Xylan (10 g) from beechwood (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 250 ml of DMSO at 55°C for 24 h. Next, 0.4 g of potassium borate was added to the Xylan solution while stirring at 55°C, followed by 200 ml acetic anhydride, which was added slowly over 5 min. After 4 h of incubation at 55°C, the mixture was dialyzed against tap water at 4°C for 5 days, followed by dialysis against distilled water for 1 day.
Xylan
Manufactured by Merck Group
Sourced in United States, Germany, China, Ireland
Xylan is a laboratory equipment product manufactured by Merck Group. It is a natural polysaccharide extracted from various plant sources. Xylan serves as a key component in various scientific and research applications, providing a versatile platform for diverse analytical and experimental procedures.
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Lab products found in correlation
Pectin, by Merck Group (9 mentions)
Laminarin, by Merck Group (7 mentions)
Carboxymethylcellulose, by Merck Group (5 mentions)
Chitosan, by Merck Group (5 mentions)
Avicel, by Merck Group (5 mentions)
Starch, by Merck Group (4 mentions)
Cellobiose, by Merck Group (4 mentions)
Inulin, by Merck Group (3 mentions)
Whatman, by Cytiva (3 mentions)
Crab shell chitin, by Merck Group (3 mentions)
Chitin beads, by New England Biolabs (3 mentions)
Gum arabic, by Merck Group (2 mentions)
Yeast extract, by Merck Group (2 mentions)
Fluoresceinamine, by Merck Group (2 mentions)
Congo red, by Merck Group (2 mentions)
Cm cellulose, by Merck Group (2 mentions)
Xylan, by Megazyme (2 mentions)
Escherichia coli top10, by Thermo Fisher Scientific (2 mentions)
Microcrystalline cellulose, by Merck Group (2 mentions)
Kraft lignin, by Merck Group (2 mentions)
80 protocols using xylan
1
Acetylation of Beechwood Xylan
2
Characterization of Aspergillus Strains
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Wild-type strains of Aspergillus candidus UCCM 00117 (Asp-C) and Aspergillus sydowii UCCM 00124 (Asp-S) were obtained from University of Calabar Collection of Microorganisms (UCCM) (www.wfcc.info/ccinfo/collection/by_id/652 ) and reactivated in Czapek-Dox agar (CDA) medium (Sigma-Aldrich, USA) for 96 h at 30 ºC. The strains were grown in minimal media containing 1% (w/v) xylan at 30 ºC and on PDA-NaCl medium containing 25% (w/v) NaCl at 55 ºC. The minimal medium contained 0.35% NaNO2, 0.15% K2HPO4, 0.05% MgSO4.7H2O, 0.05% KCl, 0.001% FeSO4.7H2O supplemented with 1% (w/v) xylan (Sigma Aldrich, Germany) (Brandt et al. 2020 (link)). xylanase production with no-growth on PDA-25% NaCl medium was characteristic of Asp-S while non-xylanase production but luxuriant growth on PDA-25% NaCl was characteristic of Asp-C.
3
Xylanase Activity: Temperature and pH
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Activity of purified xylanases was assayed at a range of temperature and pH. The enzyme reactions to be tested at different temperatures were prepared by mixing 180 μL of substrate (1% oat-spelt xylan, in Tris–HCl 50 mM buffer pH 9.0) and 20 μL of purified protein (protein concentration was adjusted to the xylanase assay conditions) and then incubated at 60, 70, 80 or 90 °C, for 10 min. The reaction was stopped by putting the tubes on ice.
Activity as a function of pH was determined using 50 mM buffered solutions, at the following pH values: 5.0 (acetate), 6.0 and 7.0 (phosphate), 8.0, 9.0 and 10.0 (Tris–HCl). The enzyme reactions were prepared by mixing 180 μL of 1% oat-spelt xylan (Sigma) in buffer and 20 μL of purified protein (diluted at a concentration suitable for the DNS assay). The reactions were incubated at 65 °C during 10 min and then stopped on ice.
Production of reducing sugars was determined by adding 100 μL of DNS solution to the reaction tubes that were then boiled for 10 min. Next, 900 μL of miliQ H2O was added and the tubes were centrifuged. 300 μL of the supernatant was transferred to 96-well plates and OD540 was measured using PowerWave HT equipment, from BioTek Instruments (Winooski, VT, USA).
Activity as a function of pH was determined using 50 mM buffered solutions, at the following pH values: 5.0 (acetate), 6.0 and 7.0 (phosphate), 8.0, 9.0 and 10.0 (Tris–HCl). The enzyme reactions were prepared by mixing 180 μL of 1% oat-spelt xylan (Sigma) in buffer and 20 μL of purified protein (diluted at a concentration suitable for the DNS assay). The reactions were incubated at 65 °C during 10 min and then stopped on ice.
Production of reducing sugars was determined by adding 100 μL of DNS solution to the reaction tubes that were then boiled for 10 min. Next, 900 μL of miliQ H2O was added and the tubes were centrifuged. 300 μL of the supernatant was transferred to 96-well plates and OD540 was measured using PowerWave HT equipment, from BioTek Instruments (Winooski, VT, USA).
4
Conversion of Xylan to Lactic Acid
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Example 1
a) To a 250 mL flask was charged xylan (3.301 g, from beech wood, Sigma-Aldrich, 96.4% purity based on HPLC area) followed by water (50 mL), concentrated H2SO4 (0.5 mL) and n-butanol (30 mL, purity determined by GC analysis, see
b) The reaction mixture was then cooled to ambient temperature and the layers were separated. The organic n-butanol layer was dark coloured, consistent with the removal of lignin residues, and the lower aqueous layer was pale straw/yellow coloured. Analysis of the n-butanol layer by GC indicated the presence of butyl acetate (
c) The separated aqueous layer (50.9 g) was then added over a period of 60 min to 50% aqueous sodium hydroxide solution (8.2 mL) at 100-120° C. The reaction mixture was then cooled to ambient temperature and acidified with 10 mL concentrated (37%) HCl to achieve a pH <3 and made up to 1 L with water in a volumetric flask and analysed by HPLC, indicating a lactic acid yield of 0.84 g (
5
Simulated Gastrointestinal Digestion
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Pepsin from porcine stomach mucosa (250 U/mg), pancreatin (8 × USP) from porcine pancreas, porcine bile extract, mucin from porcine stomach-type II, albumin, resazurin, cysteine, peptone, yeast extract, pectin, xylan, gum arabic, potato starch, casein, glucose, and inulin were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Phenolic compounds standards (2,4 dihydroxybenzoic, 3,4 dihydroxybenzoic, gallic, benzoic, caffeic, ferulic, p-coumaric, salicylic, rosmarinic, and 5-caffeoylquinic acids) were purchased from Sigma-Aldrich Chemical Co. All solvents were HPLC grade from Tedia (Fairfield, OH, USA). HPLC grade water (Milli-Q system, Millipore, Bedford, MA, USA) was used throughout the experiments.
6
Fluorescent Labeling of Polysaccharides
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Three polysaccharides (laminarin, xylan, chondroitin sulphate) obtained from Sigma-Aldrich (Munich, Germany) were fluorescently labelled with fluorescein amine (Sigma-Aldrich; isomer II) as described in Arnosti (2003) . The FLA-PS solutions are free of monosaccharides or oligosaccharides, due to the fact that they are repeatedly injected onto standardised gel permeation chromatography systems as part of the labelling procedure; any low-molecular-weight carbohydrates are thereby removed during purification. Average-molecular weights of fluorescently labelled laminarin, xylan and chondroitin sulphate are 6000, 9000 and >50 000 daltons, respectively. A single polysaccharide was added at a concentration of 1.75 μmol monomer-equivalent to each 500 ml water sample; each polysaccharide was incubated in triplicate, plus one killed control, as described above.
7
Screening and Quantification of Cellulases and Xylanases
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In order to determine the production of cellulases and Xylanases by the isolates, pure cultures of microorganisms were individually transferred onto solid medium (NaNO3 1 g L−1, K2HPO4 1 g L−1, KCl 1 g L−1, MgSO4 0.5 g L−1, yeast extract 0.5 g L−1, glucose 1 g L−1, agar 17 g L−1) containing 5 g L−1 of carboxymethylcellulose (CMC, Sigma) or Xylan (Sigma), respectively. After incubation for 2–4 days, depending on the speed of growth of the isolates, the cellulolytic/Xylanolytic strains were assayed for their ability to degrade CMC/Xylan by incubation with 0.1% Congo Red solution for 30 min, followed by washing with 5 mol L−1 NaCl as reported in Amore et al.6 (link) All the strains with a clear halo around the colonies were chosen as positive.
The liquid medium adopted for analysis of cellulase and Xylanase production levels contained 1% CMC or Xylan, respectively, 0.7% yeast extract, 4 g L−1 KH2PO4, 4 g L−1 Na2HPO4, 0.2 g L−1 MgSO4.7H2O, 0.001 g L−1 CaCl2.2H2O, 0.004 g L−1 FeSO4.7H2O.13 (link)
The liquid medium adopted for analysis of cellulase and Xylanase production levels contained 1% CMC or Xylan, respectively, 0.7% yeast extract, 4 g L−1 KH2PO4, 4 g L−1 Na2HPO4, 0.2 g L−1 MgSO4.7H2O, 0.001 g L−1 CaCl2.2H2O, 0.004 g L−1 FeSO4.7H2O.13 (link)
8
Xylanase Activity Quantification in Transformants
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Transformants carrying different numbers of copies of xynB were separately inoculated into galactose medium or YPD medium in a fermenter and incubated at 30 °C for 72 h with an agitation speed of 150 rpm. Xylanase activity in the resulting cultures was determined using the reducing sugar assay, as described previously17 (link) (Sigma Aldrich). The standard curve of the xylose regression equation was as follows: y = 0.1275x − 0.07, with R2 = 0.9935. Xylan was purchased from Sigma Aldrich (catalogue number V900513).
9
Ciliate Growth on Polysaccharide Substrates
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The growth with different polysaccharide feeds (substrates) was investigated. The ciliate growth was examined with no particulate substrate (NoPOS), with Xylan (Xylan from beech wood, Sigma-Aldrich), carboxymethyl cellulose (CMC, Sigma-Aldrich), crystalline cellulose (CC, Sigmacel 20, Sigma-Aldrich), rice starch (RS, ANALAR, Hopkins and Williams GB), and Inulin (Inulin from Dahlia tubers, Sigma-Aldrich).
10
Xylanase Activity Determination Protocol
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Endo-1,4-xylanase (xylanase) activity was determined as described previously [21 (link)]. Briefly, 50 μL of properly diluted enzyme was added to 450 μL of 20 g·L−1 oat spelt xylan (Sigma, St. Louis, MO, USA) in 50 mM citrate-phosphate buffer (pH 7.0) and placed in a thermoshaker (TS-100, BOECO, Hamburg, Germany) operating at 900 rpm and 60 °C for 15 min. Samples were placed into an ice bath and 500 μL of 3,5-dinitrosalicylic acid (DNS) reagent was added to terminate the reaction. Following the removal of residual xylan by centrifugation (3 min at 9000 rpm), the supernatant was boiled for 5 min. The concentration of the reducing sugars was estimated by measuring the absorbance at 540 nm using a xylose standard curve [22 (link)].
One unit of xylanase activity was defined as the amount of enzyme that produced 1 μmol of reducing sugar per minute, determined as xylose equivalents.
One unit of xylanase activity was defined as the amount of enzyme that produced 1 μmol of reducing sugar per minute, determined as xylose equivalents.
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