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- Detailed kinetic model describing new oligosaccharides synthesis using different β-galactosidasesPublication . Rodriguez-Fernandez, Maria; Cardelle-Cobas, Alejandra; Villamiel, Mar; Banga, Julio R.The production of prebiotic galactooligosaccharides (GOS) from lactose has been widely studied whereas the synthesis of new prebiotic oligosaccharides with improved properties as those derived from lactulose is receiving an increasing interest. Understanding the mechanism of enzymatic oligosaccharides synthesis from lactulose would help to improve the quality of the products in a rational way as well as to increase the production efficiency by optimally selecting the operating conditions. A detailed kinetic model describing the enzymatic transgalactosylation reaction during lactulose hydrolysis is presented here for the first time. The model was calibrated with the experimental data obtained in batch assays with two different B-galactosidases at various temperatures and concentrations of substrate. A complete system identification loop, including model selection, robust estimation of the parameters by means of a global optimization method and computation of confidence intervals was performed. The kinetic model showed a good agreement between experimental data and predictions for lactulose conversion and provided important insights into the mechanism of formation of new gligosaccharides with potential prebiotic properties.
- Improving Properties of a Novel β-Galactosidase from Lactobacillus plantarum by Covalent ImmobilizationPublication . Benavente, Rocio; Pessela, Benevides C.; Curiel, Jose Antonio; de las Rivas, Blanca; Muñoz, Rosario; Guisán, Jose Manuel; Mancheño, Jose M.; Cardelle-Cobas, Alejandra; Ruiz-Matute, Ana I.; Corzo, NievesA novel -galactosidase from Lactobacillus plantarum (LPG) was over-expressed in E. coli and purified via a single chromatographic step by using lowly activated IMAC (immobilized metal for affinity chromatography) supports. The pure enzyme exhibited a high hydrolytic activity of 491 IU/mL towards o-nitrophenyl -d-galactopyranoside. This value was conserved in the presence of different divalent cations and was quite resistant to the inhibition effects of different carbohydrates. The pure multimeric enzyme was stabilized by multipoint and multisubunit covalent attachment on glyoxyl-agarose. The glyoxyl-LPG immobilized preparation was over 20-fold more stable than the soluble enzyme or the one-point CNBr-LPG immobilized preparation at 50 degrees C. This -galactosidase was successfully used in the hydrolysis of lactose and lactulose and formation of different oligosaccharides was detected. High production of galacto-oligosaccharides (35%) and oligosaccharides derived from lactulose (30%) was found and, for the first time, a new oligosaccharide derived from lactulose, tentatively identified as 3'-galactosyl lactulose, has been described.