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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...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2022
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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. |
format | Online Article Text |
id | pubmed-9353140 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
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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