Enrique J. Mammarella

Improving the Properties of Chitosan as Support for the Covalent Multipoint Immobilization of Chymotrypsin

Changing gel structure and immobilization conditions led to a significant improvement in the covalent multipoint attachment of chymotrypsin on chitosan. The use of sodium alginate, gelatin, or kappa-carrageenan, activation with glutaraldehyde, glycidol, or epichlorohydrin, and addition of microorganisms followed by cellular lysis allowed the modification of the gel structure. Immobilization yields, recovered activities, and stabilization factors at 55 and 65 degrees C were evaluated. Enzyme immobilization for 72 h at pH 10.05, 25 degrees C and reduction with NaBH 4 in chitosan 2.5%-carrageenan 2.5%, with addition of S. cerevisiae 5% and activation with epichlorohydrin led to the best derivative, which was 9900-fold more stable than the soluble enzyme. This support allowed an enzyme load up to 40 mg chymotrypsin x g gel (-1). The number of covalent bonds, formed by active groups in the support and lysine residues of the enzyme, can explain the obtained results. SEM images of the gel structures corroborate these conclusions.

Crosslinking kinetics of cation-hydrocolloid gels

Abstract The gel formation of hydrocolloid (sodium alginate and kappa-carrageenan) is the result of the molecule aggregation in presence of an effective cation. The gelling process was considered as a process of diffusion–reaction between the effective cation and the hydrocolloid to produce a porous solid structure. A simple mathematical model for predicting the reaction interface position in alginate or alginate-carrageenan gelation was developed. The model represents appropriately the physicochemical phenomenon and allows to determine the time for complete crosslinking of the hydrocolloid and the amount of incorporated ions.

Prediction of the fixed-bed reactor behaviour using dispersion and plug-flow models with different kinetics for immobilised enzyme

Abstract The behaviour of fixed-bed reactors, which have an immobilised enzyme on the packing surface, was studied considering steady-state conditions and external mass transfer resistance in the fluid around catalyst spherical particles. Solutions were obtained by integration of the plug-flow model equation and by the orthogonal collocation method of the second order differential equation of the axial dispersion flow model. Both models were analysed for lactose hydrolysis with β-galactosidase immobilised on chitosan using different kinetic reaction mechanisms after determining the specific parameters. The calculated results show the importance of the hydrodynamic and kinetic reaction parameters for error reduction in the prediction of the experimental behaviour.

Evaluation of stress-strain for characterization of the rheological behavior of alginate and carrageenan gels

The stress-strain of samples deformed until failure and the relaxation response after 50% deformation of the initial height under constant stress were obtained. Uniaxial compression and stress-relaxation tests enabled satisfactory differentiation of the mechanical resistance of gels with different alginate and carrageenan concentrations. Higher values for initial force at the beginning of the relaxation test were associated with higher calcium uptake by the gels. An increment of failure stress during the uniaxial compression tests for higher concentration of calcium in the gel structure was also observed. The maximum amount of cation uptake was higher than the theoretical value for saturation of all the carboxylic groups available in alginate molecules due to structural rearrangements. Stress-relaxation tests indicated that the residual stress of the gel increased with k-carrageenan concentration.

Screening and selection of wild strains for L-arabinose isomerase production

The majority of L-arabinose isomerases have been isolated by recombinant techniques, but this methodology implies a reduced technological application. For this reason, 29 bacterial strains, some of them previously characterized as L-arabinose isomerase producers, were assayed as L-arabinose fermenting strains by employing conveniently designed culture media with 0.5% (w/v) L-arabinose as main carbon source. From all evaluated bacterial strains, Enterococcus faecium DBFIQ ID: E36, Enterococcus faecium DBFIQ ID: ETW4 and Pediococcus acidilactici ATCC ID: 8042 were, in this order, the best L-arabinose fermenting strains. Afterwards, to assay L-arabinose metabolization and L-arabinose isomerase activity, cell-free extract and saline precipitated cell-free extract of the three bacterial cultures were obtained and the production of ketoses was determined by the cysteine carbazole sulfuric acid method. Results showed that the greater the L-arabinose metabolization ability, the higher the enzymatic activity achieved, so Enterococcus faecium DBFIQ ID: E36 was selected to continue with production, purification and characterization studies. This work thus describes a simple microbiological method for the selection of L-arabinose fermenting bacteria for the potential production of the enzyme L-arabinose isomerase.

Response Surface Methodology To Optimize β‐Galactosidase Immobilization Using a Combination of Hydrocolloids as the Supporting Matrix

Response surface methodology (RSM) was applied to optimize the composition of a mixture of hydrocolloids (low‐viscosity sodium alginate, high‐viscosity sodium alginate, and κ‐carrageenan) used in the immobilization of β‐galactosidase for application in the hydrolysis of lactose. A five levels, a three‐factor design was adopted. The activity and stability of the immobilized enzyme and the strength of the supporting matrix were optimized by using a mathematical model applied in the range of process conditions. Requirements for multifactor response surface designs were satisfied, and the correlation coefficient, R2, was larger than 0.850, ensuring a good adjustment of the model to the experimental values. Best results were obtained for values of 1.00% low‐viscosity sodium alginate, 1.40−1.60% high‐viscosity sodium alginate, 0.10−0.30% κ‐carrageenan, and 10−12% enzyme.

Engineering the l-Arabinose Isomerase from Enterococcus Faecium for d-Tagatose Synthesis

l-Arabinose isomerase (EC 5.3.1.4) (l-AI) from Enterococcus faecium DBFIQ E36 was overproduced in Escherichia coli by designing a codon-optimized synthetic araA gene. Using this optimized gene, two N- and C-terminal His-tagged-l-AI proteins were produced. The cloning of the two chimeric genes into regulated expression vectors resulted in the production of high amounts of recombinant N-His-l-AI and C-His-l-AI in soluble and active forms. Both His-tagged enzymes were purified in a single step through metal-affinity chromatography and showed different kinetic and structural characteristics. Analytical ultracentrifugation revealed that C-His-l-AI was preferentially hexameric in solution, whereas N-His-l-AI was mainly monomeric. The specific activity of the N-His-l-AI at acidic pH was higher than that of C-His-l-AI and showed a maximum bioconversion yield of 26% at 50 °C for d-tagatose biosynthesis, with Km and Vmax parameters of 252 mM and 0.092 U mg−1, respectively. However, C-His-l-AI was more active and stable at alkaline pH than N-His-l-AI. N-His-l-AI follows a Michaelis-Menten kinetic, whereas C-His-l-AI fitted to a sigmoidal saturation curve.