Kinetics and modeling of fructo-oligosaccharide synthesis by immobilized fructosyltransferase from Rhodotorula sp.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)BACKGROUND: Fructosyltransferase synthesizes fructo-oligosaccharides from sucrose. Data used in this work were obtained by an enzyme produced by Rhodotorula sp., a microorganism isolated from fruit samples from the Brazilian Atlantic Forest, which was immobilized in an inorganic support, consisting of a niobium and graphite alloy. RESULT: All essays were conducted using enzymes at two purification grades, highly and partially purified enzymes, as comparison. The results were not significantly different between the two enzyme grades, mainly concerning the final fructo-oligossacharides yield, which were around 46%. Concerning the kinetics, the enzyme follows the Michaelis-Menten equation with inhibition by sucrose (above 60%). Also, a competitive inhibition by glucose was observed on sucrose, kestose and nystose uptakes. The immobilization of the enzyme was by ion exchange on the surface of the particles, since the support is a charged and compact solid, with negligible porosity. The mathematic model includes mass balances, considering the resistance to external mass transfer. A parameter sensitivity analysis and parameter fitting were performed by simulations and the model was validated by comparison with experimental data. CONCLUSION: The model fitted experimental data well, with deviations lower than 5% concerning FOS concentrations, indicating that it can be used in the design and control of bioreactors, either using purified or partially purified enzyme. (C) 2010 Society of Chemical Industry851216541662Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

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

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)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 degrees 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.1610431050Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP