Structural characterization of the family GH115 alpha-glucuronidase from Amphibacillus xylanus yields insight into its coordinated action with alpha-arabinofuranosidases

The coordinated action of carbohydrate-active enzymes has mainly been evaluated for the purpose of complete saccharification of plant biomass (lignocellulose) to sugars. By contrast, the coordinated action of accessory hemicellulases on xylan debranching and recovery is less well characterized. Here, the activity of two family GH115 alpha-glucuronidases (SdeAgu115A from Saccharophagus degradans, and AxyAgu115A from Amphibacillus xylanus) on spruce arabinoglucuronoxylan (AGX) was evaluated in combination with an alpha-arabinofuranosidase from families GH51 (AniAbf51A, aka E-AFASE from Aspergillus niger) and GH62 (SthAbf62A from Streptomyces thermoviolaceus). The alpha-arabinofuranosidases boosted (methyl)-glucuronic acid release by SdeAgu115A by approximately 50 % and 30 %, respectively. The impact of the alpha-arabinofuranosidases on AxyAgu115A activity was comparatively low, motivating its structural characterization. The crystal structure of AxyAgu115A revealed increased length and flexibility of the active site loop compared to SdeAgu115A. This structural difference could explain the ability of AxyAgu115A to accommodate more highly substituted arabinoglucuronoxylan, and inform enzyme selections for improved AGX recovery and use.Peer reviewe

Xylo- and cello-oligosaccharide oxidation by gluco-oligosaccharide oxidase from Sarocladium strictum and variants with reduced substrate inhibition

Peer reviewe

Natural Products and Their Derivatives with Antibacterial, Antioxidant and Anticancer Activities

Natural products and their derivatives have been commonly used in our daily life, as they play important roles in boosting immune systems and fighting diseases [...

Enzymatic production of 4-O-methyl d-glucaric acid from hardwood xylan

Abstract Background Dicarboxylic acids offer several applications in detergent builder and biopolymer fields. One of these acids, 4-O-methyl d-glucaric acid, could potentially be produced from glucuronoxylans, which are a comparatively underused fraction of wood and agricultural biorefineries. Results Accordingly, an enzymatic pathway was developed that combines AxyAgu115A, a GH115 α-glucuronidase from Amphibacillus xylanus, and GOOX, an AA7 gluco-oligosaccharide oxidase from Sarocladium strictum, to produce this bio-based chemical from glucuronoxylan. AxyAgu115A was able to release almost all 4-O-methyl d-glucuronic acid from glucuronoxylan while a GOOX variant, GOOX-Y300A, could convert 4-O-methyl d-glucuronic acid to the corresponding glucaric acid at a yield of 62%. Both enzymes worked effectively at alkaline conditions that increase xylan solubility. Given the sensitivity of AxyAgu115A to hydrogen peroxide and optimal performance of GOOX-Y300A at substrate concentrations above 20 mM, the two-step enzyme pathway was demonstrated as a sequential, one-pot reaction. Additionally, the resulting xylan was easily recovered from the one-pot reaction, and it was enzymatically hydrolysable. Conclusions The pathway in this study requires only two enzymes while avoiding a supplementation of costly cofactors. This cell-free approach provides a new strategy to make use of the underutilized hemicellulose stream from wood and agricultural biorefineries

Enzymatic upgrading of heteroxylans for added-value chemicals and polymers

| openaire: EC/H2020/648925/EU//BHIVE Funding Information: This work was supported by Genome Canada for the project ‘SYNBIOMICS - Functional genomics and techno-economic models for advanced biopolymer synthesis’ (LSARP, grant number 10405 ), and the European Research Council (ERC) Consolidator Grant ( BHIVE- 648925 ). Publisher Copyright: © 2021 The Author(s)Xylan is one of the most abundant, natural polysaccharides, and much recent interest focuses on upgrading heteroxylan to make use of its unique structures and chemistries. Significant progress has been made in the discovery and application of novel enzymes for debranching and modifying heteroxylans. Debranching enzymes include acetylxylan esterases, α-L-arabinofuranosidases and α-Dglucuronidases that release side groups from the xylan backbone to recover both biochemicals and less substituted xylans for polymer applications in food packaging or drug delivery systems. Besides esterases and hydrolases, many oxidoreductases including carbohydrate oxidases, lytic polysaccharide monooxygenases, laccases and peroxidases have been also applied to alter different types of xylans for improved physical and chemical properties. This review will highlight the recent discovery and application of enzymes for upgrading xylans for use as added-value chemicals and in functional polymers.Peer reviewe

Processivity, Synergism, and Substrate Specificity of Thermobifida fusca Cel6B ▿

A relationship between processivity and synergism has not been reported for cellulases, although both characteristics are very important for hydrolysis of insoluble substrates. Mutation of two residues located in the active site tunnel of Thermobifida fusca exocellulase Cel6B increased processivity on filter paper. Surprisingly, mixtures of the Cel6B mutant enzymes and T. fusca endocellulase Cel5A did not show increased synergism or processivity, and the mutant enzyme which had the highest processivity gave the poorest synergism. This study suggests that improving exocellulase processivity might be not an effective strategy for producing improved cellulase mixtures for biomass conversion. The inverse relationship between the activities of many of the mutant enzymes with bacterial microcrystalline cellulose and their activities with carboxymethyl cellulose indicated that there are differences in the mechanisms of hydrolysis for these substrates, supporting the possibility of engineering Cel6B to target selected substrates

Treatment strategies for high resveratrol induction in Vitis vinifera L. cell suspension culture

Bioprocesses capable of producing large scales of resveratrol at nutraceutical grade are in demand. This study herein investigated treatment strategies to induce the production of resveratrol in Vitis vinifera L. cell suspension cultures. Among seven investigated elicitors, jasmonic acid (JA), salicylic acid, β-glucan (GLU), and chitosan enhanced the production of intracellular resveratrol manyfold. The combined treatment of JA and GLU increased extracellular resveratrol production by up to tenfold. The application of Amberlite XAD-7 resin for in situ removal and artificial storage of secreted resveratrol further increased resveratrol production by up to four orders of magnitude. The level of resveratrol produced in response to the combined treatment with 200 g/L XAD-7, 10 μM JA and 1 mg/mL GLU was approximately 2400 mg/L, allowing the production of resveratrol at an industrial scale. The high yield of resveratrol is due to the involvement of a number of mechanisms working in concert

Fusion of a Xylan-Binding Module to Gluco-Oligosaccharide Oxidase Increases Activity and Promotes Stable Immobilization

<div><p>The xylan-binding module <i>Clostridium thermocellum</i> CBM22A was successfully fused to a gluco-oligosaccharide oxidase, GOOX-VN, from <i>Sarocladium strictum</i> via a short TP linker, allowing the fused protein to effectively bind different xylans. The presence of the <i>Ct</i>CBM22A at the N-terminal of GOOX-VN increased catalytic activity on mono- and oligo-saccharides by 2-3 fold while not affecting binding affinity to these substrates. Notably, both GOOX-VN and its CBM fusion also showed oxidation of xylo-oligosaccharides with degrees of polymerization greater than six. Whereas fusion to <i>Ct</i>CBM22A did not alter the thermostability of GOOX-VN or reduce substrate inhibition, <i>Ct</i>CBM22A_GOOX-VN could be immobilized to insoluble oat spelt xylan while retaining wild-type activity. QCM-D analysis showed that the fused enzyme remained bound during oxidation. These features could be harnessed to generate hemicellulose-based biosensors that detect and quantify the presence of different oligosaccharides.</p></div

HPAEC-PAD separation of native and oxidized xylo-oligosaccharides.

<p>(A): Xylo-oligosaccharide standard from xylose to xylohexaose (X1 to X6, respectively) before oxidation (top) and after the oxidation of <i>Ct</i>CBM22A_GOOX-VN, which released oxidized xylose to xylohexaose (ox.X1 to ox.X6, respectively) (bottom). (B): A chromatogram of soluble oat spelt xylan without any enzyme treatment. (C): Xylanase treatment of soluble oat spelt xylan (black solid), GOOX-VN treated soluble oat spelt xylan (red dashed) and <i>Ct</i>CBM22A_GOOX-VN treated soluble oat spelt xylan (green dotted dashed); peaks formed by oxidation were asterisked.</p