Application and Principle
Application and Principle This procedure is used to determine Beta glucan activity of enzyme preparations derived from Aspergillus niger var. and Bacillus subtilis var. The assay is based on a 15-min hydrolysis of lichenin substrate at 40°and at pH 6.5. The increase in reducing power due to liberated reducing groups is measured by the neocuproine method.
Reagents and Solutions
Phosphate Buffer Dissolve 13.6 g of monobasic potassium phosphate in about 1900 mL of water, add 70% sodium hydroxide solution until the pH is 6.5 ± 0.05, then transfer the solution into a 2000-mL volumetric flask, dilute to volume with water, and mix.
Neocuproine Solution A Dissolve 40.0 g of anhydrous sodium carbonate, 16.0 g of glycine, and 450 mg of cupric sulfate pentahydrate in about 600 mL of water. Transfer the solution into a 1000-mL volumetric flask, dilute to volume with water, and mix.
Neocuproine Solution B Dissolve 600 mg of neocuproine hydrochloride in about 400 mL of water, transfer the solution into a 500-mL volumetric flask, dilute to volume with water, and mix. Discard when a yellow color develops.
Lichenin Substrate Grind 150 mg of lichenin (Sigma Chemical Co., Catalog No. L-6133, or equivalent) to a fine powder in a mortar, and dissolve it in about 50 mL of water at about 85°. After solution is complete (20 to 30 min), add 90 mg of sodium borohydride and continue heating below the boiling point for 1 h. Add 15 g of Amberlite MB-3, or an equivalent ion-exchange resin, and stir continuously for 30 min. Filter with the aid of a vacuum through Whatman No. 1 filter paper, or equivalent, in a Büchner funnel, and wash the paper with about 20 mL of water. Add 680 mg of monobasic potassium phosphate to the filtrate, and refilter through a 0.22-µm Millipore filter pad, or equivalent. Wash the pad with 10 mL of water, and adjust the pH of the filtrate to 6.5 ± 0.05 with 1 N sodium hydroxide or 1 N hydrochloric acid. Transfer the filtrate into a 100-mL volumetric flask, dilute to volume with water, and mix. Store at 2° to 4° for not more than 3 days.
Glucose Standard Solution Dissolve 36.0 mg of anhydrous dextrose in Phosphate Buffer in a 1000-mL volumetric flask, dilute to volume with water, and mix.
Test Preparation Prepare a solution from the enzyme preparation sample so that 1 mL of the final dilution will contain between 0.01 and 0.02 Beta glucan units. Weigh the sample, transfer it into a volumetric flask of appropriate size, dilute to volume with Phosphate Buffer, and mix.
Pipet 2 mL of Lichenin Substrate into each of four separate test tubes graduated at 25 mL, and heat the tubes in a water bath at 40°for 10 to 15 min to equilibrate.
After equilibration, add 1 mL of Phosphate Buffer to tube 1 (substrate blank), 1 mL of Glucose Standard Solution to tube 2 (glucose standard), 4 mL of Neocuproine Solution A and 1 mL of the Test Preparation to tube 3 (enzyme blank), and 1 mL of the Test Preparation to tube 4 (sample). Prepare a fifth tube for the buffer blank, and add 3 mL of Phosphate Buffer.
Incubate the five tubes at 40°for exactly 15 min, and then add 4 mL of Neocuproine Solution A to tubes 1, 2, 4, and 5. Add 4 mL of Neocuproine Solution B to all five tubes, and cap each with a suitably sized glass marble.
[CAUTION: Do not use rubber stoppers. ]
Heat the tubes in a vigorously boiling water bath for exactly 12 min to develop color, then cool to room temperature in cold water, and adjust the volume of each to 25 mL with water. Cap the tubes with Parafilm, or other suitable closure, and mix by inverting several times.
Determine the absorbance of each solution at 450 nm in 1-cm cells, with a suitable spectrophotometer, against the buffer blank in tube 5.
One Beta glucan unit (BGU) is defined as that quantity of enzyme that will liberate reducing sugar (as glucose equivalence) at a rate of 1 µmol/min under the conditions of the assay.
Calculate the activity of the enzyme preparation taken for analysis as follows:
BGU = [(A4 A3) × 36 × 106]/[(A2 A1) × 180 × 15 × µg sample]
in which A4 is the absorbance of the sample (tube 4), A3 is the absorbance of the enzyme blank (tube 3), A2 is the absorbance of the glucose standard (tube 2), A1 is the absorbance of the substrate blank (tube 1), 36 is the micrograms of glucose in the Glucose Standard Solution, 106 is the factor converting micrograms to grams, 180 is the weight of 1 µmol of glucose, and 15 is the reaction time, in minutes.
CERTIFICATE OF ANALYSIS
*The above information is a direct translation of the information provided to Newgen Biotech from the manufacturer of this product. This Certificate of Analysis should be used for informational purposes and is not intended as a substitute for strict quality control analysis by the purchaser of this product.
Packaging and storage
Solid food packaging bag, 25 kg / barrel.
Store in dry, cool and well ventilated places away from the sun, heat.
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