elucidating-13-glucanase-peroxidase-physicochemical-properties-wheat

Elucidating &#946;-1,3-Glucanase and Peroxidase Physicochemical Properties of Wheat Cell Wall Defense Mechanism Against <i> Diuraphis noxia</i> Infestation

Wheat plants infested by Russian wheat aphids (RWA) induce a cascade of defense responses, including the hypersensitive responses (HR) and induction of ...

university of the free state

Cellulase cocktails formulated to degrade crystalline cellulose generally contain cellobiohydrolases (CBHs), referred to as CBHI (Cel7A) and CBHII (Cel6A), as the major constituents. The combined hydrolytic activities of CBHI and CBHII improve the release of fermentable sugars (β-1,4-cellobiose as the main product) from crystalline cellulose. In this study, a novel cellobiohydrolase (Exg-D) sourced from a metagenome of hindgut bacterial symbionts of a termite was heterologouly expressed, purified, and functionally characterised. Exg-D specific activity was higher on insoluble barley β-glucan (38.94 U/mg protein), soluble wheat flour β-glucan (12.71 U/mg protein) and oat β-glucan (8.89 U/mg protein) compared to cellulosic substrates; Avicel and CMC. We further explored Exg-D activity on the unpretreated or NaOH-pretreated (mercerised) Avicel and compared its activity to commercially available CBHI and CBHII on these celluloses. CBHI displayed the highest activity of 4.74 U/mg protein on mercerised cellulose followed by CBHII (2.14 U/mg protein), while Exg-D activity on untreated and mercerised cellulose was 1.66 and 1.67 U/mg protein, respectively. The high activity of CBHI was supported by binding assays, which revealed that CBHI has a higher binding capacity towards crystalline cellulose compared to Exg-D and CBHII. Only CBHI and CBHII showed synergism during the hydrolysis of mercerised Avicel, showing a degree of synergy (DS) of about 1.299 and yielded about 1.43 μmol/ml of reducing sugars higher than control. In contrast, Exg-D and CBHII displayed synergism during β-glucan degradation, displaying a DS of about 1.22. Thus, we propose that Exg-D should only be used synergistically with other CBHs to degrade mixed linked-β-(1,3)-(1,4)-glucan.

Delineating functional properties of a cello-oligosaccharide and β-glucan specific cellobiohydrolase (GH5_38): Its synergism with Cel6A and Cel7A for β-(1,3)-(1,4)-glucan degradation.

Cereal feedstocks have high arabinoxylan content as their main hemicellulose, which is linked to lignin by hydroxycinnamic acids such as ferulic acid. The ferulic acid is linked to arabinoxylan by ester bonds, and generally, the high substitution of ferulic acid leads to a loss of activity of xylanases targeting the arabinoxylan. In the current study, a feruloyl esterase (FAE-1) from a termite hindgut bacteria was functionally characterised and used in synergy with xylanases during xylan hydrolysis. The FAE-1 displayed temperature and pH optima of 60 ℃ and 7.0, respectively. FAE-1 did not release reducing sugars from beechwood xylan (BWX), wheat arabinoxylan (WAX) and oat spelt xylan (OX), however, displayed high activity of  164.74 U/mg protein on p-nitrophenyl-acetate (pNPA). In contrast, the GH10 xylanases; Xyn10 and XT6, and a GH11 xylanase, Xyn2A, showed more than two-fold increased activity on xylan substrates with low sidechain substitutions; BWX and OX, compared to the highly branched substrate, WAX. Interestingly, the FAE-1 and GH10 xylanases (Xyn10D and XT6) displayed a degree of synergy (DS) that was higher than 1 in all enzyme loading combinations during WAX hydrolysis. The 75%XT6:25%FAE-1 synergistic enzyme combination increased the release of reducing sugars by 1.34-fold from WAX compared to the control, while 25%Xyn10D:75%FAE-1 synergistic combination released about 2.1-fold of reducing sugars from WAX compared to controls. These findings suggest that FAE-1 can be used in concert with xylanases, particularly those from GH10, to efficiently degrade arabinoxylans contained in cereal feedstocks for various industrial settings such as in animal feeds and baking.

Feruloyl esterase (FAE-1) sourced from a termite hindgut and GH10 xylanases synergy improves degradation of arabinoxylan.

Enzymes classified with the same Enzyme Commission (EC) that are allotted in different glycoside hydrolase (GH) families can display different mechanisms of action and substrate specificities. Therefore, the combination of different enzyme classes may not yield synergism during biomass hydrolysis, as the GH family allocation of the enzymes influences their behavior. As a result, it is important to understand which GH family combinations are compatible to gain knowledge on how to efficiently depolymerize biomass into fermentable sugars. We evaluated GH10 (Xyn10D and XT6) and GH11 (XynA and Xyn2A) β-xylanase performance alone and in combination with various GH family α-l-arabinofuranosidases (GH43 AXH-d and GH51 Abf51A) and α-d-glucuronidases (GH4 Agu4B and GH67 AguA) during xylan depolymerization. No synergistic enhancement in reducing sugar, xylose and glucuronic acid released from beechwood xylan was observed when xylanases were supplemented with either one of the glucuronidases, except between Xyn2A and AguA (1.1-fold reducing sugar increase). However, overall sugar release was significantly improved (≥1.1-fold reducing sugar increase) when xylanases were supplemented with either one of the arabinofuranosidases during wheat arabinoxylan degradation. Synergism appeared to result from the xylanases liberating -oligomers, which are the preferred substrates of the terminal arabinofuranosyl-substituent debranching enzyme, Abf51A, allowing the exolytic β-xylosidase, SXA, to have access to the generated unbranched -oligomers. Here, it was shown that arabinofuranosidases are key enzymes in the efficient saccharification of hetero-xylan into xylose. This study demonstrated that consideration of GH family affiliations of the carbohydrate-active enzymes (CAZymes) used to formulate synergistic enzyme cocktails is crucial for achieving efficient biomass saccharification.

Unraveling Synergism between Various GH Family Xylanases and Debranching Enzymes during Hetero-Xylan Degradation.

Vegetable-type soybean, also known as edamame, was recently introduced to South Africa. However, there is lack of information on its responses to drought. The aim of this study was to investigate the photosynthetic efficiency and carbohydrates responses of six edamame cultivars under drought stress. Photosynthetic efficiency parameters, including chlorophyll fluorescence and stomatal conductance, were determined using non-invasive methods, while pigments were quantified spectrophotometrically. Non-structural carbohydrates were quantified using Megazyme kits. Structural carbohydrates were determined using Fourier-transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). Drought stress significantly increased the Fv/Fm and PIabs of AGS429 and UVE17 at pod filling stage. Chlorophyll-a, which was most sensitive to drought, was significantly reduced in AGS429 and UVE17, but chlorophyll-b was relatively stable in all cultivars, except UVE17, which showed a significant decline at flowering stage. AGS354 and AGS429 also showed reduced chlorophyll-b at pod filling. UVE17 showed a significant reduction in carotenoid content and a substantial reduction in stomatal conductance during pod filling. Drought stress during pod filling resulted in a significant increase in the contents of trehalose, sucrose and starch, but glucose was decreased. Chlorophyll-a positively correlated with starch. The FTIR and XRD results suggest that the cell wall of UVE14, followed by UVE8 and AGS429, was the most intact during drought stress. It was concluded that carotenoids, stomatal conductance, starch and hemicellulose could be used as physiological/biochemical indicators of drought tolerance in edamame. This information expands our knowledge of the drought defense responses in edamame, and it is essential for the physiological and biochemical screening of drought tolerance.

The Photosynthetic Efficiency and Carbohydrates Responses of Six Edamame (. L. Merrill) Cultivars under Drought Stress.

Russian wheat aphid (RWA) is one of the most challenging pests for wheat crops globally. In South Africa, RWA has breached the strategy of introducing resistant genes into wheat plants, and so far, five RWA biotypes with different virulence levels have been documented in the field. Our study investigated how the cell wall plays a defensive role in Tugela-Dn1 (susceptible) and-Dn5 (resistant) cultivars infested with South African RWA-biotype 2 (RWASA2). The activities of enzymes related to defense responses were measured. The cell wall's holo-cellulose content, soluble lignin and physicochemical changes were quantified in the infested susceptible and resistant cultivars. Lastly, in vitro RWASA2 saliva-associated CWDEs activity was determined on cell wall-related model substrates. The results show that apoplastic peroxidase and β-1,3-glucanase activities were significantly higher in Tugela-Dn5 relative to the control during the infestation periods. Peroxidase activity is associated with lignin cross-linking of the cell wall, which could deter RWASA2 feeding. The total phenolic and holo-cellulose contents were significantly induced in Tugela-Dn5 at 72 and 120 h post infestation (hpi). These findings were corroborated by the FTIR results, which showed that holocellulose and lignin regions of the resistant and susceptible wheat were affected by infestation at 72 hpi. However, Tugela-Dn5 reinforced cell wall content at 120 hpi. An increased crystallinity index in the resistant cultivar validated the cell wall reinforcement at 120 hpi, while Tugela-Dn1 delayed cell wall reinforcement. This study demonstrates that cell wall reinforcement's modification is part of defense responses against Russian wheat aphid infestation.

Cell-wall structural carbohydrates reinforcements are part of the defence mechanisms of wheat against Russian wheat aphid (Diuraphis noxia) infestation.

Plant cell walls are composed of a heterogeneous mixture of polysaccharides that require several different enzymes to degrade. These enzymes are important for a variety of biotechnological processes, from biofuel production to food processing. Several classical mannanolytic enzyme functions of glycoside hydrolases (GH), such as β-mannanase, β-mannosidase and α-galactosidase activities, are helpful for efficient mannan hydrolysis. In this light, we bring three enzymes into the model of mannan degradation that have received little or no attention. By linking their three-dimensional structures and substrate specificities, we have predicted the interactions and cooperativity of these novel enzymes with classical mannanolytic enzymes for efficient mannan hydrolysis. The novel exo-β-1,4-mannobiohydrolases are indispensable for the production of mannobiose from the terminal ends of mannans, this product being the preferred product for short-chain mannooligosaccharides (MOS)-specific β-mannosidases. Second, the side-chain cleaving enzymes, acetyl mannan esterases (AcME), remove acetyl decorations on mannan that would have hindered backbone cleaving enzymes, while the backbone cleaving enzymes liberate MOS, which are preferred substrates of the debranching and sidechain cleaving enzymes. The nonhydrolytic expansins and swollenins disrupt the crystalline regions of the biomass, improving their accessibility for AcME and GH activities. Finally, lytic polysaccharide monooxygenases have also been implicated in promoting the degradation of lignocellulosic biomass or mannan degradation by classical mannanolytic enzymes, possibly by disrupting adsorbed mannan residues. Modelling effective enzymatic mannan degradation has implications for improving the saccharification of biomass for the synthesis of value-added and upcycling of lignocellulosic wastes.

Mpho S. Mafa

Carbohydrates and Enzymology Laboratory (CHEM-LAB),
Department of Plant Sciences,
Carbohydrates and Enzymology Laboratory (CHEM-LAB), Department of Plant Sciences

university of the free state

Elucidating β-1,3-Glucanase and Peroxidase Physicochemical Properties of Wheat Cell Wall Defense Mechanism Against Diuraphis noxia Infestation

Mpho S. Mafa

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Carbohydrates and Enzymology Laboratory (CHEM-LAB),Department of Plant Sciences,Carbohydrates and Enzymology Laboratory (CHEM-LAB), Department of Plant Sciences

university of the free state

Elucidating β-1,3-Glucanase and Peroxidase Physicochemical Properties of Wheat Cell Wall Defense Mechanism Against Diuraphis noxia Infestation

Carbohydrates and Enzymology Laboratory (CHEM-LAB),
Department of Plant Sciences,
Carbohydrates and Enzymology Laboratory (CHEM-LAB), Department of Plant Sciences