Laccase Protocols

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Laccase Protocols

Introduction

This protocols are used to screent for and assay laccase activity.

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.

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

Bromophenol Blue Plate Screen

Materials

• Agar medium of your choice.
• Bromophenol blue

Procedure

1. Supplement the agar medium with 0.2g/L bromophenol blue.
2. Autoclave and pour plates.
3. Culture microorganism on plates
4. Observe clear halos around laccase producting colonies.

References

• Tekere et al. 2001. Growth, dye degradation and ligninolytic activity studies on Zimbabwean white rot fungi. Enzyme and Microbial Technology 28 (2001) 420–426

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

Guiacol Screen

Materials

• Guiacol (or gum guaiac)
• Ethanol
• Agar medium

Plate screen Procedure

1. Add 0.01% guiaiacol to agar medium (100μL/L).
2. Autoclave and pour plates.
3. Culture microorganisms on plates.
4. Observe orange/brown halos around laccase positive colonies.

Drop Screen 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 orange/brown.
4. Bacteria don't usually like ethanol, so now they're probably dead.

Notes

• These resutls can be difficult to observe if the medium is already brownish.

References

• Kiiskinen et al. 2004. Screening for novel laccase-producing microbes. Journal of Applied Microbiology 2004, 97, 640–646
• Lopez et al. 2005. Decolorization of industrial dyes by ligninolytic microorganisms isolated from composting environment. Enzyme and Microbial Technology 40 (2006) 42–45

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

1. Streak bacteria onto the plates and incubate for 5 days.
2. Mix equal parts Ferric Chloride Solution and Potassium Ferric Cyanide Solution.
3. Flood plates with this wash solution.
4. The was solution will stain the lignin green. Non-green halos around colonies indicate lignin degradation.

Notes

• Some protocols bump up the lignin concentration to 0.25g/L.

References

• Sundman and Nase. 1974. A simple plate test for direct visualization for biological lignin degradation. Papper och Tra 2
• Tekere et al. 2001. Growth, dye degradation and ligninolytic activity studies on Zimbabwean white rot fungi. Enzyme and Microbial Technology 28 (2001) 420–426

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