MetY kinetic parameters were determined by a coupled enzymatic assay using an acetic acid assay kit that uses pyruvate kinase and lactate dehydrogenase for the detection of acetic acid (Megazyme). The oxidation of NADH concomitant with O-acetyl-L -homoserine (Toronto Research Chemicals) transhydroxylation with either sodium sulfide (Sigma-Aldrich, 431648) or methanol (Sigma-Aldrich, 34860) was monitored at 340 nm in a SAFAS UVmc2 double-beam spectrophotometer. All kinetic parameters were determined from duplicate experiments by non-linear analysis of initial rates with SigmaPlot version 9.0 (Systat Software). Enzymatic reactions were performed at RT in 100 μL total volume reaction at pH 7.4 containing NADH at 4 mM and initiated by the addition of 2 ng of the purified enzyme. For the wildtype protein assay, we varied the concentration of one substrate and set the other substrate at a saturating concentration (O-acetyl-L -homoserine versus sulfide or methanol). The mutated proteins were tested with the saturating concentrations determined for the wildtype protein as described above.
Acetic acid assay kit
Manufactured by Megazyme
Sourced in Ireland
The Acetic Acid Assay Kit is a laboratory equipment designed to quantify the concentration of acetic acid in various samples. It provides a reliable and accurate method for the determination of acetic acid levels.
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Lab products found in correlation
Acetic acid, by Megazyme (1 mentions)
Sodium sulfide, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Sigmaplot version 9, by Grafiti LLC (1 mentions)
Glucose assay kit, by Merck Group (1 mentions)
Acid washed glass beads, by Merck Group (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
D gluconate d glucono d lactone assay kit, by Megazyme (1 mentions)
10 protocols using acetic acid assay kit
1
Determining MetY Kinetic Parameters
2
Enzymatic Assay of Chitin Deacetylase
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The enzymatic activity assay of AsCDA was performed as Acetic Acid Assay Kit (Acetate kinase analyzer format, Megazyme, Republic of Ireland) described by Bergmeyer with modification [59 ]. The Standard curve of acetic acid with ∆A340 was drawn for enzyme assay of AsCDA (Figure S2 ). The reaction mixture of 2 mg α-chitin and 10 µL AsCDA (2.11 µg/µL) was added to 190 µL phosphate buffer (pH 7.0) and incubated at 30 °C for 30 min; the enzyme reaction was terminated by boiling at 100 °C for 10 min in a sealed tube prior. The supernatant was collected by centrifugation at 12,000 rpm for 5 min, and the amount of acetic acid in 10 µL supernatant was determined to calculate the enzyme activity through the A340 according to the assay procedure of the Acetic Acid Assay Kit. The minimum differential absorbance of the Acetic Acid Assay Kit was 0.005 absorbance unit, which was equivalent to 0.063 mg/L acetic acid in the sample solution. One unit of AsCDA activity was defined as the amount of enzyme required to catalyze the release of 1µmol acetic acid per minute.
3
Characterization of Destarched Wheat Bran and Treated Wheat Straw
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The preparation of destarched wheat bran and NaClO2-treated wheat straw was described by Wang et al. [57 ] and Saarnio et al. [58 (link), 59 (link)], respectively. The carbohydrate and lignin content of destarched wheat bran, NaClO2-treated wheat straw and inAX was determined by the standard procedure of NREL [60 ]. The acetic acid content was determined after alkaline hydrolysis (2 M NaOH, 4 h, 70 °C) [61 (link)] and quantified by the Acetic Acid Assay Kit (Megazyme) according to the manufacturer’s instructions.
4
Enzymatic Hydrolysis of Lignocellulosic Biomass
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Reaction mixtures consisted of: 0.1 g destarched wheat bran, NaClO2-treated wheat straw or inAX [2 % (w/v) suspension in 100 mM sodium citrate buffer, pH 7.0] with 0.005 μmol of purified enzyme in a total volume of 5 mL. Mixtures were incubated at 50 °C for 1, 6, 12, 24 h with orbital shaking (150 r.p.m.) and then boiled at 99 °C for 10 min. All hydrolysis experiments were carried out in duplicates. After centrifugation, the acetic acid in the supernatant was quantified using the Acetic Acid Assay Kit (Megazyme) according to the manufacturer’s instructions.
5
Acetic Acid Quantification Protocol
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Acetate concentrations were measured from the supernatant using an acetic acid assay kit (Megazyme), following the protocol of the manufacturer.
6
Glucose and Acetate Measurement
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The ancestor, 6.5KS1, and 6.5KL4 clones were grown in DM250-glucose as described. Samples were taken at time 0 and every h for 9 h. After centrifugation to remove cells, we measured glucose and acetate concentrations in the supernatant using the Glucose Assay Kit (Merck Millipore, Lyon, France) and Acetic Acid Assay Kit (Megazyme, Pontcharra-sur-Turdine, France), respectively, following the manufacturers’ recommendations.
7
Phosphoketolase Activity Assay Protocol
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Cells in mid log phase (OD 0.4-0.8) were harvested by centrifugation. Cell pellets were resuspended in 500 μL 50 mM histidine-HCl buffer (pH 7.0) containing 20 mM KH2PO4-Na2HPO4, 2 mM dithiothreitol, 1 mM MgSO4 and 5 μg Read-Lyse lysozyme (Lucigen, Middleton, WI). Resuspended cells were added to a screwcap tube containing acid washed glass beads (Sigma-Aldrich, St. Louis, MO) and lysed using a Tissuelyser II (Qiagen, Hilden, Germany) at a frequency of 30 s -1 for 2 min. Cell free extract was made by spinning lyse cells at 15,000g for 3 minutes and taking the supernatant.
Phosphoketolase activity assay was adapted from previously reported protocols 29, 32 .
Briefly, cell free extracts of the wild-type and transformed phosphoketolase strain were added to buffer containing 25 mM xylulose 5-phosphate to generate acetyl-P, which was then converted to acetate by adding 1 μl of 1 M MgCl2, 1 μl of 30 mM ADP, and 0.2 U of acetate kinase to 75 μl of the assay mixture and incubating at 30°C for 30 minutes. The acetate was then determined enzymatically with the Acetic Acid Assay Kit (Megazyme, Bray, Ireland), using an assay mixture without xylulose 5-phosphate as a control.
Phosphoketolase activity assay was adapted from previously reported protocols 29, 32 .
Briefly, cell free extracts of the wild-type and transformed phosphoketolase strain were added to buffer containing 25 mM xylulose 5-phosphate to generate acetyl-P, which was then converted to acetate by adding 1 μl of 1 M MgCl2, 1 μl of 30 mM ADP, and 0.2 U of acetate kinase to 75 μl of the assay mixture and incubating at 30°C for 30 minutes. The acetate was then determined enzymatically with the Acetic Acid Assay Kit (Megazyme, Bray, Ireland), using an assay mixture without xylulose 5-phosphate as a control.
8
Quantitative Measurement of Metabolites
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Plasma and urine lactate concentrations were determined by test strips (EDGE Handheld Lactate Analyser, Red Med, Poland). The D-Gluconate/D-Glucono-d-lactone assay kit (detection limit 0.5 mg/L) and acetic acid assay kit (detection limit 0.14 mg/L) (Megazyme International Ireland, Bray Business Park, Bray, Co. Wicklow, Ireland) were used to determine the plasma urine gluconate and acetate concentrations. Briefly, this is based on an enzymatic ultraviolet (spectrophotometric at 340 nm) method using two enzymes, gluconate kinase and gluconate-6phosphate dehydrogenase.
9
Acetylated Xylan Isolation and Quantification
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Isolation of acetylated xylan was done according to Goncalves et al. [15 (link)]. Acetic acid was released by incubating xylan with NaOH [13 (link)], and the released acetyl groups was quantitated using an acetic acid assay kit (Megazyme). The data were the average of three separate pools of samples.
10
Acetate Quantification in Bacterial Cultures
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The acetate concentration in spent supernatants was measured using an Acetic Acid Assay Kit (Megazyme International Ireland) according to the manufacturer’s instructions. Bacteria were cultured overnight in LB broth, diluted into fresh LB broth to yield a starting OD600 of 0.04, and then incubated at 37°C with shaking overnight. 100 μl of this culture was centrifuged to remove bacteria, and the resulting supernatant was diluted in a 1:5 ratio with water and used in the assay. Sodium acetate was used to generate a standard curve.
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