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.

This paper aims to investigate the effects of some salts (NaCl, (NH₄)₂SO₄ and Na₂SO₄) at pH 5.0, 7.0 and 9.0 on the stability of 13 different immobilized enzymes: five lipases, three proteases, two glycosidases, and one laccase, penicillin G acylase and catalase. The enzymes were immobilized to prevent their aggregation. Lipases were immobilized via interfacial activation on octyl agarose or on glutaraldehyde-amino agarose beads, proteases on glyoxyl agarose or glutaraldehyde-amino agarose beads. The use of high concentrations of salts usually has some effects on enzyme stability, but the intensity and nature of these effects depends on the inactivation pH, nature and concentration of the salt, enzyme and immobilization protocol. The same salt can be a stabilizing or a destabilizing agent for a specific enzyme depending on its concentration, inactivation pH and immobilization protocol. Using lipases, (NH₄)₂SO₄ generally permits the highest stabilities (although this is not a universal rule), but using the other enzymes this salt is in many instances a destabilizing agent. At pH 9.0, it is more likely to find a salt destabilizing effect than at pH 7.0. Results confirm the difficulty of foreseeing the effect of high concentrations of salts in a specific immobilized enzyme.

Xylooligosaccharides (XOS) are widely used in the food industry as prebiotic components. XOS with high purity are required for practical prebiotic function and other biological benefits, such as antioxidant and inflammatory properties. In this work, we immobilized the recombinant endo-1,4-β-xylanase of Malbranchea pulchella (MpXyn10) in various chemical supports and evaluated its potential to produce xylooligosaccharides (XOS) from hydrothermal liquor of eucalyptus wood chips. Values >90% of immobilization yields were achieved from amino-activated supports for 120 min. The highest recovery values were found on Purolite (142%) and MANAE-MpXyn10 (137%) derivatives, which maintained more than 90% residual activity for 24 h at 70 °C, while the free-MpXyn10 maintained only 11%. In addition, active MpXyn10 derivatives were stable in the range of pH 4.0–6.0 and the presence of the furfural and HMF compounds. MpXyn10 derivatives were tested to produce XOS from xylan of various sources. Maximum values were observed for birchwood xylan at 8.6 mg mL⁻¹ and wheat arabinoxylan at 8.9 mg mL⁻¹, using Purolite-MpXyn10. Its derivative was also successfully applied in the hydrolysis of soluble xylan present in hydrothermal liquor, with 0.9 mg mL⁻¹ of XOS after 3 h at 50 °C. This derivative maintained more than 80% XOS yield after six cycles of the assay. The results obtained provide a basis for the application of immobilized MpXyn10 to produce XOS with high purity and other high-value-added products in the lignocellulosic biorefinery field.

The lipase from Prunus dulcis almonds has been immobilized for the first time. For this purpose, two different supports, an octadecyl methacrylate particulate support, and aminated agarose (monoaminoethyl-N-aminoethyl) have been utilized. Both immobilized biocatalysts show improved enzyme stability, but great changes in enzyme specificity were detected. The enzyme immobilized via ion exchange maintained its activity intact versus p-nitrophenyl butyrate, while the enzyme immobilized on the hydrophobic support fully lost its activity versus this substrate, which was confirmed to be due to substrate adsorption on the support. However, this biocatalyst was much more active versus triacetin (more than 10-fold), R- or S- methyl mandelate at pH 7. At pH 9, a strong effect of using phosphate or bicarbonate as reaction buffers was detected. Using bicarbonate, the interfacially immobilized enzyme presented no activity versus R-isomer, but it was very active versus the S-isomer and triacetin. Using a phosphate buffer during the reaction, all compounds were recognized as substrates. The enzyme immobilized via ion exchange was significantly more active using phosphate; in fact, using bicarbonate, the enzyme was inactive versus both methyl mandelate isomers. This paper shows for the first time a great interaction between the effects of the immobilization protocol and buffer used during reaction on the enantiospecificity of lipases.

The fungus Aspergillus caespitosus was cultivated under solid-state fermentation (SSF) using wheat bran (WB) and sugarcane bagasse (SCB) as agro-industrial substrates to produce xylanase. WB supported the highest enzymatic activity (approximately 1100 U g⁻¹ dry substrate), while pretreatment of SCB with NaOH (AT-SCB) enhanced productivity to about 1500 U g⁻¹, confirming the positive effect of lignocellulosic modification. The optimal moisture ratio (1:3 water: solid) yielded approximately 2400 U g⁻¹, and supplementation with 1% NH₄NO₃ and trace salts further increased xylanase synthesis by approximately 40%. The crude extract retained more than 80% of its activity after 72 h at 50 °C, indicating good thermal stability. In kraft pulp biobleaching, treatment with WB-derived xylanase (10 U g⁻¹ pulp, 50 °C, 3 h) resulted in a 15% reduction in the kappa number and a 2.5 ISO-point increase in brightness, with no detectable cellulose degradation. These findings demonstrate that A. caespitosus efficiently produced a thermostable and selective xylanase under SSF, highlighting the potential of agro-residue valorization for developing environmentally friendly processes in the pulp and paper industry.