trisaccharides; C008317:maltotriose; D013213:Starch; D005087:Glucan 1,4-alpha-Glucosidase