Kinetic studies and modelling of the production of fructooligosaccharides by fructosyltransferase from Rhodotorula sp

Abstract

Fructosyltransferase was produced by a strain of Rhodotorula, isolated from flowers collected in the costal Atlantic Forest located in Southern Brazil and screened according to its ability to produce the enzyme. The production was carried out in submerged fermentation and subsequently purified using the following three procedures: alcohol precipitation, Q-Sepharose ion-exchange chromatography and ultrafiltration. The studies of fructooligossacaride production were carried out in a batch stirred reactor using sucrose as the substrate and 5 U_TF mL⁻¹ of fructosyltransferase at pH 4.5 and 50 °C. Since the industrial application of this enzyme does not require a highly purified enzymatic solution, the enzyme kinetics were comparatively performed using both partially purified (only alcohol precipitated enzyme) and purified (using all steps specified above) enzyme. The kinetics showed a characteristic Michaelis–Menten behavior with substrate inhibition effects at high sucrose concentrations (up to 70% w/v). Additionally, glucose competitive inhibition relating to the sucrose, 1-kestose and nystose uptakes were verified. An inhibitory effect was also noticed with high concentrations of fructose (over 50%) but considered meaningless since the fructose concentration is always low in the actual medium reaction. The hydrolyzing activity over nystose was found to be significant, so it was included in the mathematical model. The initial values for the kinetic constants, K_m, V_m and K_i, for each substrate were obtained, and then fine-tuned by simulations, after a parameter sensitivity analysis was carried out. The model predictions fitted well the experimental data, either for the purified or partially purified enzyme, while a different set of adjusted parameters was used in each case. Model predictions for FOS production deviated by not more than 5% in both cases, so they can be used for bioreactor designs.