Laccase Protocols: Difference between revisions
Line 75: | Line 75: | ||
*DMP (2,6-dimethoxyphenol) | *DMP (2,6-dimethoxyphenol) | ||
*DMS (2,2 dimethyl succinate) | *DMS (2,2 dimethyl succinate) | ||
**Can also substitute with sodium malonate | |||
===Procedure=== | ===Procedure=== | ||
1. Prepare substrate solution: | 1. Prepare substrate solution: |
Revision as of 14:55, 10 October 2011
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Introduction
This protocols are used to screent for and assay laccase activity.
Bromophenol Blue Plate Screen
Materials
Bromophenol blue
Procedure
Notes
References
Guiacol Colony Screen
Materials
- Gum Guiac or Guiacol
- 95% Ethanol
Procedure
1. Dissolve either 17mg gum guaiac or 13mg guaiacol into 1ml of 100% ethanol.
- If your guaiacol is in liquid form then add 12μL to 1ml ethanol.
2. Drop one drops of this solution on a large colony (2 days old for bacteria).
3. Wait four hours and a laccase positive colony should turn purple.
4. Bacteria don't usually like ethanol, so now they're probably dead.
Notes
References
Lignin Plate Screen
Materials
- Plates containing:
- 20g/L agar
- 5g/L glucose
- 5g/L ammonium tartrate
- 1g/L malt extract
- 0.5g/L MgSO4 (heptahydrate)
- 0.1g/L NaCl
- 0.01g/L CaCl
- 0.01g/L FeCl3
- 0.01g/L kraft alkaline lignin
- 1mg/L thiamine
- Ferric Chloride Solution
- 10g/L FeCl3
- Potassium Ferric Cyanide Solution
- 10g/L K3[Fe(CN)6]
Procedure
- Streak bacteria onto the plates and incubate for 5 days.
- Mix equal parts Ferric Chloride Solution and Potassium Ferric Cyanide Solution.
- Flood plates with this wash solution.
- The was solution will stain the lignin green. Non-green halos around colonies indicate lignin degradation.
Notes
References
Sundman and Nase. 1974. A simple plate test for direct visualization for biological lignin degradation. Papper och Tra 2
ABTS Plate Screen
Materials
- ABTS 20mM (10mg/mL) stock solution
- CuSO4 100mM (25mg/mL) stock solution
Procedure
1. Make a liter of your favorite agar growth medium and autoclave.
2. After cooling to 65°C add:
- 10ml/ABTS (0.2 mM)
- 1mL CuSO4
3. Swirl to mix and pour plates.
4. Once plates are hard:
- Drop 5μL of overnight culture onto the plate (when this dries it will form a little circle).
- Culture plate 24-48 hours
- Observe green halos around ABTS oxidizing cultures.
Notes
References
- Soden et al. (2002) Molecular cloning of a laccase isozyme gene from Pleurotus sajor-caju and expression in the heterologous Pichia pastoris host. Microbiology 148 (2002), 4003-4014
DMP Assay
Materials
- DMP (2,6-dimethoxyphenol)
- DMS (2,2 dimethyl succinate)
- Can also substitute with sodium malonate
Procedure
1. Prepare substrate solution:
- 4mM DMP (620mg/L)
- 200mM DMS (29.2g/L)
2. Centrifuge 1ml of overnight culture.
3. Place 750μL of cleared supernatant in a microcentrifuge tube.
4. Add 250μL of substrate solution.
5. Incubate for 10 minutes.
6. Measure absorbance at 468nm every minute.
- An increae in absorbance is positive for laccase activity.
- Extinction coefficienct = = 49.6 AU/mM*cm
Notes
References
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