Improving β-Galactosidase-Catalyzed Transglycosylation Yields by Cross-Linked Layer-by-Layer Enzyme Immobilization

Abstract

The biotransformation of lactose into gut-bioactive glycans catalyzed by βgalactosidase can give economic value to lactose-rich side streams generated in the food or dairy industry. Herein, we study the immobilization of the commercially used β-galactosidase from Bacillus circulans onto silica particles using an enzyme immobilization technology involving a cross-linked layer-by-layer encapsulation method. The immobilized β-galactosidase was used for the synthesis of Nacetyllactosamine (LacNAc) as an important precursor for numerous bioactive compounds and a prebiotic in itself. Techniques including molecular analysis, enzyme activity determination, secondary structure analysis, thermodynamic characterization as well as the determination of thermal and operational stability were conducted to characterize the immobilized enzyme. Changes in the activity of the enzyme after immobilization were attributed to possible changes in electrostatic, covalent and protein-protein interactions. Immobilization significantly improved enzymatic LacNAc yield compared to the free enzyme. In turn, this improved the economics and the sustainability of the process. The immobilized enzyme encapsulated in multilayer films was significantly more stable in the presence of divalent cations and its thermostability also substantially increased, with the thermal denaturation activation energy increasing from 53 kJ mol -1 to 294 kJ mol -1 . The immobilized enzyme was successfully reused in eight consecutive reaction cycles with no significant reduction in the LacNAc yield. The improved transgalactosylation yield and productivity, higher stability and reusability obtained with this immobilization method provide new opportunities for industrial applications.

Synthesis of N-Acetyllactosamine and N-Acetyllactosamine-Based Bioactives

“…We recently used a layer-by-layer encapsulation method to immobilize β-galactosidase onto silica particles. 190 In this technique, a positively charged layer of polyallylamine hydrochloride (PAH) was first coated on the negatively charged silica particles, followed by the electrostatic adsorption of the Bacillus circulans β-galactosidase (negatively charged at pH of 6), glutaraldehyde crosslinking, as well as the additional deposition of oppositely charged polyelectrolytes (PAH and polystyrene sulfonate (PSS)). This immobilized enzyme was used in at least eight successive cycles of LacNAc synthesis from lactose with no significant decrease in the LacNAc yield.…”

“…We recently used a layer-by-layer encapsulation method to immobilize β-galactosidase onto silica particles . In this technique, a positively charged layer of poly­(allylamine hydrochloride) (PAH) was first coated on the negatively charged silica particles, followed by the electrostatic adsorption of the B.…”

Bioactives from Whey: A Sustainable Approach to Enzymatic Production of Sialyl-N-acetyllactosamine

“…In particular, whey lactose can be used as a galactosyl donor and whey casein glycomacropeptide (CGMP) as a sialyl donor in enzymatic glycosylation and sialylation reactions. 3,4 Sialic acidterminated glycans containing two trisaccharide units of sialylated N-acetyllactosamine (LacNAc), namely, Neu5Ac-α2-6-Gal-β1-4-GlcNAc (6′-sialyl-LacNAc; 6′-SLN) and Neu5Ac-α2-3-Gal-β1-4-GlcNAc (3′-sialyl-LacNAc; 3′-SLN), on the host cell surface act as receptors for binding of influenza virus hemagglutinin glycoprotein, leading to the initiation of viral infections. 5,6 Thus, several researchers have developed antiviral analogs based on sialylated LacNAcs that inhibit the binding of sialic-acid-targeting viruses.…”

“…Only half of this whey is utilized to produce value-added compounds, and so, the unused whey represents a substantial disposal problem given its high organic content. , Thus, the development of sustainable routes for transforming whey into higher value ingredients is of great significance. In particular, whey lactose can be used as a galactosyl donor and whey casein glycomacropeptide (CGMP) as a sialyl donor in enzymatic glycosylation and sialylation reactions. , Sialic acid-terminated glycans containing two trisaccharide units of sialylated N -acetyllactosamine (LacNAc), namely, Neu5Ac-α2-6-Gal-β1-4-GlcNAc (6′-sialyl-LacNAc; 6′-SLN) and Neu5Ac-α2-3-Gal-β1-4-GlcNAc (3′-sialyl-LacNAc; 3′-SLN), on the host cell surface act as receptors for binding of influenza virus hemagglutinin glycoprotein, leading to the initiation of viral infections. , Thus, several researchers have developed antiviral analogs based on sialylated LacNAcs that inhibit the binding of sialic-acid-targeting viruses. − Efficient synthesis methods that facilitate cheap mass production of these new antiviral drugs can play a key role in the feasibility and marketability of these products. In addition, sialylated LacNAcs have application in the fabrication of glycomaterials and for immobilization onto biosensors. , …”

Pickering Droplet-Derived Silica Microreactors with a Biomimetic Aqueous Environment for Continuous-Flow Enzymatic Reactions

“…Enzymes, as fascinating biocatalysts in living systems, are attracting increasing concerns in chemical and pharmaceutical industries, due to the sustainable chemistry they can accomplish with high reactivity and specificity under mild conditions. − However, for most of the enzymes, some serious problems are often encountered outside their native environments, like structural denaturation, lowered activity, and limited stability. − Although great efforts including enzyme or medium engineering, chemical modification, and enzyme immobilization have been devoted to overcome these obstacles, the enzymatic efficiency is still severely restricted by the fact that the inherent structure and function of most enzymes are only well deserved in biologically friendly aqueous environments. − …”