## 4 Conclusions  
These findings highlight the advantages and limitations of different enzyme immobilization strategies, in particular when considering an enzyme targeting the release of glycans from macromolecular substrates such as glycoproteins. The immobilization process shifted the optimum pH of the enzyme to a neutral pH, which is of relevance when considering the immobilization of this enzyme for industrial applications, such as in dairy products or other food products having a pH close to neutral. The use of moderate temperatures favored the reuse of the immobilized enzyme. Immobilized enzyme reusability may compensate for its initial reduced activity. When using a complex and diverse source of glycoproteins as bovine colostrum, the immobilized enzyme released a higher abundance of neutral \(N\)-glycans than the free enzyme, which yielded a different overall composition of glycans by class. This difference suggests that specific glycan release can be achieved by potentially tailoring immobilized enzyme reactions conditions in future studies.  
Further investigation of the different rates of glycan release, possibly due to limited enzyme accessibility to specific glycan sites on glycoproteins, may enhance our understanding of the role of certain glycosylation sites and structures on their function, in particular for therapeutic glycoproteins such as monoclonal antibodies. Investigation on alternative glycoprotein substrates of plant origin is warranted to understand substrate specificity. This study presents a scalable technique for enzymatic deglycosylation of industrially relevant glycoproteins. Optimized reactor configurations, including flow-through column reactors and fluidized beds, could favor glycan release and subsequent isolation from deglycosylated proteins in downstream isolation, enabling future processing scale-up and commercialization of bioactive glycans and novel therapeutics.  
The following are available online at [[http://www.mdpi.com/2073-4344/8/7/278/s1](http://www.mdpi.com/2073-4344/8/7/278/s1)]([http://www.mdpi.com/2073-4344/8/7/278/s1](http://www.mdpi.com/2073-4344/8/7/278/s1)), Figure S1: Sodium dodecyl sulfate polyacrylamide gel electrophoresis of immobilization washings from amino, adsorption, and epoxy methods on a 4-15% acrylamide gel. Endo BI-1 weighs approximately 53 kDa, Figure S2: Normalized MALDI-ToF MS intensity vs. N glycan: LS. ratio for glycans released from RNase B, Table S1: Mean relative quantities (\(\pm\)S.D.) of \(N\)-glycans released by free and immobilized Endo BI-1 from bovine colostrum whey proteins as determined by nano-LC Chip Q-ToF MS/MS Compound formula is signified by the number of residues of each monosaccharide in the following order: Hex_HexNAc_Fuc_NeuAc_NeuGc:  
**Author Contributions:** [PERSON] and [PERSON] came up with the original idea. [PERSON] and [PERSON] designed the experiments. [PERSON] expressed and isolated the enzyme. [PERSON] performed the experiments and analyzed the data. [PERSON], S.K., [PERSON], and [PERSON] wrote the paper.  
This research was supported by the Center for Advanced Processing and Packaging Studies (CAPPS), the National Institutes of Health awards R01 AT007079 and R01 AT008759, the USDA:NIFA Hatch project 232719, and was partially supported by an industry/campus supported fellowship under the Training Program in Biomolecular Technology (T32-GM008799) at the University of California, Davis. The authors would like to thank [PERSON] for providing the endo- \(\beta\)-\(N\)-acetylglucosamineaminidase enzyme and [PERSON] from Bruker Daltonics for assistance with MALDI-ToF MS applications.  
[PERSON] is a cofounder of Evolve Biosystems, a company focused on diet-based manipulation of the gut microbiota. Evolve Biosystems played no role in the funding, design, execution, interpretation, or publication of this work. The other authors declare no conflict of interest.