The isolation of cell wall and analysis of carbohydrate composition was performed as previously described51 (link). In brief, cells were harvested during stationary-phase at 48 h and washed once with deionized water. The cell pellet was resuspended with 1 ml TE buffer and broken with 1 g acid-washed glass beads. The supernatant was transferred into a 15 ml falcon tube. The glass beads were washed five times and the washing solutions were also collected in 15 ml falcon tube. Samples were centrifuged at 4,800 g for 15 min and the pellets were resuspended in 1 mL TE buffer for centrifugation at 3,000 g for 5 min. The supernatant was dried at 110 °C. Cell walls were hydrolyzed with 72% H2SO4 and the carbohydrate was analyzed by CarboPacTM PA1 anion-exchange column (2 × 250 mm, Dionex). Elution was performed with 18 mM NaOH at a flow rate of 0.5 mL/min.
Carbopac pa 1 anion exchange column
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CarboPac PA-1 anion-exchange column is designed for the separation and analysis of carbohydrates. The column utilizes a polymeric resin with quaternary ammonium functional groups to facilitate the separation of a wide range of monosaccharides, disaccharides, and oligosaccharides through ion-exchange chromatography.
Automatically generated - may contain errors
Lab products found in correlation
Carbopac pa 1 guard column, by Thermo Fisher Scientific (1 mentions)
Mega calc, by Megazyme (1 mentions)
K dlate, by Megazyme (1 mentions)
K etoh, by Megazyme (1 mentions)
Pherastar, by BMG Labtech (1 mentions)
Ics 5000 system, by Thermo Fisher Scientific (1 mentions)
Ultrapure water, by Merck Group (1 mentions)
Chromeleon 7.2 cds, by Thermo Fisher Scientific (1 mentions)
5 protocols using carbopac pa 1 anion exchange column
1
Cell Wall Carbohydrate Composition Analysis
2
HPAEC-PAD Analysis of LPMO Reaction Products
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Enzyme reaction products were analyzed using a CARBOPACTM PA-1 anion exchange column (Dionex) with a CARBOPACTM PA-1 guard column and run at a rate of 1 ml/min. The loaded samples were usually 40 μl of reaction mixed with 160 μl of filtered water. The exception to this was the synergy samples, which were 5 μl of sample mixed with 195 μl of filtered water. The column was equilibrated with 100 mm NaOH. The reaction products from the LPMO reactions were eluted with a 0–300 mm sodium acetate gradient in 100 mm NaOH. The column was cleaned with 1 m sodium acetate in 100 mm NaOH and then with 500 mm NaOH. For measuring glucuronic acid, the chromatography was performed in 66.7 mm NaOH, and elution of oligosaccharides was achieved using a 0–200 mm sodium acetate gradient in 66.7 mm NaOH.
3
Analytical Methods for Biomass Characterization
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Reducing sugars concentration was analyzed using the dinitrosalicylic (DNS) method97 (link). The absorbance was measured at 575 nm, using Novaspec II spectrophotometer.
BE was determined using commercial kits (K-ETOH) from Megazyme International (Wicklow, Ireland). The calculations were done with Megazyme Mega-Calc and expressed as g ethanol/L. LA was determined using commercial kits (KDLATE) from Megazyme International (Wicklow, Ireland). The calculations were done with Megazyme Mega-Calc and expressed as g lactic acid/L. The monomeric sugars content was determined by high performance anion-exchange chromatography (HPAEC; DX-300 series chromatography system, Dionex, USA). The effluent was monitored with pulsed amperometric detection detector (PAD, Dionex, CA, USA). Filtered samples (10 μL) were injected into a CarboPac PA-1 anion-exchange column (0.4 × 250 mm, Dionex, CA, USA) that was pre-equilibrated with 18 mM KOH. The isocratic method was employed with 18 mM KOH at a flow rate of 1.0 mL/min in 20 min. Protein content was determined by the Bradford method with OD measured at 595 nm98 . Each measurement was repeated at least three times and averaged.
BE was determined using commercial kits (K-ETOH) from Megazyme International (Wicklow, Ireland). The calculations were done with Megazyme Mega-Calc and expressed as g ethanol/L. LA was determined using commercial kits (KDLATE) from Megazyme International (Wicklow, Ireland). The calculations were done with Megazyme Mega-Calc and expressed as g lactic acid/L. The monomeric sugars content was determined by high performance anion-exchange chromatography (HPAEC; DX-300 series chromatography system, Dionex, USA). The effluent was monitored with pulsed amperometric detection detector (PAD, Dionex, CA, USA). Filtered samples (10 μL) were injected into a CarboPac PA-1 anion-exchange column (0.4 × 250 mm, Dionex, CA, USA) that was pre-equilibrated with 18 mM KOH. The isocratic method was employed with 18 mM KOH at a flow rate of 1.0 mL/min in 20 min. Protein content was determined by the Bradford method with OD measured at 595 nm98 . Each measurement was repeated at least three times and averaged.
4
Enzymatic Performance Evaluation of CapF
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The enzymatic performance of CapF was evaluated by examining the consumption of NADPH spectrophotometrically, or by monitoring the disappearance of substrate and the formation of product by HPLC9 (link)12 (link). First, the time-dependent consumption of NADPH (monitored from a decrease of adsorption at 340 nm) was carried out in 384-well plates at a final volume of 50 μL. The solution contained 1.0 μM CapE, 0.02 μM CapF, 125 μM UDP-D-GlcNAc (substrate), 250 μM NADPH and fragment compounds at various concentrations in HBS supplemented with 0.005% (v/v) Tween-20 and 5% (v/v) DMSO. Each reaction mixture was incubated at 37 °C and the absorption at 340 nm was measured with a plate reader (PHERAstar, BMG Labtech, Durham, NC).
For the HPLC-based inhibition assay, a solution containing 1.0 μM CapE, 0.02 μM CapF, 250 μM UDP-D-GlcNAc, 250 μM NADPH, and 1 mM compound in HBS buffer supplemented with 0.005% (v/v) Tween-20 and 5% (v/v) DMSO were incubated at 37 °C for 2 hours. The reaction was stopped by addition of 100 μL of ice-cold phenol/chloroform/isoamyl alcohol in a 25:24:1 molar ratio. The supernatant containing the products of the reaction was mixed with 100 μL of chloroform and examined by HPLC using a CarboPac PA1 anion-exchange column (Dionex) as described previously9 (link)12 (link). Identification of monosaccharides in the elution profile was based on a previous report9 (link).
For the HPLC-based inhibition assay, a solution containing 1.0 μM CapE, 0.02 μM CapF, 250 μM UDP-D-GlcNAc, 250 μM NADPH, and 1 mM compound in HBS buffer supplemented with 0.005% (v/v) Tween-20 and 5% (v/v) DMSO were incubated at 37 °C for 2 hours. The reaction was stopped by addition of 100 μL of ice-cold phenol/chloroform/isoamyl alcohol in a 25:24:1 molar ratio. The supernatant containing the products of the reaction was mixed with 100 μL of chloroform and examined by HPLC using a CarboPac PA1 anion-exchange column (Dionex) as described previously9 (link)12 (link). Identification of monosaccharides in the elution profile was based on a previous report9 (link).
5
Quantitative Analysis of Soluble Sugars
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About 10 mg of freeze-dried sample powder was placed in a 2 ml tube containing 0.7 ml 80 % ethanol (v/v). This was mixed by vortexing before incubation at 50 °C for 2 h. To this, 0.7 ml ultra-pure water (Millipore, Bedford, MA, USA) was added before centrifugation at 12000 rpm for 5 min. The extracts were then injected into a high-performance anion-exchange chromatography (HPAEC) system equipped with a CarboPac PA-1 anion-exchange column (4.0 * 250 mm; Dionex) and a pulsed amperometric detector (PAD; Dionex ICS 5000 system). The flow rate was 1.0 ml/min and injection volume was 10 μl. The column temperature was 30 °C. The gradient programs (H2O:0.1 M NaOH) were: 95:5V/V from 0 min, 95:5 V/V at 9 min, 0:100 V/V at 20 min, 0:100 V/V at 40 min, 95:5 V/V at 40.1 min, 95:5 V/V at 60 min. Data were acquired with an ICS5000 system (Thermo Scientific) and processed using Chromeleon 7.2 CDS (Thermo Scientific). Identification of soluble sugars was based on the retention times of pure standards and quantification was done by external calibration (Table S2 ).
To ascertain and compare sweetness levels in the samples, sweetness (sucrose equivalent) was calculated using this equation: Sucrose Equivalent (SE) = 1.2 fructose + 1 sucrose + 0.64 glucose + 0.43 maltose (Kays, Wang, & McLaurin, 2005 (link)).
To ascertain and compare sweetness levels in the samples, sweetness (sucrose equivalent) was calculated using this equation: Sucrose Equivalent (SE) = 1.2 fructose + 1 sucrose + 0.64 glucose + 0.43 maltose (Kays, Wang, & McLaurin, 2005 (link)).
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