Characterizing the release of bioactiveN-glycans from dairy products by a novel endo-β-N-acetylglucosaminidase

Karav, Sercan; Bell, Juliana Maria Leite Nóbrega de Moura; Parc, Annabelle Le; Liu, Yan; Mills, David A.; Block, David E.

doi:10.1002/btpr.2135

Cited by 33 publications

(30 citation statements)

References 32 publications

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“…14,15 Its activity is not affected by a fucosylated N -glycan core and it was also shown to be heat resistant, which enables use during 95°C heat treatments, in contrast to the currently available N -acetylglucosaminidases such as PNGase F. We recently demonstrated that temperature, pH, reaction time, and enzyme/protein ratio significantly affected the yield of released N -glycans by EndoBI-1. 16 In this work, we demonstrate that varying these conditions (pH, temperature, reaction time, and enzyme/protein ratio) affects the structural diversity of released N -glycans from bovine colostrum whey protein concentrate as determined by advanced mass spectrometry. This selective release of N -glycans from bovine colostrum whey glycoproteins allows for further study of their biological activity.…”

Section: Introductionmentioning

confidence: 77%

“…Signal peptide and transmembrane domains were not amplified to facilitate protein expression and purification from E. coli . Gene amplification and protein expression in E. coli BL21* strain were performed as described by Garrido et al 16 …”

Section: Methodsmentioning

confidence: 99%

“…Two discontinuous diafiltrations by volume reduction were performed to increase the removal of residual of simple sugars and free oligosaccharides from the ultrafiltration retentate. In this study, we used three different reaction condition combinations (Table 1), which produced varying yields of released glycans from concentrated bovine colostrum whey by EndoBI-1 in a previous study 16 and the reactions were terminated by 1 M Na 2 CO 3 .…”

Section: Methodsmentioning

confidence: 99%

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A novel endo-β-N-acetylglucosaminidase releases specificN-glycans depending on different reaction conditions

Parc

Karav

Bell

et al. 2015

Biotechnol Progress

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Milk glycoproteins are involved in different functions and contribute to different cellular processes, including adhesion and signaling, and shape the development of the infant micro-biome. Methods have been developed to study the complexities of milk protein glycosylation and understand the role of N-glycans in protein functionality. Endo-β-N-acetylglucosaminidase (EndoBI-1) isolated from Bifidobacterium longum subsp. infantis ATCC 15697 is a recently isolated heat-stable enzyme that cleaves the N-N′-diacetyl chitobiose moiety found in the N-glycan core. The effects of different processing conditions (pH, temperature, reaction time, and enzyme/protein ratio) were evaluated for their ability to change EndoBI-1 activity on bovine colostrum whey glycoproteins using advanced mass spectrometry. This study shows that EndoBI-1 is able to cleave a high diversity of N-glycan structures. Nano-LC-Chip–Q-TOF MS data also revealed that different reaction conditions resulted in different N-glycan compositions released, thus modifying the relative abundance of N-glycan types. In general, more sialylated N-glycans were released at lower temperatures and pH values. These results demonstrated that EndoBI-1 is able to release a wide variety of N-glycans, whose compositions can be selectively manipulated using different processing conditions.

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Section: Introductionmentioning

confidence: 77%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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A novel endo-β-N-acetylglucosaminidase releases specificN-glycans depending on different reaction conditions

Parc

Karav

Bell

et al. 2015

Biotechnol Progress

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“…1. Briefly, EndoBI-1 was incubated with concentrated bovine colostrum whey under conditions previously determined to be optimal for enzyme activity (pH 5, overnight, 37°C) (44). Cleavage was performed under nondenaturing conditions because the goal of this study was to produce the N-glycans in a native state and to preserve biological function.…”

Section: Resultsmentioning

confidence: 99%

“…We recently determined the optimal conditions and kinetic parameters of N-glycan release from concentrated bovine whey for large-scale N-glycan production using EndoBI-1 (13,44,45). In this work, EndoBI-1 treatment of milk glycoproteins demonstrated that freed glycans, as opposed to deglycosylated milk protein, are a good substrate for rapid and selective growth, similar to HMOs.…”

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confidence: 99%

Oligosaccharides Released from Milk Glycoproteins Are Selective Growth Substrates for Infant-Associated Bifidobacteria

Karav

Parc

Bell

et al. 2016

Appl Environ Microbiol

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Milk, in addition to nourishing the neonate, provides a range of complex glycans whose construction ensures a specific enrichment of key members of the gut microbiota in the nursing infant, a consortium known as the milk-oriented microbiome. Milk glycoproteins are thought to function similarly, as specific growth substrates for bifidobacteria common to the breast-fed infant gut. Recently, a cell wall-associated endo-␤-N-acetylglucosaminidase (EndoBI-1) found in various infant-borne bifidobacteria was shown to remove a range of intact N-linked glycans. We hypothesized that these released oligosaccharide structures can serve as a sole source for the selective growth of bifidobacteria. We demonstrated that EndoBI-1 released N-glycans from concentrated bovine colostrum at the pilot scale. EndoBI-1-released N-glycans supported the rapid growth of Bifidobacterium longum subsp. infantis (B. infantis), a species that grows well on human milk oligosaccharides, but did not support growth of Bifidobacterium animalis subsp. lactis (B. lactis), a species which does not. Conversely, B. infantis ATCC 15697 did not grow on the deglycosylated milk protein fraction, clearly demonstrating that the glycan portion of milk glycoproteins provided the key substrate for growth. Mass spectrometry-based profiling revealed that B. infantis consumed 73% of neutral and 92% of sialylated N-glycans, while B. lactis degraded only 11% of neutral and virtually no (<1%) sialylated N-glycans. These results provide mechanistic support that N-linked glycoproteins from milk serve as selective substrates for the enrichment of infant-associated bifidobacteria capable of carrying out the initial deglycosylation. Moreover, released N-glycans were better growth substrates than the intact milk glycoproteins, suggesting that EndoBI-1 cleavage is a key initial step in consumption of glycoproteins. Finally, the variety of N-glycans released from bovine milk glycoproteins suggests that they may serve as novel prebiotic substrates with selective properties similar to those of human milk oligosaccharides. IMPORTANCEIt has been previously shown that glycoproteins serve as growth substrates for bifidobacteria. However, which part of a glycoprotein (glycans or polypeptides) is responsible for this function was not known. In this study, we used a novel enzyme to cleave conjugated N-glycans from milk glycoproteins and tested their consumption by various bifidobacteria. The results showed that the glycans selectively stimulated the growth of B. infantis, which is a key infant gut microbe. The selectivity of consumption of individual N-glycans was determined using advanced mass spectrometry (nano-liquid chromatography chip-quadrupole time of flight mass spectrometry [nano-LC-Chip-Q-TOF MS]) to reveal that B. infantis can consume the range of glycan structures released from whey protein concentrate. P rotein glycosylation is a common modification that adds a major dimension of complexity to protein structure and has been linked to significant roles in protein fun...

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Characterizing the release of bioactiveN-glycans from dairy products by a novel endo-β-N-acetylglucosaminidase

Karav¹,

Bell²,

Parc³

et al. 2015

Biotechnol Progress

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Endo-β-N-acetylglucosaminidase isolated from B. infantis ATCC 15697 (EndoBI-1) is a novel enzyme that cleaves N-N′-diacetyl chitobiose moieties found in the N-glycan core of high mannose, hybrid, and complex N-glycans. These conjugated N-glycans are recently shown as a new prebiotic source that stimulates the growth of a key infant gut microbe, Bifi-dobacterium longum subsp. Infantis. The effects of pH (4.45–8.45), temperature (27.5–77.5°C), reaction time (15–475 min), and enzyme/protein ratio (1:3,000–1:333) were evaluated on the release of N-glycans from bovine colostrum whey by EndoBI-1. A central composite design was used, including a two-level factorial design (24) with four center points and eight axial points. In general, low pH values, longer reaction times, higher enzyme/protein ratio, and temperatures around 52°C resulted in the highest yield. The results demonstrated that bovine colostrum whey, considered to be a by/waste product, can be used as a glycan source with a yield of 20 mg N-glycan/g total protein under optimal conditions for the ranges investigated. Importantly, these processing conditions are suitable to be incorporated into routine dairy processing activities, opening the door for an entirely new class of products (released bioactive glycans and glycan-free milk). The new enzyme’s activity was also compared with a commercially available enzyme, showing that EndoBI-1 is more active on native proteins than PNGase F and can be efficiently used during pasteurization, streamlining its integration into existing processing strategies.

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Characterizing the release of bioactiveN-glycans from dairy products by a novel endo-β-N-acetylglucosaminidase

Cited by 33 publications

References 32 publications

A novel endo-β-N-acetylglucosaminidase releases specificN-glycans depending on different reaction conditions

A novel endo-β-N-acetylglucosaminidase releases specificN-glycans depending on different reaction conditions

Oligosaccharides Released from Milk Glycoproteins Are Selective Growth Substrates for Infant-Associated Bifidobacteria

Characterizing the release of bioactiveN-glycans from dairy products by a novel endo-β-N-acetylglucosaminidase

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