Control of the association state of tetrameric glucose-fructose oxidoreductase from Zymomonas mobilis as the rationale for stabilization of the enzyme in biochemical reactors.
Tetrameric, NADP-containing glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis catalyzes the oxidation of glucose into glucono-delta-lactone coupled to the reduction of fructose to sorbitol. GFOR is inactivated during substrate turnover in vitro, the long-term stability of the enzyme during conversions in biochemical reactors thereby being drastically reduced. The process of inactivation is triggered by structural transitions that are induced by the lactone product and involves aggregation as the ultimate cause of irreversible inactivation. Guanidinium hydrochloride-induced changes in the conformation of GFOR seem to be similar to those observed in the presence of lactone, and are manifested by incubation time-dependent increases in protein fluorescence and the solvent-exposed hydrophobic surface. The formation of high-order protein associates in solution in the presence of this denaturant proceeds from the native tetramer to a reversibly inactivated octamer and then to a dodecameric form that cannot be reactivated through spontaneous or assisted refolding. Therefore, stabilization of GFOR during turnover requires that the marked tendency of the enzyme to form aggregates is prevented efficiently. This goal has been accomplished in the presence of low urea concentrations (1.0 M), which led to a 10-fold increase in the half-life of GFOR under operational conditions.
Division of Biochemical Engineering, Institute of Food Technology, Universität für Bodenkultur Wien (BOKU), A-1190 Vienna, Austria.
M
M
Fürlinger
M
M
Satory
D
D
Haltrich
K D
KD
Kulbe
B
B
Nidetzky
D004794:Enzyme Repression; D004795:Enzyme Stability; D019791:Guanidine / Q000378:metabolism; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D013997:Time Factors; D014508:Urea / Q000494:pharmacology; D017355:Zymomonas / Q000201:enzymology*
D014508:Urea; D010088:Oxidoreductases; C074291:glucose-fructose oxidoreductase; D019791:Guanidine