Manuela Poletto Klein

Effect of the support size on the properties of β-galactosidase immobilized on chitosan: advantages and disadvantages of macro and nanoparticles.

The effect of the support size on the properties of enzyme immobilization was investigated by using chitosan macroparticles and nanoparticles. They were prepared by precipitation and ionotropic gelation, respectively, and were characterized by Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), transmission electron microscopy (TEM), light scattering analysis (LSA), and N(2) adsorption-desorption isotherms. β-Galactosidase was used as a model enzyme. It was found that the different sizes and porosities of the particles modify the enzymatic load, activity, and thermal stability of the immobilized biocatalysts. The highest activity was shown by the enzyme immobilized on nanoparticles when 204.2 mg protein·(g dry support)(-1) were attached. On the other hand, the same biocatalysts presented lower thermal stability than macroparticles. β-Galactosidase immobilized on chitosan macro and nanoparticles exhibited excellent operational stability at 37 °C, because it was still able to hydrolyze 83.2 and 75.93% of lactose, respectively, after 50 cycles of reuse.

High operational stability of invertase from Saccharomyces cerevisiae immobilized on chitosan nanoparticles

Invertase (E.C.3.2.1.26) from Saccharomyces cerevisiae was covalently immobilized on chitosan nanoparticles prepared by the ionotropic gelation method and activated with glutaraldehyde. The support was characterized and it was studied its load capacity, the influence of the presence of substrate during immobilization, and determined the biocatalyst kinetic parameters and stabilities. The light scattering analysis (LSA) and transmission electron microscopy (TEM) techniques indicated a mixture of chitosan nano and aggregated nanoparticles, providing high superficial area for enzyme immobilization. The thermal and storage stabilities, the optimal pH and temperature of the enzyme were not altered. K(m) increased 3-fold, while V(max) remained unaltered. The immobilized biocatalyst was reused for 59 batches with maximal invertase activity, the highest operational stability so far described in the literature. These results fulfill some important aspects for the enzyme immobilization: the simplicity of the protocols, the conservation of the enzyme activity, and the high operational stability.

Continuous production of β-cyclodextrin from starch by highly stable cyclodextrin glycosyltransferase immobilized on chitosan.

Cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacter sp. was covalently immobilized on glutaraldehyde-activated chitosan spheres and used in a packed bed reactor to investigate the continuous production of β-cyclodextrin (β-CD). The optimum temperatures were 75 °C and 85 °C at pH 6.0, respectively for free and immobilized CGTase, and the optimum pH (5.0) was the same for both at 60 °C. In the reactor, the effects of flow rate and substrate concentration in the β-CD production were evaluated. The optimum substrate concentration was 4% (w/v), maximizing the β-CD production (1.32 g/L) in a flow rate of 3 mL/min. In addition, the biocatalyst had good operational stability at 60 °C, maintaining 61% of its initial activity after 100 cycles of batch and 100% after 100 h of continuous use. These results suggest the possibility of using this immobilized biocatalyst in continuous production of CDs.

Fructooligosaccharides synthesis by highly stable immobilized β-fructofuranosidase from Aspergillus aculeatus

The enzymatic synthesis of fructooligosaccharides (FOS) was carried out using a partially purified β-fructofuranosidase from the commercial enzyme preparation Viscozyme L. Partial purification of β-fructofuranosidase from Viscozyme L was done by batch adsorption using ion-exchange resin DEAE-Sepharose, showing a 6-fold increase in specific activity. The biocatalyst was then covalently immobilized on glutaraldehyde-activated chitosan particles. Thermal stability of the biocatalyst was evaluated at 50 °C and 60 °C, being around 100 times higher at 60 °C when compared to the free enzyme. The immobilized biocatalyst was reused 50 times for FOS production (100 min per batch at 50 °C and pH 5.5) without significant loss of activity. The average yield (grams of FOS per grams of initial sucrose) was 55%. The immobilization process combined with partial purification method resulted in a derivative with activity of 1230 Ut/g, which is among the best for FOS production.

Silver Nanoparticle Thin Films Deposited on Glass Surface Using an Ionic Silsesquioxane as Stabilizer and as Crosslinking Agent

Thin films containing silver nanoparticles homogeneously dispersed, with narrow size distribution below 10 nm, were synthesized on flat glass surface, by using an ionic silsesquioxane as stabilizer and crosslinking agent. The films can be prepared without previous functionalization of substrate surfaces and without addition of other components. The films were heat treated up to 200 oC and characterized by ultraviolet-visible, transmission electron microscopy, atomic force microscopy, thermogravimetric analysis and ellipsometry. The films were thermally stable when heated up to 200 oC, presenting the same thickness, and maintaining both optical and morphological properties of silver nanoparticles. The antibacterial activity of the films, containing the silver nanoparticles, was evaluated against Staphylococcus aureus by using the film applicator coating method, showing an excellent performance even after the third cycle of sterilization.

Chitosan/Carboxymethylcellulose/Ionic Liquid/Ag(0) Nanoparticles Form a Membrane with Antimicrobial Activity

Silver metal nanoparticles were immobilized in chitosan/carboxymethylcellulose/BMI.BF4(1-n-butyl-3-methylimidazolium tetrafluoroborate ionic liquid) (CS/CMC/IL) to form polymeric membrane with 20 μm thickness. The CS/CMC/IL polymeric membrane was prepared using a simple solution blending method. Irregularly shaped Ag(0) nanoparticles with monomodal size distributions of  nm Ag(0) were immobilized in the membrane. The presence of small Ag(0) nanoparticles induced an augmentation in the CS/CMC/IL film surface areas. The CS/CMC/IL membrane containing Ag(0) showed increase antimicrobial activity the Ag(0) concentration increased up to saturation at 10 mg. CS/CMC/IL membrane that contains Ag(0) nanoparticles has enhanced durability of the membrane and exhibited stronger antimicrobial activity against Escherichia coli and Staphylococcus aureus.

Magnetic biocatalysts of pectinase and cellulase: Synthesis and characterization of two preparations for application in grape juice clarification

In the present study, we prepared two different magnetic biocatalysts of pectinase and cellulase: carrier-free magnetic CLEAs (CLEA-MP*) and immobilization on glutaraldehyde-activated magnetite (Enz-Glu-MP*). The biocatalysts were compared to their magnetic properties, immobilization parameters, stability and grape juice clarification. Enz-Glu-MP* presented higher magnetic properties than CLEA-MP*, whereas this presented higher surface area and pore volume. The KM of the enzyme immobilized on Enz-Glu-MP* was 25.65mM, lower in comparison to the CLEA-MP* (33.83mM). On the other hand, CLEA-MP* was the most active and stable biocatalyst, presenting higher recovered activity (33.4% of cellulase), higher thermal stability (2.39 stabilization factor) and improved reusability (8cycles). The integration of magnetic technology with enzymatic immobilization emerges as a possibility to increase the recover and reuse of biocatalysts for application in juice technology.

Efficient Enzyme-Assisted Extraction of Genipin from Genipap ( Genipa americana L.) and its Application as a Crosslinker for Chitosan Gels

Enzyme-assisted extraction in liquid-liquid two-phase aqueous system was applied for the first time in order to extract genipin from genipap. The effect of different commercial enzymes, their concentrations, and extraction parameters were investigated. Moreover, chitosan gels were prepared, crosslinked with glutaraldehyde or genipin and characterized by their textural and rheological properties. The crosslinked chitosan was used as support for the immobilization of model β-galactosidases. Among the different commercial enzymes tested for extraction, Celluclast 10% (36 °C and pH 3.7) provided an extraction of 196 mg.g-1 of genipin. Chitosan gels crosslinked with genipin 0.5% showed better textural and similar rheological properties when compared to the chitosan crosslinked with glutaraldehyde 3%. The percentage of lactose hydrolysis by the immobilized K. lactis β-galactosidase using genipin as a crosslinker was 87%. Thus, the genipin obtained in this work proved to be an excellent alternative to the use of glutaraldehyde in chitosan crosslinking applications.

Utilização da β-galactosidase para prevenção da cristalização em doce de leite

Este estudo avaliou o efeito da utilizacao da enzima β-galactosidase sobre a cristalizacao da lactose no doce de leite. As concentracoes de lactase testadas variaram de 0 a 0,4 g/L. O grau de cristalizacao do produto foi avaliado sensorialmente (teste de ordenacao em funcao de uma escala especifica), apos 30, 60, 90 e 180 dias de armazenamento a temperatura ambiente, por provadores previamente treinados. O teste de ordenacao indicou nao haver diferenca estatistica significativa (P>0,05) entre as amostras em relacao a cristalizacao, tanto aos 30 quanto aos 60 dias de armazenamento, porem aos 90 e 180 dias essa diferenca foi significativa entre o controle (T0) e os tratamentos. A utilizacao de 0,2 g/L de β-galactosidase (23,16% de hidrolise da lactose) foi suficiente para que a arenosidade no doce de leite nao fosse percebida sensorialmente pelos provadores, durante todo o periodo considerado.