## 2 Results and Discussion
### Temperature and pH Sensitivity of Immobilized Enzyme Using RNase B  
Enzyme properties can change with respect to pH and temperature sensitivity when immobilized [31; 32]. Temperature sensitivity was evaluated at pH 5 with 20 mM Na\({}_{2}\)HPO\({}_{4}\) buffer at temperatures from 45 to 85 \({}^{\circ}\)C (Figure 4a). The optimal temperature for both free and immobilized Endo BI-1 was 65 \({}^{\circ}\)C. These results differ from what was previously reported by [PERSON] et al. [33], in which a lower optimum temperature of 52 \({}^{\circ}\)C was identified for the free enzyme. This discrepancy could be attributed to different reaction conditions used in both studies. While RNase B and a short reaction time (20 min) were used in the present work, bovine colostrum whey protein and longer reaction times (15-475 min) were evaluated in the previous study. At the optimum temperature identified (65 \({}^{\circ}\)C), the immobilized enzyme retained 63% of the activity compared with the free enzyme. In each experimental condition of both temperature and pH, the activity of immobilized Endo BI-1 was lower than that of the free enzyme, at all conditions. For both free and immobilized enzymes, activities decreased at temperatures above 65 \({}^{\circ}\)C.  
To evaluate pH sensitivity, 20 mM Na\({}_{2}\)HPO\({}_{4}\) buffer was adjusted to pH 3, 5, 7, and 9. Reactions with free and immobilized Endo BI-1 and RNase B were carried out at 45 \({}^{\circ}\)C for 20 min, and the normalized activity can be seen in Figure 4b. The optimal pH for free Endo BI-1 was 5, in agreement with previous reports [9], while for the immobilized enzyme a shift to a neutral pH (7.0) was observed. At the optimal pH for the free (pH 5) and immobilized enzyme (pH 7), the immobilized enzyme retained approximately 62% of the activity of the free enzyme at 20 min of reaction. However, the immobilized enzyme was more resilient in the range of pH 7 to 9, where reduced differences in the activities of the free and immobilized enzyme were observed. Indeed, at pH 9, the activities of free and immobilized Endo BI-1 were not statistically different (Figure 4a). Our results are in agreement with several reports in the literature where a shift on the working pH of the immobilized enzyme to a neutral or more alkaline pH was observed, with the same being attributed to changes in the amine group during the covalent binding [31; 34]. In the context of scaling up \(N\)-glycan release using bovine  
Figure 3: Relative activity of free or immobilized Endo BI-1 (on amino, epoxy, and adsorption resins) on the glycoprotein RNase B. Error bars represent one standard deviation. Means followed by different letters (a, b, and c) are statistically different at \(p\) < 0.05.  
milk proteins, the optimum pH of the immobilized enzyme (pH 7), presents a clear advantage over the free enzyme, considering that milk and dairy streams naturally have a pH close to neutral and would not necessitate the use of buffers for pH adjustment, which can become problematic and costly at large scale. Literature suggests that immobilization alters temperature and pH sensitivity by stabilizing the enzyme conformation and creating stable microenvironments around the enzyme or close to its active site [35]. The data here presented provides valuable information for future scale-up of the process and testing other substrates for glycan release.