A kinetic model for the solvent-free lipase-catalyzed acetylation of phlorizin with triacetin

Little attention has been given to the kinetics of enzyme-catalyzed modification of flavonoids and flavonoid glycosides, especially the successive addition of multiple functional groups. The only currently available model is not consistent with the principles of enzyme kinetics. We developed a simple kinetic model, using the lipase-catalyzed acetylation of phlorizin in a solvent-free system as a case study. Our model assumes that the acetylation is irreversible, the concentration of the solvent/acetyl-donor remains constant and the lipase inactivates in a first-order process. We estimated the parameters of the model by fitting it to literature data for the lipase-catalyzed acetylation of phlorizin, with triacetin as the acetyl-donor. Our model fitted well to profiles for phlorizin, phlorizin-6”-O-acetate and phlorizin-3”,6”-O-diacetate obtained at several initial phlorizin concentrations. The estimated parameters were a specificity constant of 0.380 h⁻¹%(w/v)⁻¹ for phlorizin, a selectivity of 0.362 for the second acetylation in relation to the first, saturation constants of 22.3 mmol L⁻¹ for phlorizin and 76.5 mmol L⁻¹ for phlorizin-6”-O-acetate, an inhibition constant for phlorizin-3”,6”-O-diacetate of 3.5 mmol L⁻¹ and a first-order denaturation constant of 0.0125 h⁻¹. Our work not only presents a model that describes well the acetylation of phlorizin, it also demonstrates a strategy for modelling and parameter estimation that can be applied to other enzyme-catalyzed modifications of flavonoids and flavonoid glycosides.