GB/T 23874-2009 Determination of xylanase activity in feed additives - Spectrophotometric method
1 Scope
This standard specifies the determination of xylanase activity in feed additives using the spectrophotometric method.
This standard is applicable to xylanase products for feed additives, with a minimum detectable amount of 10.0 U/g.
2 Normative references
The following standards contain provisions which, through reference in this text, constitute provisions of this standard. For dated references, subsequent amendments (excluding corrections) to, or revisions, of any of these publications do not apply. However, parties to agreements based on this standard are encouraged to investigate the possibility of applying the most recent editions of the normative documents indicated below. For undated references, the latest edition of the normative document applies.
GB/T 6682 Water for analytical laboratory use - Specification and test methods
3 Principle
Xylanase can degrade xylan into oligosaccharides and monosaccharides. Under boiling water bath conditions, reducing oligosaccharides and monosaccharides can undergo a chromogenic reaction with 3,5-dinitrosalicylic acid (DNS) reagent. The color depth of the reaction solution is proportional to the amount of reducing sugars produced by enzymatic hydrolysis, and the amount of reducing sugars produced is proportional to the xylanase activity in the reaction solution. Therefore, the xylanase activity in the reaction solution can be calculated by determining the color intensity of the reaction solution using spectrophotometry.
Under conditions of 37°C and a pH of 5.50, the amount of enzyme required to release 1 μmol of reducing sugar per minute from a xylan solution with a concentration of 5 mg/mL is defined as one enzyme activity unit (U).
4 Reagents and solutions
Unless otherwise specified, all reagents used are of analytical grade, and water is Grade 2 water as specified in GB/T 6682.
4.1 Sodium hydroxide solution, 200 g/L
Weigh 20.0g of sodium hydroxide, dissolve in water, and dilute to 100 mL.
4.2 Acetic acid solution, 0.1 mol/L
Pipette 0.60 mL of glacial acetic acid, dissolve in water, and dilute to 100 mL.
4.3 Sodium acetate solution, 0.1 mol/L
Weigh 1.36g of sodium acetate trihydrate, dissolve in water, and dilute to 100 mL.
4.4 Acetic acid-sodium acetate buffer solution, 0.1 mol/L, pH 5.50
Weigh 23.14g of sodium acetate trihydrate, and add 1.70 mL of glacial acetic acid. Then, dissolve in water and dilute to 2 000 mL. Measure the pH of the solution. If the pH deviates from 5.50, adjust to pH 5.50 using acetic acid solution (4.2) or sodium acetate solution (4.3).
4.5 Xylose solution (C5H10O5), 10.0 mg/mL
Weigh 1.000g of anhydrous xylose, dissolve in acetic acid-sodium acetate buffer solution (4.4), and dilute to 100 mL.
4.6 Xylan solution, 100 mg/mL
Contents
Foreword i
1 Scope
2 Normative references
3 Principle
4 Reagents and solutions
5 Apparatus
6 Plotting of standard curve
7 Preparation of enzyme solution for reaction
8 Determination procedures
9 Calculation of enzyme activity of the specimen
10 Repeatability
Annex A (Informative) Correspondence between the sample enzyme activity and the recommended weighing amount
