Constructing arabinofuranosidases for dual arabinoxylan debranching activity

Wang, Weijun; Andric, Nikola; Sarch, Cody; Silva, Bruno T.; Tenkanen, Maija; Master, Emma R.

doi:10.1002/bit.26445

Cited by 6 publications

(3 citation statements)

References 37 publications

(53 reference statements)

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“…42,43 The signal of the (1→2)-linked ⊍-L-Araf linked to the monosubstituted xylose unit (5.29 ppm) overlapped with that of the (1→3)-linked ⊍-L-Araf residue in the doubly substituted xylose unit. [44][45][46] A comparison of signal intensities in the MBAX 1 H-NMR spectrum shows that the primary substitution was that of ⊍-L-Araf (1→3)-linkage to monosubstituted ⊎-D-Xylp residues of the main chain (A in Fig. 4).…”

Section: H-nmrmentioning

confidence: 99%

Fine structural features and antioxidant capacity of ferulated arabinoxylans extracted from nixtamalized maize bran

et al. 2023

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BACKGROUND The nixtamalization process improves the nutritional and technological properties of maize. This process generates nixtamalized maize bran as a by‐product, which is a source of arabinoxylans (AX). AX are polysaccharides constituted of a xylose backbone with mono‐ or di‐arabinose substitutions, which can be ester‐linked to ferulic acid (FA). The present study investigated the fine structural features and antioxidant capacity (AC) of nixtamalized maize bran arabinoxylans (MBAX) to comprehend the structure–radical scavenging capacity relationship in this polysaccharide deeply. RESULTS MBAX presented a molecular weight, intrinsic viscosity, and hydrodynamic radius of 674 kDa, 1.8 dL g−1, and 24.6 nm, respectively. The arabinose‐to‐xylose ratio (A/X) and FA content were 0.74 and 0.25 g kg−1 polysaccharide, respectively. MBAX contained dimers (di‐FA) and trimer (tri‐FA) of FA (0.14 and 0.07 g kg−1 polysaccharide, respectively). The main di‐FA isomer was the 8–5′ structure (80%). Fourier transform infrared spectroscopy confirmed MBAX molecular identity, and the second derivate of the spectral data revealed a band at 958 cm−1 related to the presence of arabinose disubstitution. 1H‐Nuclear magnetic resonance spectroscopy showed mono‐ and di‐arabinose substitution in the xylan backbone with more monosubstituted residues. MBAX registered an AC of 25 and 20 μmol Trolox equivalents g−1 polysaccharide despite a low FA content, using ABTS (2,2′‐azino‐bis‐3‐ethylbenzthiazoline‐6‐sulfonic acid) and DPPH (1,1‐diphenyl‐2‐picrylhydrazyl) methods, respectively. CONCLUSION AC in MBAX could be related to the high A/X ratio (mainly monosubstitution) and the high 8–5′ di‐FA proportion in this polysaccharide. © 2023 Society of Chemical Industry.

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Section: H-nmrmentioning

confidence: 99%

Fine structural features and antioxidant capacity of ferulated arabinoxylans extracted from nixtamalized maize bran

et al. 2023

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“…Arabinoxylan degradation requires β-1,4-endo-xylanases for xylan depolymerization, but arabinose substitutions generally interfere with such activity [43]. Therefore, AFases are key enzymes for degradation of arabinoxylan before the backbone can be hydrolyzed by β-1,4-endo-xylanases [44][45].…”

Section: Regioselectivity Of Swuabf62a Towards Wheat Arabinoxylanmentioning

confidence: 99%

Identification and characterization of GH62 bacterial α-l-arabinofuranosidase from thermotolerant Streptomyces sp. SWU10 that preferentially degrades branched l-arabinofuranoses in wheat arabinoxylan

Phuengmaung

Kunishige

Sukhumsirichart

et al. 2018

Enzyme and Microbial Technology

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We previously described thermotolerant Streptomyces sp. SWU10, which produced four endo-xylanases and one xylosidase able to digest xylan backbones. To achieve arabinoxylan degradation, the swu62A gene was cloned and overexpressed in Escherichia coli, and the recombinant enzyme, termed SWUAbf62A, was characterized. The 438 amino acids of SWUAbf62A revealed Glyco_hydro_62 and closely related with putative α-l-arabinofuranosidases belonging to glycoside hydrolase family 62. SWUAbf62A was purified in two steps, Ni-affinity and size-exclusion column chromatographies, and its molecular mass without signal peptide was determined to be 49 kDa. SWUAbf62A showed optimum activity at pH 5.0 and 50 °C, and more than 70% of its initial enzymatic activity remained after incubation at pH 4.1-10.5, while SWUAbf62A lost all activity after 1 h at 60 °C. SWUAbf62A activity was stimulated by Ba, Ca, and Mn and decreased by Ag, Cu, Fe, and EDTA. SWUAbf62A had no activity towards p-nitrophenyl-α-l-arabinofuranoside or p-nitrophenyl-β-d-xylopyranoside synthetic substrates. On the other hand, SWUAbf62A had the highest activity against wheat arabinoxylan, with a specific activity of 1.29 U/mg, and was also active against sugar beet arabinan, with a specific activity of 0.14 U/mg; these results indicated that SWUAbf62A is an arabinoxylan arabinofuranohydrolase. Using H-NMR analysis, SWUAbf62A was found to release l-arabinofuranoses singly linked to O-3 of wheat arabinoxylan. In addition, SWUAbf62A acted synergistically with endo-xylanase (XynSW3) and α-l-arabinofuranosidase, which releases arabinose linked to O-3 of double-substituted xylose residues on arabinoxylan, to digest the wheat arabinoxylan. SWUAbf62A is an important debranching enzyme for hydrolysis of hemicelluloses to monosaccharides and can be applied in various industrial biotechnologies.

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“…Analysis of (arabino)xylan-oligosaccharides ((A)XOS) produced by ABFs is generally done by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) and nuclear magnetic resonance (NMR) (Pastell et al 2008;Lagaert et al 2010;Pouvreau et al 2011;Borsenberger et al 2014;Mccleary et al 2015;Koutaniemi and Tenkanen 2016;Wang et al 2017). Although these techniques are very useful for the identification of (A)XOS structures, MS-and NMR-based techniques require dedicated instrumentation, in-depth instrumental knowledge and expertise (Duus et al 2000;Mantovani et al 2018).…”

Section: Introductionmentioning

confidence: 99%

Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis

Fonseca

Jurak

Kataja

et al. 2018

Appl Microbiol Biotechnol

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Carbohydrate-active enzymes discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of carbohydrate-active enzymes in an efficient manner. DNA sequencer-aided fluorophoreassisted carbohydrate electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different α-L-arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two α-L-arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 α-L-arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 α-L-arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two α-L-arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 α-L-arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted β-D-xylosyl residues, whereas a GH43 α-L-arabinofuranosidase from a metagenomic sample only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), handson and analysis time and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.

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Constructing arabinofuranosidases for dual arabinoxylan debranching activity

Cited by 6 publications

References 37 publications

Fine structural features and antioxidant capacity of ferulated arabinoxylans extracted from nixtamalized maize bran

Fine structural features and antioxidant capacity of ferulated arabinoxylans extracted from nixtamalized maize bran

Identification and characterization of GH62 bacterial α-l-arabinofuranosidase from thermotolerant Streptomyces sp. SWU10 that preferentially degrades branched l-arabinofuranoses in wheat arabinoxylan

Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis

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