Xylanase Protocols

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Contents

Introduction

These protocols are used to screen for and measure xylanase activity.

Plate Screen

This is a quick procedure to screen for xylanase activity.

Materials

  • 2.5g Xylan
  • 2.5g RBB (Remazol Brilliant Blue)
  • 60ml Water
  • 20ml Sodium Hydroxide Solution (1.5g in 20ml)
  • 20ml Sodium Acetate Solution (0.675g in 20ml)
  • 200ml 96% Ethanol
  • 1L Wash solution (660ml Ethanol, 330ml Water, 1.35g Sodium Acetate)
  • Acetone

Preparing the Dyed Xylan

  1. Add 2.5g RBB dye and 2.5g Xylan to 60 ml Water.
  2. Add 20ml Sodium Acetate solution to RBB-Xylan solution.
    1. Add dropwise over 5 minutes while stirring at room temp.
  3. After mixing add 20ml Sodium Hydroxide solution.
  4. Stir for 90 minutes at room temperature.
  5. Add two volumes of 96% Ethanol to precipitate the xylan-RBB.
  6. Filter using a vacuum flask and watman filter paper.
  7. Wash the precipitate sequentially with 1L of wash solution.
    1. The filtrate should now be colorless.
  8. Wash the precipitate with 100ml 75% Ethanol.
  9. Wash the precipitate with 50ml Acetone.
  10. Dry at room temp overnight.

Procedure

  1. Add 1% RBB-Xylan (weight per volume) to your agar media of choice after autoclaving.
  2. Pour the plates immediately being sure to swirl frequently.
  3. Drop 5μL of overnight culture onto the plates
  4. Incubate for two days
  5. Observe clearing zones around xylanase producing cultures.

Assay

This assay measures the release of reducing sugars (including xylose and arabinose) during the enzymatic treatment of xylan. If you have reducing sugars (e.g. glucose) in your media, it will also be detected, so you will always have to compare to a control culture with no xylanase.

Materials

  • Sodium Acetate
  • Purified Xylan
  • Purified Xylose
  • Copper Sulfate
  • Sodium Sulfate
  • Sodium Carbonate
  • Sodium Bicarbonate
  • Sodium Potassium Tartrate
  • Molybdic Acid
  • Arsenic Acid
  • Sulfuric Acid

Preparing the Reagents

=Substrate Solution

Copper Solution

Acid Solution

Procedure

Standard Curve

Samples

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