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

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...

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Autores principales: Pekdemir, Burcu, Duman, Hatice, Arslan, Ayşenur, Kaplan, Merve, Karyelioğlu, Melda, Özer, Tolgahan, Kayılı, Hacı Mehmet, Salih, Bekir, Henrick, Bethany M., Duar, Rebbeca M., Karav, Sercan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9353140/
https://www.ncbi.nlm.nih.gov/pubmed/35935492
http://dx.doi.org/10.3389/fbioe.2022.922423
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author Pekdemir, Burcu
Duman, Hatice
Arslan, Ayşenur
Kaplan, Merve
Karyelioğlu, Melda
Özer, Tolgahan
Kayılı, Hacı Mehmet
Salih, Bekir
Henrick, Bethany M.
Duar, Rebbeca M.
Karav, Sercan
author_facet Pekdemir, Burcu
Duman, Hatice
Arslan, Ayşenur
Kaplan, Merve
Karyelioğlu, Melda
Özer, Tolgahan
Kayılı, Hacı Mehmet
Salih, Bekir
Henrick, Bethany M.
Duar, Rebbeca M.
Karav, Sercan
author_sort Pekdemir, Burcu
collection PubMed
description 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.
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spelling pubmed-93531402022-08-06 Immobilization of a Bifidobacterial Endo-ß-N-Acetylglucosaminidase to Generate Bioactive Compounds for Food Industry Pekdemir, Burcu Duman, Hatice Arslan, Ayşenur Kaplan, Merve Karyelioğlu, Melda Özer, Tolgahan Kayılı, Hacı Mehmet Salih, Bekir Henrick, Bethany M. Duar, Rebbeca M. Karav, Sercan Front Bioeng Biotechnol Bioengineering and Biotechnology 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. Frontiers Media S.A. 2022-07-22 /pmc/articles/PMC9353140/ /pubmed/35935492 http://dx.doi.org/10.3389/fbioe.2022.922423 Text en Copyright © 2022 Pekdemir, Duman, Arslan, Kaplan, Karyelioğlu, Özer, Kayılı, Salih, Henrick, Duar and Karav. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Pekdemir, Burcu
Duman, Hatice
Arslan, Ayşenur
Kaplan, Merve
Karyelioğlu, Melda
Özer, Tolgahan
Kayılı, Hacı Mehmet
Salih, Bekir
Henrick, Bethany M.
Duar, Rebbeca M.
Karav, Sercan
Immobilization of a Bifidobacterial Endo-ß-N-Acetylglucosaminidase to Generate Bioactive Compounds for Food Industry
title Immobilization of a Bifidobacterial Endo-ß-N-Acetylglucosaminidase to Generate Bioactive Compounds for Food Industry
title_full Immobilization of a Bifidobacterial Endo-ß-N-Acetylglucosaminidase to Generate Bioactive Compounds for Food Industry
title_fullStr Immobilization of a Bifidobacterial Endo-ß-N-Acetylglucosaminidase to Generate Bioactive Compounds for Food Industry
title_full_unstemmed Immobilization of a Bifidobacterial Endo-ß-N-Acetylglucosaminidase to Generate Bioactive Compounds for Food Industry
title_short Immobilization of a Bifidobacterial Endo-ß-N-Acetylglucosaminidase to Generate Bioactive Compounds for Food Industry
title_sort immobilization of a bifidobacterial endo-ß-n-acetylglucosaminidase to generate bioactive compounds for food industry
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9353140/
https://www.ncbi.nlm.nih.gov/pubmed/35935492
http://dx.doi.org/10.3389/fbioe.2022.922423
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