Effect of the Support Size on the Properties of β-Galactosidase Immobilized on Chitosan: Advantages and Disadvantages of Macro and Nanoparticles

Departamento de Engenharia Química e Alimentos, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil.
Biomacromolecules (Impact Factor: 5.75). 06/2012; 13(8):2456-64. DOI: 10.1021/bm3006984
Source: PubMed


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.

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Available from: Manuela Poletto Klein, May 31, 2015
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    • "M a n u s c r i p t 6 2.2.1. Preparation of β-D-galactosidase immobilized on genipin-crosslinked chitosan particles Chitosan particles (CS) were prepared by the precipitation method as described in a previous work (Klein et al., 2012). Then, one hundred chitosan particles (0.5 g) were incubated with β-D-galactosidase solution (2 mL, 20 U mL -1 ) prepared in 0.02 M of sodium phosphate buffer (pH 7.0), during 8 h at room temperature. "
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    ABSTRACT: Abstract In order to develop safer processes for the food industry, we prepared a chitosan support with the naturally occurring crosslinking reagent, genipin, for enzyme. As application model, it was tested the immobilization of β-D-galactosidase from Aspergillus oryzae. Chitosan particles were obtained by precipitation followed by adsorption of the enzyme and crosslinking with genipin. The particles were characterized by Fourier transform infrared (FTIR) spectroscopy and thermogravimetric analysis (TGA). The immobilization of the enzyme by crosslinking with genipin provided biocatalysts with satisfactory activity retention and thermal stability, comparable with the ones obtained with the traditional methodology of immobilization using glutaraldehyde. β-D-galactosidase-chitosan-genipin particles were applied to galactooligosaccharides synthesis, evaluating the initial lactose concentration, pH and temperature, and yields of 30% were achieved. Moreover, excellent operational stability was obtained, since the immobilized enzyme maintained 100% of its initial activity after 25 batches of lactose hydrolysis. Thus, the food grade chitosan-genipin particles seem to be a good alternative for application in food process.
    Carbohydrate Polymers 10/2015; DOI:10.1016/j.carbpol.2015.10.069 · 4.07 Impact Factor
    • "Sometimes the support may be an ionic exchanger, and may behave as a buffer, generating a pH inside the biocatalyst bead that may greatly differ from the pH value in the reaction medium (Rodrigues, Ortiz, Berenguer-Murcia, Torres, & Fernández- Lafuente, 2013). However, this explanation could be discarded since chitosan is a polyaminosaccharide without ionic strength and the enzyme molecules probably get immobilized mostly in their outer surfaces (Klein et al., 2012). This wider pH range may be probably due to the stabilization of enzyme 3D structure by the attachment on the support. "
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    ABSTRACT: 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.
    Carbohydrate Polymers 03/2014; 103:193–197. DOI:10.1016/j.carbpol.2013.12.038 · 4.07 Impact Factor
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    • ") and activated with glutaraldehyde (5% v/v) as previously described (Klein et al., 2012). In order to test the support loading, 10 mL of enzyme solution with different protein concentrations, in sodium phosphate buffer (0.1 M, pH 6.0) was added to 100 chitosan spheres (representing 20, 60, 120 and 200 mg/g of dry support), and incubated , overnight, at room temperature under gentle shaking in an orbital shaker (200 rpm). "
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    ABSTRACT: 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 (1.32g/L) in a flow rate of 3mL/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 100h of continuous use. These results suggest the possibility of using this immobilized biocatalyst in continuous production of CDs.
    11/2013; 98(2):1311-6. DOI:10.1016/j.carbpol.2013.07.044
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