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Development of Biocatalytic Approach Using Fungal Laccase to Produce Novel Functional Ingredients via Protein Crosslinking and Protein/Polysaccharide Conjugation.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Development of Biocatalytic Approach Using Fungal Laccase to Produce Novel Functional Ingredients via Protein Crosslinking and Protein/Polysaccharide Conjugation./
Author:	Li, Mingqin.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2020,
Description:	221 p.
Notes:	Source: Dissertations Abstracts International, Volume: 82-10, Section: B.
Contained By:	Dissertations Abstracts International82-10B.
Subject:	Chemistry. -
Online resource:	https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=28383659
ISBN:	9798708707154

Development of Biocatalytic Approach Using Fungal Laccase to Produce Novel Functional Ingredients via Protein Crosslinking and Protein/Polysaccharide Conjugation.
Li, Mingqin.

Development of Biocatalytic Approach Using Fungal Laccase to Produce Novel Functional Ingredients via Protein Crosslinking and Protein/Polysaccharide Conjugation. - Ann Arbor : ProQuest Dissertations & Theses, 2020 - 221 p.

Source: Dissertations Abstracts International, Volume: 82-10, Section: B.

Thesis (Ph.D.)--McGill University (Canada), 2020.

This item must not be sold to any third party vendors.

Laccase (E.C 1.3.10.2) is a versatile biocatalyst, which can be used for protein modification. Kinetics of biocatalytic conversions of protein-related substrate models by laccases were studied in order to contribute to the understanding of mechanistic actions of laccase in protein cross-linking. Fungal laccases exhibited higher substrate specificity towards the peptide models (Km = 0.17-0.67 mM, kcat/Km = 0.56-0.81 s-1mM-1) ST-10 (SYMTDYYLST), derived from potato patatin, and AG-10 (AKKIVSDGNG), derived from egg white lysozyme (LZM), than tyrosine (Y) (Km = 1.10-1.12 mM, kcat/Km = 0.01-0.31 s-1mM-1). This was confirmed by the molecular docking study, in which substrate/enzyme binding modes with better affinity were observed for laccase/peptides than for laccase/tyrosine. Laccase-catalyzed reaction resulted in the cross-linking of tyrosine-containing peptide ST-10 via the formation of di- and oligotyrosine linkages; while the cross-linking of peptide AG-10, which does not contain tyrosine residues, was achieved only in the presence of ferulic acid (FA) as reaction mediator.Grafting of FA reactive sites on proteins via amidation followed by Trametes versicolor laccase -catalyzed cross-linking was performed on compact globular proteins, LZM and ovalbumin (OVA). The substrate reactivity and cross-linking efficiency of FA-modified LZM and OVA was improved as compared to their native forms. Polymerization was achieved by FA-modified LZM, whereas oligomers were the main cross-linked products of FA-OVA. The grafted FA-associated cross-links of the products were characterized using mass spectrometry, which were predominated by the form of 8-5' dehydrodiferulic acid. FA grafting/laccase-cross-linking treatment enhanced the emulsification performance of LZM and the foaming capacity of OVA. Furthermore, the cross-linked proteins (particularly LZM) exhibited lower immunoglobulin E binding capacity (up to 50% reduction) than the native forms, indicating current cross-linking treatment is able to decrease their allergenicity.In light of our findings on the cross-linking of ST-10 peptides derived from potato patatin, a study on the cross-linking of potato proteins was carried out using laccases from T. versicolor and Coriolus hirsutus with or without the presence of FA. Higher catalytic efficiency of laccases was shown towards potato protein fraction (PAT) containing patatin, whereas smaller-sized potato protease inhibitor fraction (PIs) were faster in forming oxidative cross-linked products. Reaction with Coriolus hirsutus laccase or with FA resulted in higher cross-linking extent. The proportion of high molecular weight cross-linked products increase as function of reaction time up to 24h, while the extended treatments (48h) led to oxidative fragmentation.The conjugation of PAT with selected pectic polysaccharides via laccase-catalyzed reaction was further investigated and modulated using response surface methodology (RSM) to achieve product with defined conjugation extent and emulsification performance. The highest heteroconjugation efficiency was shown by C. hirsutus laccase-PAT/sugar beet pectin (SBP) reaction system. The developed predictive models revealed that PAT ratio in PAT/SBP mixture was correlated negatively with the conjugation extent but positively with the emulsification performance, and these correlations were affected by the interaction between PAT ratio and enzyme concentration or reaction time.The effect of laccase-catalyzed modification on functionality profile of potato proteins was evaluated in correlation with the structural and chemical properties. The oxidative cross-linking improved the emulsifying property of both PAT and PIs, while short time treatment and product profile with enriched 60-80 kDa fraction was associated with enhanced foaming property in PAT. The PATs conjugated with SBP had relatively low surface tension as compared to the native and cross-linked PATs at neutral pH; however, the formed interface were characterized by low elasticity. The highest surface dilatational elasticity of the protein samples at neutral pH was observed with cross-linked PAT obtained from 24h reaction without FA. Extended treatments for 48h led to both cross-linking and fragmentation of PAT; the resulted products showed a higher structural stability and contributed to the low susceptibility of the interfacial behaviour of PAT to acidic pH and to relatively high surface dilatational elasticity.

ISBN: 9798708707154Subjects--Topical Terms:

516420
Chemistry.
Subjects--Index Terms:

Biocatalytic

Development of Biocatalytic Approach Using Fungal Laccase to Produce Novel Functional Ingredients via Protein Crosslinking and Protein/Polysaccharide Conjugation.
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245 1 0 $a Development of Biocatalytic Approach Using Fungal Laccase to Produce Novel Functional Ingredients via Protein Crosslinking and Protein/Polysaccharide Conjugation.
260 1 $a Ann Arbor : $b ProQuest Dissertations & Theses, $c 2020
300 $a 221 p.
500 $a Source: Dissertations Abstracts International, Volume: 82-10, Section: B.
500 $a Advisor: Kermasha, Selim.
502 $a Thesis (Ph.D.)--McGill University (Canada), 2020.
506 $a This item must not be sold to any third party vendors.
520 $a Laccase (E.C 1.3.10.2) is a versatile biocatalyst, which can be used for protein modification. Kinetics of biocatalytic conversions of protein-related substrate models by laccases were studied in order to contribute to the understanding of mechanistic actions of laccase in protein cross-linking. Fungal laccases exhibited higher substrate specificity towards the peptide models (Km = 0.17-0.67 mM, kcat/Km = 0.56-0.81 s-1mM-1) ST-10 (SYMTDYYLST), derived from potato patatin, and AG-10 (AKKIVSDGNG), derived from egg white lysozyme (LZM), than tyrosine (Y) (Km = 1.10-1.12 mM, kcat/Km = 0.01-0.31 s-1mM-1). This was confirmed by the molecular docking study, in which substrate/enzyme binding modes with better affinity were observed for laccase/peptides than for laccase/tyrosine. Laccase-catalyzed reaction resulted in the cross-linking of tyrosine-containing peptide ST-10 via the formation of di- and oligotyrosine linkages; while the cross-linking of peptide AG-10, which does not contain tyrosine residues, was achieved only in the presence of ferulic acid (FA) as reaction mediator.Grafting of FA reactive sites on proteins via amidation followed by Trametes versicolor laccase -catalyzed cross-linking was performed on compact globular proteins, LZM and ovalbumin (OVA). The substrate reactivity and cross-linking efficiency of FA-modified LZM and OVA was improved as compared to their native forms. Polymerization was achieved by FA-modified LZM, whereas oligomers were the main cross-linked products of FA-OVA. The grafted FA-associated cross-links of the products were characterized using mass spectrometry, which were predominated by the form of 8-5' dehydrodiferulic acid. FA grafting/laccase-cross-linking treatment enhanced the emulsification performance of LZM and the foaming capacity of OVA. Furthermore, the cross-linked proteins (particularly LZM) exhibited lower immunoglobulin E binding capacity (up to 50% reduction) than the native forms, indicating current cross-linking treatment is able to decrease their allergenicity.In light of our findings on the cross-linking of ST-10 peptides derived from potato patatin, a study on the cross-linking of potato proteins was carried out using laccases from T. versicolor and Coriolus hirsutus with or without the presence of FA. Higher catalytic efficiency of laccases was shown towards potato protein fraction (PAT) containing patatin, whereas smaller-sized potato protease inhibitor fraction (PIs) were faster in forming oxidative cross-linked products. Reaction with Coriolus hirsutus laccase or with FA resulted in higher cross-linking extent. The proportion of high molecular weight cross-linked products increase as function of reaction time up to 24h, while the extended treatments (48h) led to oxidative fragmentation.The conjugation of PAT with selected pectic polysaccharides via laccase-catalyzed reaction was further investigated and modulated using response surface methodology (RSM) to achieve product with defined conjugation extent and emulsification performance. The highest heteroconjugation efficiency was shown by C. hirsutus laccase-PAT/sugar beet pectin (SBP) reaction system. The developed predictive models revealed that PAT ratio in PAT/SBP mixture was correlated negatively with the conjugation extent but positively with the emulsification performance, and these correlations were affected by the interaction between PAT ratio and enzyme concentration or reaction time.The effect of laccase-catalyzed modification on functionality profile of potato proteins was evaluated in correlation with the structural and chemical properties. The oxidative cross-linking improved the emulsifying property of both PAT and PIs, while short time treatment and product profile with enriched 60-80 kDa fraction was associated with enhanced foaming property in PAT. The PATs conjugated with SBP had relatively low surface tension as compared to the native and cross-linked PATs at neutral pH; however, the formed interface were characterized by low elasticity. The highest surface dilatational elasticity of the protein samples at neutral pH was observed with cross-linked PAT obtained from 24h reaction without FA. Extended treatments for 48h led to both cross-linking and fragmentation of PAT; the resulted products showed a higher structural stability and contributed to the low susceptibility of the interfacial behaviour of PAT to acidic pH and to relatively high surface dilatational elasticity.
520 $a La laccase (E.C 1.3.10.2) est un biocatalyseur polyvalent, qui peut etre utilise pour la modification des proteines. Les cinetiques des bioconversions des substrats-modeles derives des proteines par les laccases ont ete etudiees, afin de contribuer a la comprehension du mecanisme d'action de la laccase pendant la reticulation des proteines. Les laccases fongiques ont ete caracterisees par une specificite plus elevee vis-a-vis des substrats modeles peptidiques (Km = 0,17-0,67mM, kcat/Km = 0,56-0,81s-1mM-1) ST-10 (SYMTDYYLST), derive de la patatine de pomme de terre, et AG-10 (AKKIVSDGNG), derive du lysozyme de blanc d'oeuf (LZM), en comparaison avec la tyrosine (Y) (Km = 1,10-1,12 mM, kcat/Km = 0,01-0,31 s-1mM-1). Ces resultats ont ete confirmes par l'etude moleculaire « Docking », dans laquelle des modes de liaison substrat/enzyme ayant une meilleure affinite ont ete observes pour laccase/peptides que pour laccase/tyrosine. La reaction catalysee par la laccase a conduit a une reticulation du peptide ST-10 contenant de la tyrosine via la formation des liaisons de di- et oligo-tyrosine ; tandis que la reticulation du peptide AG-10, qui ne contient pas de residu tyrosine, n'a pas pu etre accomplis qu'en presence d'acide ferulique (AF) comme mediateur de reaction.Le greffage des sites reactifs du AF sur les proteines via amidation suivi d'une reticulation catalysee par la laccase de Trametes versicolor ont ete realises sur des proteines globulaires compactes, LZM et ovalbumine (OVA). La reactivite du substrat et l'efficacite de la reticulation des LZM et OVA modifiees par AF etaient plus elevees en comparaison avec leurs formes natives. LZM modifiee par AF a ete polymerisee, tandis que des oligomeres etaient les principaux produits reticules de AF-OVA. Les reticulations formees au niveau des greffes AF ont ete caracterisees par la spectrometrie de masse, par laquelle la forme d'acide 8-5' dehydrodiferulique etait identifiee comme etant la plus predominante. Le traitement de greffage avec AF suivi de la reticulation avec laccase a ameliore la performance emulsifiante du LZM et la capacite moussante de l'OVA. De plus, les proteines reticulees presentaient une capacite de liaison aux immunoglobulines E plus faible (jusqu'a 50% de reduction pour LZM) que les formes natives, ce qui indique que le traitement de reticulation des proteines modifies est capable de diminuer l'allergenicite.A la lumiere de nos resultats sur la reticulation des peptides ST-10 derives de la patatine de pomme de terre, une etude sur la reticulation des proteines de pomme de terre a ete realisee en utilisant des laccases de T. versicolor et Coriolus hirsutus avec ou sans presence d'AF. Les laccases ont montre une efficacite catalytique plus elevee vis-a-vis de la fraction de proteine de pomme de terre (PAT) contenant de la patatine, alors que l'utilisation de la fraction contenant des inhibiteurs de protease de pomme de terre (IP) a conduit a une formation rapide des produits de la reticulation oxydative. Le degree de reticulation a ete affectee par le type de laccase et la presence de AF. L'augmentation du temps de reaction jusqu'a 24h a entraine le deplacement des produits reticules vers un poids moleculaire plus eleve, tandis qu'un traitement prolonge (48h) a entraine une precipitation ou une fragmentation oxydative.La conjugaison de PAT avec des polysaccharides pectiques selectionnes a travers une reaction catalysee par la laccase a ete etudiee et modulee en utilisant une methodologie de surface de reponse (RSM) pour obtenir un produit avec un degree de conjugaison et de performance emulsifiante bien definie. L'hetero-conjugaison la plus elevee a ete obtenue lors de l'utilisation du systeme reactionnel compose de C. hirsutus laccase-PAT/pectine de betterave sucriere (SBP). Les modeles predictifs developpes ont revele que la proportion de PAT dans le melange PAT/SBP etait correlee negativement avec l'etendue de la conjugaison mais positivement avec la performance emulsifiante, et ces correlations etaient affectees par l'interaction entre la proportion de PAT et la concentration enzymatique ou le temps de reaction.L'effet de la modification catalysee par la laccase sur le profil de fonctionnalite des proteines de pomme de terre a ete evalue en correlant les proprietes interfaciales avec l'etendue de la reticulation/conjugaison, le profil du produit, le deploiement des proteines, la composition de la structure secondaire et l'incorporation d'acide ferulique. La reticulation oxydative a ameliore la propriete emulsifiante du PAT et d'IP. Un traitement de courte duree et l'enrichissement avec des produits ayant 60 - 80 kDa ont ete associes avec l'amelioration de la propriete moussante de PAT. A pH neutre, PAT conjugue avec SBP a montre une tension superficielle relativement faible par rapport aux autres PAT natif et reticules. Cependant, les interfaces resultantes ont ete caracterises par une faible elasticite. L'elasticite de dilatation de surface proteique la plus elevee a pH neutre a ete observee pour le PAT reticule obtenu a partir d'une reaction de 24h sans AF. Les traitements prolonges pendant 48 h ont entraine a la fois la reticulation et la fragmentation du PAT. Les produits de PAT resultants de ces modifications ont demontre une stabilite structurelle plus elevee, et ont contribue a reduire la sensibilite des proprietes interraciales du PAT au pH acide et a l'elasticite relativement elevee du dilatation de surface.
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