Fucosylated and non-fucosylated <i>N</i>-glycans released from hLF, rhLF and bLF by PNGase F.

Tukey’s test was used to indicate significant differences (p<0.05) between groups. Same letters indicate no significant difference.</p

<i>N-</i>glycan compositions of bLF, rhLF and hLF.

N-glycan compositions of bLF, rhLF and hLF.</p

Comparison of bLF, rhLF and hLF <i>N-</i>glycan compositions.

<p>Comparison of bLF, rhLF and hLF <i>N-</i>glycan compositions.</p

PCA plot of composition of released <i>N-</i>glycans of human lactoferrin (hLF), recombinant human lactoferrin (rhLfa) and bovine lactoferrin (bLf).

<p>Of the variance, 62.45% was explained by the first principal component and 36.97% was explained by the second principal component.</p

Extracted compound chromatograms (ECCs) of N-glycans from recombinant human lactoferrin.

Extracted compound chromatograms (ECCs) of N-glycans from recombinant human lactoferrin.</p

Recombinant human lactoferrin purification.

<p>(A) Skim milk protein profile analyzed by 12% SDS-PAGE. (B) Elution of rhLF purification by heparin Sepharose. (C) Elution of rhLF purification by ion-exchange chromatography.</p

Deconvoluted tandem spectrum of the neutral N-glycan 5Hex2HexNAc from recombinant human lactoferrin.

<p>This glycan corresponds to 1235.44 m/z. Green circles and blue squares represent mannose and HexNAc residues, respectively.</p

Heatmap of compound abundances associated with lactoferrin from different sources.

<p>Compound relative abundances were standardized (Z score, shown in legend) prior to unsupervised hierarchical clustering of samples (rows). Compound identity is noted below each column.</p

High mannose, sialylated and neutral complex/hybrid <i>N</i>-glycans released from hLF, rhLF and bLF by PNGase F.

<p>Tukey’s test was used to indicate significant differences (p<0.05) between groups. Same letters indicate no significant difference.</p

DataSheet1_Immobilization of a Bifidobacterial Endo-ß-N-Acetylglucosaminidase to Generate Bioactive Compounds for Food Industry.PDF

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.</p