The enzyme activities were measured with their respective substrates, which are cited between parentheses: endo-1,5-α-L-arabinanase (debranched arabinan) (Megazyme®), endoglucanase (carboxymethylcellulose—CMC) (Sigma-Aldrich®), β-glucanase (β-glucan) (Megazyme®), lichenase (lichenan) (Megazyme®), mannanase (locust bean) (Sigma-Aldrich®), polygalacturonase (polygalacturonic acid sodium salt) (Sigma-Aldrich®), endo-1,4-β-xylanase (xylan beechwood) (Sigma-Aldrich®), and xyloglucanase (xyloglucan) (Megazyme®). The activities were determined by the quantification of reducing sugars using the 3,5-dinitrosalicylic acid (DNS), according to the Miller method [34 (link)]. The assay mixture consisted of 25 µL of 1% (w/v) substrate solution in distilled water, 10 µL of 50 mM sodium acetate buffer, pH 5.0, and 15 µL of enzyme extract. A blank was carried out for each enzymatic assay by adding the enzyme extract after the assay time had elapsed with 50 µL of DNS. The absorbance was measured at 540 nm, and reducing sugars were quantified using standard curves of arabinose, cellobiose, glucose, mannose, galacturonic acid, and xylose (0–1 mg/mL), respectively. The detection limit was 3 µg of reducing sugar. The activity unit was defined as the amount of enzyme that releases one µmol of reducing sugar per minute per mL, and it was expressed as milliunity per mL (mU/mL).
Polygalacturonase
Manufactured by Merck Group
Sourced in United States
Polygalacturonase is an enzyme that catalyzes the hydrolytic cleavage of α-1,4-glycosidic bonds in polygalacturonic acid, a major component of plant cell walls. It is used in various industrial applications, such as in the food and beverage industry.
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Lab products found in correlation
3 protocols using polygalacturonase
1
Enzymatic Activity Quantification Protocol
2
Optimizing Pectin Hydrolysis Conditions
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A commercial citrus pectin with a high degree of esterification (67.0%) was purchased (Vetec, Brazil) . Sulfuric acid (Vetec, Brazil) was employed in the acid hydrolysis and a polygalacturonase (Sigma-Aldrich, St. louis, US, e.C.3.2.1.15), with an enzymatic activity of 1.32 UI per mg of enzyme, was used in the enzymatic hydrolysis, the value of enzymatic activity was confirmed experimentally (data not shown).
In order to refine the conditions for pectin hydrolysis and maximize the yield of reducing compounds (RCs), two independent variables (defined separately for each hydrolysis method) were evaluated using a full 2 2 experimental design (rotational central composite design -RCCD), with three central points (level 0) and four axial points (levels ± α, where α = 1.4142), totaling 11 experiments. This experimental design model, it allows a greater comprehension of the parameters tested, minimizing the experiments number. The experiments were performed randomly, and the data were analyzed using Statistica 8.0 software (StatSoft, Dell Software, US), with a 95.0% confidence level. The experimental error was obtained from the mean and standard deviation of the central points. The software calculate an empirical model described by equation, through the experimental data, allowing prediction of the experimental value at any point within the study area.
In order to refine the conditions for pectin hydrolysis and maximize the yield of reducing compounds (RCs), two independent variables (defined separately for each hydrolysis method) were evaluated using a full 2 2 experimental design (rotational central composite design -RCCD), with three central points (level 0) and four axial points (levels ± α, where α = 1.4142), totaling 11 experiments. This experimental design model, it allows a greater comprehension of the parameters tested, minimizing the experiments number. The experiments were performed randomly, and the data were analyzed using Statistica 8.0 software (StatSoft, Dell Software, US), with a 95.0% confidence level. The experimental error was obtained from the mean and standard deviation of the central points. The software calculate an empirical model described by equation, through the experimental data, allowing prediction of the experimental value at any point within the study area.
3
Enzymatic Hydrolysis of Fruit Extracts
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Fruit extracts were adjusted to pH 4,0 with NaOH 50% (m/v), followed by addition of citrate buffer (25 mM) and submitted to enzymatic hydrolysis in triplicate using polygalacturonase (EC 3.2.1.15, Sigma®) in concentration of 10,0 UI/g of substrate, 300 rpm of agitation for 24 hours (Locatelli 2012). The flasks were incubated in a rotary shaker incubator at 50 ºC and pH 4,0 (optimal conditions of enzyme activity, according to the supplier -Sigma®). Samples of both fruit extracts were collected at the end process for analysis in triplicate to determination of reducing compounds (RC) by DNS method. The hydrolyzed extracts were adjusted to pH 7,0 with NaOH 50% (m/v), sterilized by autoclave for 15 minutes and used in formulation of culture media.
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