Advanced glycosidases as ingenious biosynthetic instruments

Biotechnology Advances, Journal Year: 2021, Volume and Issue: 49, P. 107733 - 107733

Published: March 27, 2021

Language: Английский

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Glycosynthase reaction meets the flow: Continuous synthesis of lacto‐N‐triose II by engineered β‐hexosaminidase immobilized on solid support

Biotechnology and Bioengineering, Journal Year: 2020, Volume and Issue: 117(5), P. 1597 - 1602

Published: Feb. 4, 2020

Abstract The D746E variant of Bifidobacterium bifidum β‐ N ‐acetyl‐hexosaminidase is a promising glycosynthase (engineered glycosidase deficient in hydrolase activity) for the synthesis lacto‐ ‐triose II (LNT II), core structural unit human milk oligosaccharides. Here, we develop flow process reaction, which regioselective β‐1,3‐glycosylation lactose from d ‐glucosamine 1,2‐oxazoline donor. Using immobilized on agarose beads (∼30 mg/g) packed into fixed bed (1 ml), show stable continuous production LNT (145–200 mM) at quantitative yield donor substrate. wild‐type used under exactly comparable conditions gives primarily (∼85%) hydrolysis product ‐glucosamine. By enabling short residence times (2 min) that are challenging mixed‐vessel types reactor to establish, succeeds an effective uncoupling formation (∼80–100 mM/min) slower side reactions (decomposition substrate, enzymatic II) obtain optimum synthetic efficiency. Our study thus provides strong case application chemistry principles and by that, it reveals important synergy between enzyme reaction engineering biocatalytic

Language: Английский

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β-N-Acetylhexosaminidases for Carbohydrate Synthesis via Trans-Glycosylation

Catalysts, Journal Year: 2020, Volume and Issue: 10(4), P. 365 - 365

Published: March 29, 2020

β-N-acetylhexosaminidases (EC 3.2.1.52) are retaining hydrolases of glycoside hydrolase family 20 (GH20). These enzymes catalyze hydrolysis terminal, non-reducing N-acetylhexosamine residues, notably N-acetylglucosamine or N-acetylgalactosamine, in N-acetyl-β-D-hexosaminides. In nature, bacterial mainly involved cell wall peptidoglycan synthesis, analogously, fungal act on chitin. The work via a distinct substrate-assisted mechanism that utilizes the 2-acetamido group as nucleophile. Curiously, possess an inherent trans-glycosylation ability which is potentially useful for biocatalytic synthesis functional carbohydrates, including biomimetic human milk oligosaccharides and other glycan-functionalized compounds. this review, we summarize reaction engineering approaches (donor substrate activation, additives, conditions) have proven enhancing activity GH20 β-N-acetylhexosaminidases. We provide comprehensive overviews reported reactions with enzymes, tables list specific enzyme used, donor acceptor substrates, conditions, details products yields obtained. also describe active site traits mutations appear to favor Finally, discuss novel protein strategies suggest potential “hotspots” promote efficient carbohydrates glyco-engineered products.

Language: Английский

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High-Affinity N-(2-Hydroxypropyl)methacrylamide Copolymers with Tailored N-Acetyllactosamine Presentation Discriminate between Galectins

Biomacromolecules, Journal Year: 2020, Volume and Issue: 21(2), P. 641 - 652

Published: Jan. 6, 2020

N-Acetyllactosamine (LacNAc; Galβ4GlcNAc) is a typical disaccharide ligand of galectins. The most abundant members these human lectins, galectin-1 (Gal-1) and galectin-3 (Gal-3), participate in number pathologies including cancerogenesis metastatic formation. In this study, we synthesized series fifteen N-(2-hydroxypropyl)methacrylamide (HPMA)-based glycopolymers with varying LacNAc amounts presentations evaluated the impact their architecture on binding affinity to Gal-1 Gal-3. controlled radical reversible addition-fragmentation chain transfer copolymerization technique afforded linear polymer precursors comparable molecular weight (Mn ≈ 22,000 g mol-1) narrow dispersity (D̵ 1.1). were conjugated functionalized (4-22 mol % content glycopolymer) prepared by enzymatic synthesis under catalysis β-galactosidase from Bacillus circulans. structure-affinity relationship study based enzyme-linked immunosorbent assay revealed that type presentation, individual or clustered bi- trivalent linkers, brings clear discrimination (almost 300-fold) between Gal-3, reaching avidity nanomolar range. Whereas strongly preferred dense presentation individually distributed epitopes, Gal-3 presentation. Such strong galectin preference just structure multivalent glycopolymer exceptional. nontoxic, nonimmunogenic, biocompatible are prospective for therapeutic applications requiring selectivity one particular galectin.

Language: Английский

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Immunoprotective neo-glycoproteins: Chemoenzymatic synthesis of multivalent glycomimetics for inhibition of cancer-related galectin-3

European Journal of Medicinal Chemistry, Journal Year: 2021, Volume and Issue: 220, P. 113500 - 113500

Published: April 27, 2021

Language: Английский

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Structural and biochemical analyses of β-N-acetylhexosaminidase Am0868 from Akkermansia muciniphila involved in mucin degradation

Biochemical and Biophysical Research Communications, Journal Year: 2020, Volume and Issue: 529(4), P. 876 - 881

Published: July 28, 2020

Language: Английский

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Engineered Glycosidases for the Synthesis of Analogs of Human Milk Oligosaccharides

International Journal of Molecular Sciences, Journal Year: 2022, Volume and Issue: 23(8), P. 4106 - 4106

Published: April 7, 2022

Enzymatic synthesis is an elegant biocompatible approach to complex compounds such as human milk oligosaccharides (HMOs). These are vital for healthy neonatal development with a positive impact on the immune system. Although HMOs may be prepared by glycosyltransferases, this pathway often complicated high price of sugar nucleotides, stringent substrate specificity, and low enzyme stability. Engineered glycosidases (EC 3.2.1) represent good synthetic alternative, especially if variations in structure desired. Site-directed mutagenesis can improve process higher yields and/or increased reaction selectivity. So far, has mostly been limited analytical reactions mass spectrometry detection. The present work reveals potential library engineered preparative three tetrasaccharides derived from lacto-N-tetraose (Galβ4GlcNAcβ3Galβ4Glc), employing sequential cascade catalyzed β3-N-acetylhexosaminidase BbhI Bifidobacterium bifidum, β4-galactosidase BgaD-B Bacillus circulans, β4-N-acetylgalactosaminidase Talaromyces flavus, β3-galactosynthase BgaC B. circulans. products were isolated structurally characterized. This expands insight into multi-step catalysis shows path modified derivatives carbohydrates that cannot standard glycosyltransferase methods.

Language: Английский

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A fungal pathogen secretes a cell wall‐associated β‐N‐acetylhexosaminidase that is co‐expressed with chitinases to contribute to infection of insects

Pest Management Science, Journal Year: 2024, Volume and Issue: 80(9), P. 4699 - 4713

Published: May 21, 2024

Abstract BACKGROUND β‐ N ‐acetylhexosaminidases (HEXs) are widely distributed in fungi and involved cell wall chitin metabolism utilization of chitin‐containing substrates. However, details the fungal pathogens‐derived HEXs interaction with their hosts remain limited. RESULTS An insect nutrients‐induced ‐acetylhexosaminidase, BbHex1, was identified from entomopathogenic fungus Beauveria bassiana , which modification degradation cuticle. BbHex1 localized to secreted, displayed enzyme activity degrade chitinase‐hydrolyzed product (GlcNAc) 2 . Disruption resulted a significant decrease level presence nutrients during infection insects, impaired ability penetrate cuticle, accompanying downregulated metabolism‐involved cuticle‐degrading chitinase genes. opposite phenotypes were examined gene overexpression strain. Distinctly altered structures caused by mutation led easy activation evasion (respectively) immune response infection. As result, contributed virulence. Bioinformatics analysis revealed that promoters some co‐expressed genes promoter shared conserved transcription factors Skn7, Msn2 Ste12, CreA‐binding motifs, implying co‐regulation those CONCLUSION These data support mechanism pathogen specifically expresses is chitinases modify for recognition contributes virulence against insects. © 2024 Society Chemical Industry.

Language: Английский

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Access to both anomers of rutinosyl azide using wild-type rutinosidase and its catalytic nucleophile mutant

Catalysis Communications, Journal Year: 2020, Volume and Issue: 149, P. 106193 - 106193

Published: Oct. 10, 2020

Rutinosidases hydrolyze β-rutinosylated flavonoids. As retaining glycosidases they also have a transglycosylation activity. Here we show that two newly identified wild-type rutinosidases, which are members of the glycoside hydrolase family 5–23, capable glycosylation an inorganic azide with rutin as glycosyl donor, yielding rutinosyl β-azide. On other hand, α-azide was synthesized by catalytic nucleophile mutant rutinosidase from Aspergillus niger, belongs to GH5–23. Thus, were able synthesize at preparatory scale both anomers using either or rutinosidases

Language: Английский

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Dual Substrate Specificity of the Rutinosidase from Aspergillus niger and the Role of Its Substrate Tunnel

International Journal of Molecular Sciences, Journal Year: 2020, Volume and Issue: 21(16), P. 5671 - 5671

Published: Aug. 7, 2020

Rutinosidases (α-l-rhamnopyranosyl-(1-6)-β-d-glucopyranosidases, EC 3.2.1.168, CAZy GH5) are diglycosidases that cleave the glycosidic bond between disaccharide rutinose and respective aglycone. Similar to many retaining glycosidases, rutinosidases can also transfer rutinosyl moiety onto acceptors with a free –OH group (so-called transglycosylation). The recombinant rutinosidase from Aspergillus niger (AnRut) is selectively produced in Pichia pastoris. It catalyze transglycosylation reactions as an unpurified preparation directly cultivation. This enzyme exhibits catalytic activity towards two substrates; addition activity, it β-d-glucopyranosidase activity. As result, new compounds formed by β-glucosylation or rutinosylation of such alcohols strong inorganic nucleophiles (NaN3). Transglycosylation products aliphatic aglycones resistant cleavage rutinosidase, therefore, their side hydrolysis does not occur, allowing higher yields. Fourteen were synthesized glucosylation selected acceptors. isolated structurally characterized. Interactions AnRut analyzed molecular modeling. We revealed role substrate tunnel structure AnRut, which explained unusual properties this glycosidase its specific potential. attractive for biosynthetic applications, especially use inexpensive substrates (rutin isoquercitrin).

Language: Английский

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