Abstract β-Galactosidase enzymes catalyze the hydrolysis of terminal non-reducing β-D-galactose residues in β-galactosides. These enzymes are important in producing lactose-free dairy products, reducing the lactose content of whey in dairy products, and for production of galactooligosaccharides (GOS) as prebiotic additives to infant formula. To use β-galactosidases in industrial settings, enzyme immobilization procedures are used to enhance their activity and stability and to minimize enzyme quantities and cost. In this study, recombinant Bifidobacterium adolescentis β-galactosidase BgaC was immobilized in calcium alginate and gelatin cross-linked with glutaraldehyde. The kinetic parameters and stability properties of immobilized BgaC were characterized in comparison with free soluble enzyme. The KM for immobilized BgaC using ortho-nitrophenyl-β-galactoside (ONPG) was 810 ± 220 μM and the KM of free BgaC was 2500 ± 3 μM. The kcat and kcat/KM of immobilized BgaC were 802 s−1 and 990 s−1 mM−1, respectively, compared to kcat and kcat/KM values of 209 s−1 and 84 s−1 mM−1, respectively, for free BgaC. Immobilized BgaC β-galactosidase was active at all tested pH (pH 4–10), while the free enzyme had decreased activity at pH < 5.5 and > 8.0. The immobilized enzyme had optimum activity at 40 °C, while the free enzyme was most active at 37 °C. In addition, immobilization enhanced acidic pH and temperature stability compared to the free enzyme. Reutilization of the BgaC beads was assessed and the enzyme maintained 69% activity after 12 rounds of reutilization. Therefore, the enhanced performance properties of immobilized BgaC make it a promising candidate for industrial applications. Key points • Bifidobacterium adolescentis β-galactosidase BgaC was successfully immobilized • Immobilized BgaC has enhanced enzymatic activity and stability and allows recycling • Sustained activity of immobilized BgaC is advantageous for industrial applications Graphical Abstract

Immobilized β-galactosidase BgaC from Bifidobacterium adolescentis retains stability and activity during repeated cycles of use

期刊论文
出版模式：

开放获取
发表日期：

2025年7月30日

查看全文

Daniel Mehabie Mulualem,
Orla Dwan,
Michelle Kilcoyne,
Conor O’Byrne,
Aoife Boyd

摘要

Abstract

β-Galactosidase enzymes catalyze the hydrolysis of terminal non-reducing β-D-galactose residues in β-galactosides. These enzymes are important in producing lactose-free dairy products, reducing the lactose content of whey in dairy products, and for production of galactooligosaccharides (GOS) as prebiotic additives to infant formula. To use β-galactosidases in industrial settings, enzyme immobilization procedures are used to enhance their activity and stability and to minimize enzyme quantities and cost. In this study, recombinant Bifidobacterium adolescentis β-galactosidase BgaC was immobilized in calcium alginate and gelatin cross-linked with glutaraldehyde. The kinetic parameters and stability properties of immobilized BgaC were characterized in comparison with free soluble enzyme. The K_M for immobilized BgaC using ortho-nitrophenyl-β-galactoside (ONPG) was 810 ± 220 μM and the K_M of free BgaC was 2500 ± 3 μM. The k_cat and k_cat/K_M of immobilized BgaC were 802 s⁻¹ and 990 s⁻¹ mM⁻¹, respectively, compared to k_cat and k_cat/K_M values of 209 s⁻¹ and 84 s⁻¹ mM⁻¹, respectively, for free BgaC. Immobilized BgaC β-galactosidase was active at all tested pH (pH 4–10), while the free enzyme had decreased activity at pH < 5.5 and > 8.0. The immobilized enzyme had optimum activity at 40 °C, while the free enzyme was most active at 37 °C. In addition, immobilization enhanced acidic pH and temperature stability compared to the free enzyme. Reutilization of the BgaC beads was assessed and the enzyme maintained 69% activity after 12 rounds of reutilization. Therefore, the enhanced performance properties of immobilized BgaC make it a promising candidate for industrial applications.

Key points

• Bifidobacterium adolescentis β-galactosidase BgaC was successfully immobilized

• Immobilized BgaC has enhanced enzymatic activity and stability and allows recycling

• Sustained activity of immobilized BgaC is advantageous for industrial applications

Graphical Abstract