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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    • ") 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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    • ". Preparation ofˇ-d-galactosidase immobilized on chitosan macroparticleš -d-galactosidase was covalently immobilized on glutaraldehyde-activated chitosan macroparticles as described in a previous work (Klein et al., 2012). The amount of protein attached on chitosan macroparticles was 46. "
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    ABSTRACT: β-d-Galactosidase from Kluyveromyces lactis was immobilized on glutaraldehyde-activated chitosan and used in a packed-bed reactor for the continuous hydrolysis of lactose and the synthesis of galactooligosaccharides (GOS). The biocatalyst was tested for its optima pH and temperature, thermal stability in the presence of substrate and products, and operational stability. Immobilization increased the range of operational pH and temperature, and the enzyme thermal stability was sharply increased in the presence of lactose. Almost complete lactose hydrolysis was achieved for both milk whey and lactose solution at 37°C at flow rates up to 2.6mLmin(-1). Maximal GOS concentration of 26gL(-1) was obtained at a flow rate of 3.1mLmin(-1), with a productivity of 186gL(-1)h(-1). Steady-state operation for 15 days showed the reactor stability concerning lactose hydrolysis.
    06/2013; 95(1):465-70. DOI:10.1016/j.carbpol.2013.02.044
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    ABSTRACT: Invertase (E.C. 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.
    01/2013; 92(1):462-8. DOI:10.1016/j.carbpol.2012.09.001
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