Immobilization of a Bifidobacterial Endo-ß-N-Acetylglucosaminidase to Generate Bioactive Compounds for Food Industry

Abstract

Conjugated N-glycans are considered next-generation bioactive prebiotic compounds due to their selective stimulation of beneficial microbes. These compounds are glycosidically attached to proteins through N-acetylglucosamines via specific asparagine residue (AsN-X-Ser/Thr). Certain bacteria such as Bifidobacterium longum subspecies infantis (B. infantis) have been shown to be capable of utilizing conjugated N-glycans, owing to their specialized genomic abilities. B. infantis possess a unique enzyme, Endo-ß-N-acetylglucosaminidase (EndoBI-1), which cleaves all types of conjugated N-glycans from glycoproteins. In this study, recombinantly cloned EndoBI-1 enzyme activity was investigated using various immobilization methods: 1) adsorption, 2) entrapment-based alginate immobilization, 3) SulfoLink-, and 4) AminoLink-based covalent bonding immobilization techniques were compared to develop the optimum application of EndoBI-1 to food processes. The yield of enzyme immobilization and the activity of each immobilized enzyme by different approaches were investigated. The N-glycans released from lactoperoxidase (LPO) using different immobilized enzyme forms were characterized using MALDI-TOF mass spectrometry (MS). As expected, regardless of the techniques, the enzyme activity decreased after the immobilization methods. The enzyme activity of adsorption and entrapment-based alginate immobilization was found to be 71.55% ± 0.6 and 20.32% ± 3.18, respectively, whereas the activity of AminoLink- and SulfoLink-based covalent bonding immobilization was found to be 58.05 ± 1.98 and 47.49% ± 0.30 compared to the free form of the enzyme, respectively. However, extended incubation time recovery achieved activity similar to that of the free form. More importantly, each immobilization method resulted in the same glycan profile containing 11 different N-glycan structures from a model glycoprotein LPO based on MALDI-TOF MS analysis. The glycan data analysis suggests that immobilization of EndoBI-1 is not affecting the enzyme specificity, which enables full glycan release without a limitation. Hence, different immobilization methods investigated in this study can be chosen for effective enzyme immobilization to obtain bioactive glycans. These findings highlight that further optimization of these methods can be a promising approach for future processing scale-up and commercialization of EndoBI-1 and similar enzymes.

Enhancing Enzymatic Activity Through Co-Immobilization of Protease and Lipase Enzymes on Magnetic Nano-Iron Oxides Coated with Gum Arabic

“…Co-immobilization of multiple enzymes on magnetic nanoparticles has gained great interest in recent years due to its potential applications in various fields such as biocatalysis, bioprocessing, and food industry .Several studies have highlighted the advantages of using magnetic nanoparticles for enzyme immobilization, including enhanced stability, reusability, and ease of separation from reaction mixtures [22,31,32].The use of magnetic nanoparticles and porous materials to immobilize enzyme movement has been widely explored, demonstrating their effectiveness in enhancing enzyme stability and activity [18,19,21]. Furthermore, covalent immobilization of enzymes onto magnetic nanoparticles has been shown to improve the catalytic efficiency and operational stability of the immobilized enzymes [23,24,28,40]. Various techniques, such as adsorption, entrapment, covalent bonding, and cross-linking, have been used to immobilize enzymes on magnetic nanoparticles, each offering unique advantages in terms of IOP Publishing doi:10.1088/1755-1315/1371/6/062046 2 enzyme activity and stability [20,23,29].In addition, the use of immobilization methods based on coated magnetic nanoparticles has shown promising results in achieving high enzyme activity and stability [21].…”