The role of cations in regulating reaction pathways driven by Bacillus circulans β-galactosidase

Alavijeh, Masih Karimi; Meyer, Anne S.; Gras, Sally L.; Kentish, Sandra E.

doi:10.1016/j.cej.2020.125067

Cited by 7 publications

(7 citation statements)

References 39 publications

(49 reference statements)

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“…As previously shown, high concentrations of divalent cations, i.e., 100 mM Ca 2+ or 100 mM Mg 2+ can lead to the formation of salt bridges between the negatively charged β-galactosidases and subsequent protein aggregation. Thus, in the present study, we also examined the effect of these cations on the product formation when the immobilized enzyme is used.…”

Section: Results and Discussionmentioning

confidence: 81%

“…Changes in the transgalactosylation yield and reaction productivity upon immobilization of β-galactosidases have been reported by several researchers. 17,66−68 As previously shown, 69 high concentrations of divalent cations, i.e., 100 mM Ca 2+ or 100 mM Mg 2+ can lead to the formation of salt bridges between the negatively charged βgalactosidases and subsequent protein aggregation. Thus, in the present study, we also examined the effect of these cations on the product formation when the immobilized enzyme is used.…”

Section: ■ Introductionmentioning

confidence: 95%

“…In our previous work, 69 we developed a kinetic model to describe the multipathway reactions including transgalactosylation and hydrolysis involved in the conversion of lactose to LacNAc. Based on this model (see the Supporting Information), the reaction rate constants were estimated for the free enzyme and the immobilized enzyme (Table S1).…”

Section: Transgalactosylation Reactionmentioning

confidence: 99%

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Improving β-Galactosidase-Catalyzed Transglycosylation Yields by Cross-Linked Layer-by-Layer Enzyme Immobilization

Alavijeh

Meyer

Gras

et al. 2020

ACS Sustainable Chem. Eng.

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The biotransformation of lactose into gut-bioactive glycans catalyzed by βgalactosidase can give economic value to lactose-rich side streams generated in the food or dairy industry. Herein, we study the immobilization of the commercially used β-galactosidase from Bacillus circulans onto silica particles using an enzyme immobilization technology involving a cross-linked layer-by-layer encapsulation method. The immobilized β-galactosidase was used for the synthesis of Nacetyllactosamine (LacNAc) as an important precursor for numerous bioactive compounds and a prebiotic in itself. Techniques including molecular analysis, enzyme activity determination, secondary structure analysis, thermodynamic characterization as well as the determination of thermal and operational stability were conducted to characterize the immobilized enzyme. Changes in the activity of the enzyme after immobilization were attributed to possible changes in electrostatic, covalent and protein-protein interactions. Immobilization significantly improved enzymatic LacNAc yield compared to the free enzyme. In turn, this improved the economics and the sustainability of the process. The immobilized enzyme encapsulated in multilayer films was significantly more stable in the presence of divalent cations and its thermostability also substantially increased, with the thermal denaturation activation energy increasing from 53 kJ mol -1 to 294 kJ mol -1 . The immobilized enzyme was successfully reused in eight consecutive reaction cycles with no significant reduction in the LacNAc yield. The improved transgalactosylation yield and productivity, higher stability and reusability obtained with this immobilization method provide new opportunities for industrial applications.

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Section: Results and Discussionmentioning

confidence: 81%

Section: ■ Introductionmentioning

confidence: 95%

Section: Transgalactosylation Reactionmentioning

confidence: 99%

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Improving β-Galactosidase-Catalyzed Transglycosylation Yields by Cross-Linked Layer-by-Layer Enzyme Immobilization

Alavijeh

Meyer

Gras

et al. 2020

ACS Sustainable Chem. Eng.

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“…These changes were attributed to the formation of enzyme aggregates, which further influenced the hydrolysis and transgalactosylation pathways in the conversion of lactose to LacNAc using this β-galactosidase. 150 Water Activity. When water activity decreases, the hydrolysis of substrates or secondary hydrolysis of products is less favored, thereby increasing the selectivity of transgalactosylation.…”

Section: ■ Biochemical Synthesismentioning

confidence: 99%

“…When divalent cations Mg 2+ and Ca 2+ were added at concentrations of 100 mM, however, there was a significant reduction in the β-galactosidase activity, with a concurrent change in product selectivity and yield. These changes were attributed to the formation of enzyme aggregates, which further influenced the hydrolysis and transgalactosylation pathways in the conversion of lactose to LacNAc using this β-galactosidase …”

Section: Biochemical Synthesismentioning

confidence: 99%

Synthesis of N-Acetyllactosamine and N-Acetyllactosamine-Based Bioactives

Alavijeh

Meyer

Gras

et al. 2021

J. Agric. Food Chem.

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N-Acetyllactosamine (LacNAc), or more specifically β-D-galactopyranosyl-1,4-N-acetyl-Dglucosamine, is a unique acyl-amino sugar and a key structural unit in human milk oligosaccharides, an antigen component of many glycoproteins and an antiviral active component for the development of effective drugs against viruses. LacNAc is useful itself and as a basic building block for producing various bioactive oligosaccharides, notably because this synthesis may be used to add value to dairy lactose. Despite a significant amount of information in the literature on the benefits, structures and types of different LacNAc-derived oligosaccharides, knowledge about their effective synthesis for large scale production is still in its infancy. This work provides a comprehensive analysis of existing production strategies of LacNAc and important LacNAc-based structures, including sialylated LacNAc, as well as poly-and oligo-LacNAc. We conclude that direct extraction from milk is too complex, while chemical synthesis is also impractical at an industrial scale. Microbial routes have application when multiple step reactions are needed but the major route to large scale biochemical production will likely lie with enzymatic routes, particularly those using β-galactosidases (for LacNAc synthesis), sialidases (for sialylated LacNAc synthesis) and β-N-acetylhexosaminidases (for oligo-LacNAc synthesis).Glycosyltransferases, especially for the biosynthesis of extended complex LacNAc structures, could also play a major role in the future. In these cases, immobilization of the enzyme can increase stability and reduce cost. Processing parameters, such as substrate concentration and purity, acceptor to donor ratio, water activity and temperature can affect product selectivity and yield.More work is needed to optimize these reaction parameters and in the development of robust, thermally stable enzymes to facilitate commercial production of these important bioactive substances. Abbreviations: 39 β3GalT β1-3 galactosyltransferase β4GalT β1-4 galactosyltransferase CMP-NeuNAc cytidine-5'-monophosphate-5-N-acetylneuraminic acid DS'LNT α2-6-linked disialyllacto-N-tetraose DSLNnT α2-6-linked disialyllacto-N-neotetraose EPEC enteropathogenic Escherichia coli GalNAc N-acetylgalactosamine GfCoV Guinea fowl coronavirus GlcNAc N-acetylglucosamine GLP-1 glucagon-like peptide 1 HEp-2 human epithelial type 2 cells iGnT β1-3 N-acetylglucosaminyltransferase IGnT β1-6 N-acetylglucosaminyltransferase iLNO iso-Lacto-N-octaose LacNAc N-acetyllactosamine LacNAc-ITag imidazolium-tagged LacNAc LacY β-galactoside permease lacZ β-galactosidase gene lgtA β1-3 N-acetylglucosaminyltransferase gene lgtB β1-4 galactosyltransferase gene LND Lacto-N-decaose LNH Lacto-N-hexaose LNnD Lacto-N-neo-decaose LNnH Lacto-N-neo-hexaose LNnO Lacto-N-neo-octaose LNnT Lacto-N-neotetraose LNO Lacto-N-octaose LNT Lacto-N-tetraose ManNAc N-acetylmannosamine MERS-CoV Middle East respiratory syndrome coronavirus Neu5Ac N-acetylneuraminic acid oNPG ortho-nitrophenyl-β-galactoside pLNH para-Lacto-N...

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Biochemical characterization of a novel β-galactosidase from Pedobacter sp. with strong transglycosylation activity at low lactose concentration

Miao,

Yao,

Yan

et al. 2024

Folia Microbiol

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The role of cations in regulating reaction pathways driven by Bacillus circulans β-galactosidase

Cited by 7 publications

References 39 publications

Improving β-Galactosidase-Catalyzed Transglycosylation Yields by Cross-Linked Layer-by-Layer Enzyme Immobilization

Improving β-Galactosidase-Catalyzed Transglycosylation Yields by Cross-Linked Layer-by-Layer Enzyme Immobilization

Synthesis of N-Acetyllactosamine and N-Acetyllactosamine-Based Bioactives

Biochemical characterization of a novel β-galactosidase from Pedobacter sp. with strong transglycosylation activity at low lactose concentration

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