Xylanase Protocols

From OpenWetWare

Revision as of 15:06, 22 August 2011 by Michael A. Speer (Talk | contribs)
Jump to: navigation, search

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

  • Arsenic Acid
  • Copper Sulfate
  • Molybdic Acid
  • Sodium Acetate
  • Sodium Bicarbonate
  • Sodium Carbonate
  • Sodium Potassium Tartrate
  • Sodium Sulfate
  • Sulfuric Acid
  • Xylan (purified)
  • Xylose (purified)

Preparing the Reagents

If you prepare the amounts listed you will be able to run 10 samples (not including the standard curve)

Substrate Solution

1. Dissolve the following in 10ml of water:
  • 82mg Sodium Acetate
  • 0.2g Xylan
2. Bring the volume up to 20ml by adding water.

Copper Solution

1. Dissolve the following in 10ml of water:
  • 80mg Copper Sulfate
  • 0.32g Sodium Bicarbonate
  • 0.48g Sodium Carbonate
  • 0.25g Sodium Potassium Tartrate
  • 3.7g Sodium Sulfate
2. Bring the volume up to 20ml by adding water.

Acid Solution

1. Dissolve the following in 10ml of water:
  • 54mg Arsenic Acid
  • 0.13g Molybdic Acid
  • 1.48 Sulfuric Acid
2. Bring the volume up to 20ml by adding water.

Procedure

1. Swirl standards and blank sample and bring to 30°C.
2. Add 100ul of water to the blank sample, swirl, and incubate for 10 minutes at 30°C.
3. Add 2ml of copper solution to each standard and blank sample and swirl each solution.
4. Place a marble over each tube and place each tube in a boiling water bath for 10 minutes.
5. Allow tubes to cool to room temperature.
6. Add 2ml of acid solution to each tube and vortex until all precipitate is dissolved and foaming has stopped. Centrifuge tubes.
7. Transfer solutions to cuvettes and measure the absorbance at 540nm.

Standard Curve

Prepare the following samples:
Blank sample : 1.90 ml of xylan substrate
Standard 1 : 20ul of xylan standard and 1.88ml of water and 100ul of MRS
Standard 2 : 50ul of xylan standard and 1.85ml of water and 100 ul of MRS
Standard 3 : 70ul of xylan standard and 1.83ml of water and 100 ul of MRS
Standard 4 : 100ul of xylan standard and 1.80ml of water and 100 ul of MRS

Samples

Add 100ul of cell culture (in MRS) to 1.9ml of xylan substrate.
Personal tools