Laccase Protocols

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Laccase Protocols

Introduction

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

Bromophenol Blue Plate Screen

Materials

Bromophenol blue

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.

Lignin Plate Screen

Materials

• Alkaline Lignin
• Ferric Chloride
• Potassium ferric cyanide
• Apropriate agar growth medium

ABTS Assay

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 oxidising 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

DMP Assay

Materials

• DMP (2,6-dimethoxyphenol)
• DMS (2,2 dimethyl succinate)

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

References

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