In the last decade, new trends for enzyme attachment to solid carriers have emerged in an attempt to rationalize the classical methods for enzyme immobilization. In silico analysis is becoming a powerful tool to predict the orientation of the enzyme covalently-attached to the carrier or the protein regions involved in the adsorption to the support. Significantly, an array of algorithms has been established for the Rational Design of Immobilized Derivatives (RDID), which comprises both the protein size and the textural properties of the support. Ordered mesoporous materials open a challenging pathway to tailor immobilized enzymes with high volumetric activity and minimum lixiviation. In addition, fluorescence confocal microscopy is being successfully employed to understand the diffusional restrictions and the distribution of biomolecules within the support.

The formation of galactooligosaccharides (GOS) in skim milk during treatment with several commercial β-galactosidases (Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae) was analysed in detail, at 4 and 40°C. The maximum GOS concentration was obtained at a lactose conversion of approximately 40-50% with B. circulans and A. oryzae β-galactosidases, and at 95% lactose depletion for K. lactis β-galactosidase. Using an enzyme dosage of 0.1% (v/v), the maximum GOS concentration with K. lactis β-galactosidase was achieved in 1 and 5h at 40 and 4 °C, respectively. With this enzyme, it was possible to obtain a treated milk with 7.0 g/L GOS - the human milk oligosaccharides (HMOs) concentration is between 5 and 15 g/L--and with a low content of residual lactose (2.1g/L, compared with 44-46 g/L in the initial milk sample). The major GOS synthesised by this enzyme were 6-galactobiose [Gal-β(1 → 6)-Gal], allolactose [Gal-β(1 → 6)-Glc] and 6'-O-β-galactosyl-lactose [Gal-β(1 → 6)-Gal-β(1 → 4)-Glc].

The transgalactosylation activity of Kluyveromyces lactis cells was studied in detail. Cells were permeabilized with ethanol and further lyophilized to facilitate the transit of substrates and products. The resulting biocatalyst was assayed for the synthesis of galacto-oligosaccharides (GOS) and compared with two soluble β-galactosidases from K. lactis (Lactozym 3000 L HP G and Maxilact LGX 5000). Using 400 g/L lactose, the maximum GOS yield, measured by HPAEC-PAD analysis, was 177 g/L (44% w/w of total carbohydrates). The major products synthesized were the disaccharides 6-galactobiose [Gal-β(1→6)-Gal] and allolactose [Gal-β(1→6)-Glc], as well as the trisaccharide 6-galactosyl-lactose [Gal-β(1→6)-Gal-β(1→4)-Glc], which was characterized by MS and 2D NMR. Structural characterization of another synthesized disaccharide, Gal-β(1→3)-Glc, was carried out. GOS yield obtained with soluble β-galactosidases was slightly lower (160 g/L for Lactozym 3000 L HP G and 154 g/L for Maxilact LGX 5000); however, the typical profile with a maximum GOS concentration followed by partial hydrolysis of the newly formed oligosaccharides was not observed with the soluble enzymes. Results were correlated with the higher stability of β-galactosidase when permeabilized whole cells were used.

The synthesis of galactooligosaccharides (GOS) catalyzed by β-galactosidase from Aspergillus oryzae (Enzeco) was studied. Using 400 g/L of lactose and 15 U/mL, maximum GOS yield, measured by HPAEC-PAD, was 26.8% w/w of total carbohydrates, obtained at approximately 70% lactose conversion. No less than 17 carbohydrates were identified; the major transgalactosylation product was 6'-O-β-galactosyl-lactose, representing nearly one-third (in weight) of total GOS. In contrast with previous reports, the presence of at least five disaccharides was detected, which accounted for 40% of the total GOS at the point of maximum GOS concentration (allolactose and 6-galactobiose were the major products). A. oryzae β-galactosidase showed a preference to form β(1→6) bonds, followed by β(1→3) and β(1→4) linkages. Results were compared with those obtained with β-galactosidases from Kluyveromyces lactis and Bacillus circulans. The highest GOS yield and specific productivity were achieved with B. circulans β-galactosidase. The specificity of the linkages formed and distribution of di-, tri-, and higher GOS varied significantly among the three β-galactosidases.

The synthesis of galacto-oligosaccharides (GOS) catalyzed by a novel commercial preparation of β-galactosidase from Bacillus circulans (Biolactase) was studied, and the products were characterized by MS and NMR. Using 400 g/L lactose and 1.5 enzyme units per milliliter, the maximum GOS yield, measured by HPAEC-PAD analysis, was 165 g/L (41% w/w of total carbohydrates in the mixture). The major transgalactosylation products were the trisaccharide Gal-β(1→4)-Gal-β(1→4)-Glc and the tetrasaccharide Gal-β(1→4)-Gal-β(1→4)-Gal-β(1→4)-Glc. The GOS yield increased to 198 g/L (49.4% w/w of total carbohydrates) using a higher enzyme concentration (15 U/mL), which minimized the enzyme inactivation under reaction conditions. Using skim milk (with a lactose concentration of 46 g/L), the enzyme also displayed transgalactosylation activity: maximum GOS yield accounted for 15.4% (7.1 g/L), which was obtained at 50% lactose conversion.