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

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.

Keywords: BgaC; Bifidobacterium; Calcium alginate; Entrapment; Immobilization; β-Galactosidase.

Fig. 1

Time course progress curve and non-linear regression fit of immobilized BgaC. Activity was monitored at 2 min intervals over an 18 min period with various concentrations of ONPG (0.25–10 mM). Three independent experiments were conducted, each with three replicates. a Time course progress curve of ONPG hydrolysis. Error bars indicate ± 1 standard deviation. b Non-linear regression fit model of Michaelis–Menten plot for determination of the kinetic parameters

Fig. 2

Effect of pH on activity and stability of free and immobilized BgaC. Values represent the mean ± 1 SD of three independent experiments (i.e., three separate batches of immobilized beads). The specific activity value at pH 7 was taken as 100% relative activity. Statistical significance of free or immobilized BgaC at various pH was determined by ANOVA with Tukey’s HSD post hoc test (p < 0.05) (Supplementary Tables 1 and 2). There was no statistical difference in the activity of immobilized BgaC at different pH and therefore statistical significance values are presented only for free BgaC. a Effect of pH on the activity of free BgaC (purple circles) and immobilized BgaC (red triangles). Letters (a, b, c) indicate a statistically significant difference between mean enzyme activities at pH 4.0, 7.0, and 10.0, respectively. Statistical values for other pH values are presented in Supplementary Table 1c. b pH stability of free BgaC (purple circles) and immobilized BgaC (red triangles). The activity at pH values sharing a common letter is not significantly different. Statistical values for other pH values are presented in Supplementary Table 2c

Fig. 3

Effect of temperature on activity and stability of free and immobilized BgaC. The values represent the mean ± 1 SD of three independent experiments (three batches of beads for immobilized enzyme). The specific activity value at 37 °C was taken as 100% relative activity. Based on Tukey’s HSD post hoc test, means with different lowercase letters (for free enzyme) or numbers (for immobilized enzyme) are statistically significantly different (p < 0.05), while means sharing the same letter or number are not. a Effect of temperature on the activity of free BgaC (purple circles) and immobilized BgaC (red triangles). b Effect of temperature on stability of free BgaC (purple circles) and immobilized BgaC (red triangles)

Fig. 4

Reutilization of immobilized BgaC and AO.β-Gal. a The specific activity of immobilized BgaC and (b) the immobilized AO.β-Gal determined for the twelve rounds of reutilization assay using ONPG as a substrate. For both panels the data represent the mean ± SD of relative specific activity values from three independent biological experiments (i.e., three batches of beads for immobilized enzyme). The specific activity value of reuse cycle 1 was taken as 100% relative activity. Statistical significance analysis by repeated measure ANOVA with Greenhouse–Geisser correction showed no statistical differences in enzyme activity throughout the cycles of reutilization. Post hoc Bonferroni-corrected pairwise comparisons revealed that, while the overall effect was not significant, specific activity in Round 3 (mean = 80.88) was significantly higher than in Round 8 (mean = 34.19) (p = 0.002) for immobilized BgaC