Competitive substrate inhibition of amyloglucosidase from Rhizopus sp. by vanillin and curcumin

Abstract

Kinetic studies of two glucosylation reactions catalyzed by an amyloglucosidase from Rhizopus sp. leading to the synthesis of vanillin-α/β-D-glucoside from D-glucose and vanillin and curcumin-bis-α-D-glucoside from D-glucose and curcumin were investigated in detail. Initial reaction rates were determined from kinetic runs involving different concentrations of D-glucose and vanillin (5 mM to 0.1 M) or D-glucose and curcumin (5 mM to 0.1 M). Graphical double reciprocal plots showed that the kinetics of the two enzyme catalyzed reactions exhibited Ping-Pong Bi-Bi mechanism where competitive substrate inhibition by vanillin/curcumin led to dead-end amyloglucosidase–vanillin/curcumin complexes at higher concentrations of vanillin/curcumin. An attempt to obtain the best fit of this kinetic model through computer simulation yielded in good approximation, the values of four important kinetic parameters, vanillin-α/β-D-glucoside: k_cat=35.0±3.2 10⁻⁵M h⁻¹·mg, K_i=10.5±1.1 mM, K_mD-glucose=60.0±6.2 mM, K_mvanillin=50.0±4.8 mM; curcumin-bis-α-D-glucoside: k_cat=6.07±0.58 10⁻⁵M h⁻¹·mg, K_i=3.0±0.28 mM, K_mD-glucose=10.0±0.9 mM, K_mcurcumin=4.6±0.5 mM.

• Amyloglucosidase–vanillin/curcumin complexes
• competitive substrate inhibition
• dead-end inhibition by vanillin/curcumin
• hydrogen bonding interactions
• phenolic OH and carbonyl groups
• Ping-Pong Bi-Bi mechanism