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 2.6 mL min(-1). Maximal GOS concentration of 26 g L(-1) was obtained at a flow rate of 3.1 mL min(-1), with a productivity of 186 g L(-1) h(-1). Steady-state operation for 15 days showed the reactor stability concerning lactose hydrolysis.

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.

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Glycoside hydrolases (GH) are enzymes capable to hydrolyze the glycosidic bond between two carbohydrates or even between a carbohydrate and a non-carbohydrate moiety. Because of the increasing interest for industrial applications of these enzymes, the immobilization of GH has become an important development in order to improve its activity, stability, as well as the possibility of its reuse in batch reactions and in continuous processes. In this review, we focus on the broad aspects of immobilization of enzymes from the specific GH families. A brief introduction on methods of enzyme immobilization is presented, discussing some advantages and drawbacks of this technology. We then review the state of the art of enzyme immobilization of families GH1, GH13, and GH70, with special attention on the enzymes β-glucosidase, α-amylase, cyclodextrin glycosyltransferase, and dextransucrase. In each case, the immobilization protocols are evaluated considering their positive and negative aspects. Finally, the perspectives on new immobilization methods are briefly presented.

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