Immobilization and specific activity of recombinant BgaC

The E. coli T7 express (pDMg1a) strain which harbors the B. adolescentis β-galactosidase BgaC was grown to induce recombinant protein expression, and His-BgaC purification was conducted using Ni2+-NTA columns (Mulualem et al. 2021). Purified recombinant BgaC was immobilized with a blend of calcium-alginate and gelatin in the presence of the cross-linker glutaraldehyde. Commercial AO.β-Gal was immobilized in a similar manner for comparative purposes. The average weight of individual beads ranged from 60 to 70 mg, with an average diameter of 4.4 mm and incorporated approximately 1.34 μg enzyme.

The β-galactosidase activity of immobilized BgaC was compared to the activity of free BgaC using ONPG and lactose as substrates (Table 1). Beads generated with water in the absence of enzyme were used as a negative control and, as expected, displayed no β-galactosidase activity. The immobilized BgaC enzyme showed a 132% increase (2.3-fold increase) in specific activity compared to the free enzyme when lactose was used as a substrate and a 41% increase when ONPG was the substrate (Table 1). The specific β-galactosidase activity of immobilized AO.β-Gal with ONPG as substrate was similar to that of immobilized BgaC (Table 1). These data indicated that the β-galactosidase activity of BgaC was not only retained after immobilization but also increased.

The activity yield of immobilized BgaC compared to the free enzyme was 153%. This could be attributed to many factors. The immobilization may stabilize the active form of the enzyme with higher activity compared to the native enzyme as in the case of lipase immobilization (Mateo et al. 2007). Alternatively, immobilization may reduce substrate or product inhibition, as the free enzyme was inhibited by the substrate ONPG at concentrations ≥ 2.5 mM (Mulualem et al. 2021).

Kinetic parameters of immobilized β-galactosidase BgaC

The kinetic parameters of immobilized BgaC were determined using different concentrations of ONPG (0.25–10 mM) as substrate in a time course (0–18 min) enzyme assay. No substrate or product inhibition was observed at the tested concentrations of ONPG within the progress curve of the assay (Fig. 1a). The KM and Vmax of the immobilized enzyme were 0.81 ± 0.22 mM and 7.4 ± 0.63 μmol/min/mg, respectively. The kcat and kcat/KM of the immobilized enzyme were 802 s⁻1 and 990 s⁻1 mM⁻1, respectively (Table 1). The kcat and kcat/KM values of immobilized BgaC are much higher compared to a previous report for free BgaC (Table 1) which suggests the immobilized BgaC demonstrates high catalytic efficiency. Immobilization of BgaC improved its tolerance to ONPG inhibition, which was previously observed for the free enzyme, where the hydrolytic activity markedly declined at ONPG concentrations higher than 2.5 mM (Mulualem et al. 2021). The KM of the immobilized BgaC for ONPG reduced three-fold compared to the KM of the free enzyme (Table 1), which indicated that the immobilization increased the affinity of the enzyme towards ONPG. This suggested that this immobilization method did not restrict the movement of substrates and buffer into the carrier matrix, which can happen in many immobilization procedures (Tischer and Kasche 1999).

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

Table 1 Specific β-galactosidase activity, KM, Vmax, kcat, and kcat/KM for free and immobilized BgaC with ONPG and lactose as substrateEffect of pH and temperature on specific activity and stability of free and immobilized BgaC

The impact of different pH and temperature ranges on the catalytic activity and stability of free and immobilized BgaC was determined. The specific enzyme activities were examined at pH values between 4 and 10 and temperatures ranging from 0 to 60 °C using ONPG as substrate (Figs. 2 and 3). The optimum pH for β-galactosidase activity of free BgaC was pH 7.0, and the enzyme retained 58% and 52% of its activity at pH 4 and at pH 8.5, respectively (Fig. 2a). However, at pH 10, the activity was nearly abolished. These data were in line with the previous report, although decreased activity at pH 4 and 4.5 was detected here, rather than abolished (Mulualem et al. 2021). On the other hand, immobilized BgaC retained its activity at all the tested pH and retained 93% and 81% activity at pH 4.5 and pH 10, respectively (Fig. 2a). One-Way ANOVA analysis revealed no statistically significant effect of pH on the immobilized enzyme activity, F(12, 26) = 0.19, p = 0.998 (Supplementary Table 1a). This indicates that the immobilized enzyme maintained steady activity across the pH range of 4.0 to 10.0 under the experimental conditions. Therefore, the immobilization procedure extended the pH range for enzyme activity. In contrast, for the free enzyme, One-Way ANOVA revealed a highly statistically significant effect of pH on enzyme activity, F(12,26) = 163.40, p < 0.001, indicating that the mean enzyme activity of the free enzyme differs significantly across at least some of the pH levels tested (Supplementary Table 1b). Further post hoc comparisons using Tukey’s HSD test were performed to identify specific pH levels with significant differences in free enzyme activity (Supplementary Table 1c). The results indicated a distinct optimal pH range, with enzyme activity significantly higher at central pH values compared to acidic (pH 4.0, pH 4.5) and alkaline (pH 9.0, pH 9.5, pH 10.0) conditions. For instance, enzyme activity at pH 7.0 was significantly greater (p < 0.001) than at pH 4.0, 8.5, 9.0, 9.5, and 10.0.

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)

In the case of pH stability, the immobilized BgaC retained activity after 24 h pre-incubation at all tested pH values (Fig. 2b). The free enzyme retained ≥ 66% of residual activity after pre-incubation at acidic pH values (pH 4.0–6.5) and showed increased stability (with some variation detected) following pre-incubation at alkaline pH ranges (pH 7.5–10) compared to pH 7.0 (Fig. 2b). In the previous report, the free enzyme was stable at all tested pH values, with 87% residual activity detected at pH 4 (Mulualem et al. 2021). One-way ANOVA analysis showed no statistically significant effect of different pH conditions on the retained activity of the immobilized enzyme, F(12, 26) = 1.64, p = 0.142 (Supplementary Table 2a). This suggests that the immobilization process conferred robust pH stability to the enzyme, allowing it to maintain consistent activity across the tested pH range (pH 4.0 to 10.0). In contrast, one-way ANOVA conducted for the free enzyme revealed a highly statistically significant effect of pH conditions on its stability, F(12,26) = 57.12, p < 0.001, indicating that the free enzyme loses a significant portion of its activity when exposed to non-optimal pH conditions for extended periods (Supplementary Table 2b). The Tukey’s HSD post hoc test results for the free enzyme pH stability indicate that there were no statistically significant differences in the retained activity of the free enzyme within the acidic pH range (pH 4.0–5.5, with p > 0.05) (Supplementary Table 2c). However, the activity at all acidic pH was significantly lower than at pH where the enzyme exhibited highest stability (e.g., pH 8.0, 8.5 or 10.0) (p < 0.001 for comparisons between pH 4.0–5.5 and pH 8.0, 8.5 or 10.0). In summary, immobilized BgaC showed higher stability than free BgaC at low and high pH ranges, and the immobilization procedure enhanced enzyme stability at acidic pH.

Regarding the effect of temperature, the highest activity for the immobilized BgaC was detected at 40 °C, while the free enzyme had the highest activity at 37 °C (Fig. 3a). Immobilized BgaC retained 81% of its activity at 50 °C, but showed relatively low activity between 0 and 20 °C. The free enzyme retained 68% and 41% of its activity at 40 and 50 °C, respectively. ANOVA analysis revealed a highly significant effect of temperature on the immobilized enzyme, with a large F-statistic [F(9,20) = 7.75] and a very low P-value (p = 7.48 × 10−5) (Supplementary Table 3a and 3b). Similarly, free enzyme activity displayed a highly significant effect of temperature, with a larger F-statistic [F(8,18) = 14.50] and an extremely low P-value (p = 2.13 × 10−6) (Supplementary Table 3c and 3 d). The higher F-statistic for the free enzyme compared to the immobilized enzyme (F free = 14.50 vs. F immobilized = 7.75) might suggest that the free enzyme is more sensitive to temperature changes than its immobilized counterpart within the tested temperature range.

The maximum temperature stability of both the immobilized and free BgaC was 37 °C, yet 48% and 74% of activity were retained following 1 h pre-incubation at 40 °C by the free and immobilized enzyme, respectively (Fig. 3b). Both the free and immobilized enzymes were inactive after pre-incubation at 45 °C and above; however, ≥ 61% and ≥ 75% residual activity remained following pre-incubation at temperatures of 0–37 °C for the free enzyme and immobilized enzyme, respectively.

ANOVA analysis for the effect of temperature on the stability of immobilized BgaC showed an F-statistic of F(9,20) = 204.06 and a p-value of 1.11 × 10−17 (or p < 0.001) (Supplementary Table 4a). In addition, the Tukey’s HSD test revealed that the optimal stability range for immobilized BgaC is 0 °C to 37 °C (Supplementary Table 4b). Most temperatures within this range are not significantly different from each other in terms of stability (e.g., 0 °C vs 20 °C: p-adj = 0.492; 20 °C vs 30 °C: p-adj = 1.0; 30 °C vs 37 °C: p-adj = 0.2582). The ANOVA on the effect of temperature on the stability of free BgaC indicated an F-statistic of F(9,20) = 110.10, with an extremely low p-value of 4.72 × 10 −15 (or p < 0.001) (Supplementary Table 4c). The Tukey’s HSD test revealed that the optimal stability range for free BgaC also extends from 0 °C up to 37 °C (Supplementary Table  4 d). Within this range, temperatures generally do not show statistically significant differences in stability (e.g., 0 °C vs 20 °C: p-adj = 0.7442; 20 °C vs 30 °C: p-adj = 0.5961). In summary, Tukey’s HSD revealed that both free and immobilized BgaC maintain their stability well up to around 37 °C, but then experience a sharp and significant decline starting at 40 °C. However, immobilized BgaC appears to maintain a statistically similar level of stability at 40 °C as it does at 20 °C p-adj (0.2142) or 30 °C with p-adj (0.0921), whereas the free enzyme has already experienced a statistically significant drop at 40 °C compared to those same lower temperatures with p-adj = 0.0076 for 20 °C and p-adj = 0.0001 for 30 °C.

With regard to the effect of storage temperature on the activity of BgaC, storage at 4 °C for 24 h did not affect the activity of the free enzyme or the immobilized BgaC. It was previously reported that free BgaC can be stored at 4 °C for 5 weeks without loss of activity (Mulualem et al. 2021).

Reutilization of immobilized BgaC and AO.β-Gal

The specific activity assay was repeated twelve times sequentially on the same immobilized enzyme beads within the same day. After each assay, the beads were thoroughly washed before reutilization. Both the BgaC and AO.β-Gal showed minimal reduction in activity when the first use was compared to the successive 11 rounds (from 52 ± 9 to 42 ± 15 and from 61 ± 6 to 46 ± 17 μmol/min/mg, respectively) (Fig. 4a and b). There was a trend in declining activity with successive assays overall, although a linear decline in activity was not detected. The activity of the immobilized enzymes showed a slight decrease after the 8th reutilization for BgaC and after the 9th reutilization for AO.β-Gal.

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

Repeated measures ANOVA was conducted to assess the effect of reuse on specific activity (Supplementary Tables 5a and 5b). For the overall effect of reuse on each immobilized enzyme, Mauchly's Test indicated a significant violation of sphericity (p < 0.001), leading to the application of Greenhouse–Geisser correction. The ANOVA results showed no statistically significant overall effect of reutilization cycle on BgaC specific activity [F(1.243,2.486) = 7.134, p = 0.093]. While not statistically significant, the effect size was large (ηp2 = 0.781), suggesting a notable proportion of variance was associated with the reuse rounds, though statistical power might have been a limiting factor given the small sample size (N = 3). Despite the non-significant overall effect, Bonferroni-corrected pairwise comparisons revealed one statistically significant difference: the specific activity in Round 3 (mean, 80.88) was significantly higher than in Round 8 (mean, 34.19) (p = 0.002). All other pairwise comparisons were not statistically significant (p > 0.05). This specific finding would typically be interpreted with caution, acknowledging the non-significant overall test.

The ANOVA results similarly demonstrated no statistically significant overall effect of reuse round on AO.β-gal specific activity [F(1.056,2.112) = 0.857, p = 0.455, ηp2 = 0.300]. Tests of within-subjects contrasts revealed no statistically significant linear, quadratic, or higher-order trends. Bonferroni-corrected pairwise comparisons showed no statistically significant differences between any of the reuse rounds (p > 0.05 for all comparisons).

This analysis consistently indicated no significant effect of reuse rounds on the specific activity of immobilized BgaC and immobilized AO.β-gal. It can be concluded that the immobilization procedure using calcium alginate and gelatin cross-linked with glutaraldehyde enabled the retention of enzyme activity for at least 12 successive reutilizations.