Laccase Protocols: Difference between revisions
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==Introduction== | ==Introduction== | ||
This protocols are used to screent for and assay laccase activity. | This protocols are used to screent for and assay laccase activity. | ||
==ABTS Plate Screen== | |||
===Materials=== | |||
*ABTS 20mM (10mg/mL) stock solution | |||
*CuSO<sub>4</sub> 100mM (25mg/mL) stock solution | |||
===Procedure=== | |||
1. Make a liter of your favorite agar growth medium and autoclave.<br> | |||
2. After cooling to 65°C add: | |||
:*10ml/ABTS (0.2 mM) | |||
:*1mL CuSO<sub>4</sub> | |||
3. Swirl to mix and pour plates. <br> | |||
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 | |||
==Bromophenol Blue Plate Screen== | ==Bromophenol Blue Plate Screen== | ||
===Materials=== | ===Materials=== | ||
*Agar medium of your choice. | *Agar medium of your choice. | ||
*Bromophenol blue | *Bromophenol blue | ||
===Procedure=== | ===Procedure=== | ||
#Supplement the agar medium with 0.2g/L bromophenol blue. | #Supplement the agar medium with 0.2g/L bromophenol blue. | ||
Line 14: | Line 31: | ||
#Culture microorganism on plates | #Culture microorganism on plates | ||
#Observe clear halos around laccase producting colonies. | #Observe clear halos around laccase producting colonies. | ||
===Notes=== | |||
===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.<br> | |||
3. Place 750μL of cleared supernatant in a microcentrifuge tube.<br> | |||
4. Add 250μL of substrate solution.<br> | |||
5. Incubate for 10 minutes.<br> | |||
6. Measure absorbance at 468nm every minute. | |||
:*An increae in absorbance is positive for laccase activity. | |||
:*Extinction coefficienct = = 49.6 AU/mM*cm | |||
===Notes=== | ===Notes=== | ||
===References=== | ===References=== | ||
==Guiacol | |||
==Guiacol Screen== | |||
===Materials=== | ===Materials=== | ||
* | *Guiacol (or gum guaiac) | ||
* | *Ethanol | ||
*Agar medium | |||
===Procedure=== | ===Plate screen Procedure=== | ||
1. Add 0.01% guiaiacol to agar medium (100μL/L).<br> | |||
2. Autoclave and pour plates.<br> | |||
3. Culture microorganisms on plates.<br> | |||
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. | 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. | :*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). <br> | 2. Drop one drops of this solution on a large colony (2 days old for bacteria). <br> | ||
3. Wait four hours and a laccase positive colony should turn | 3. Wait four hours and a laccase positive colony should turn orange/brown. <br> | ||
4. Bacteria don't usually like ethanol, so now they're probably dead. <br> | 4. Bacteria don't usually like ethanol, so now they're probably dead. <br> | ||
===Notes=== | ===Notes=== | ||
*These resutls can be difficult to observe if the medium is already brownish. | |||
===References=== | ===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== | ==Lignin Plate Screen== | ||
Line 49: | Line 93: | ||
*'''Potassium Ferric Cyanide Solution''' | *'''Potassium Ferric Cyanide Solution''' | ||
**10g/L K<sub>3</sub>[Fe(CN)<sub>6</sub>] | **10g/L K<sub>3</sub>[Fe(CN)<sub>6</sub>] | ||
===Procedure=== | ===Procedure=== | ||
#Streak bacteria onto the plates and incubate for 5 days. | #Streak bacteria onto the plates and incubate for 5 days. | ||
Line 57: | Line 100: | ||
===Notes=== | ===Notes=== | ||
*Some protocols bump up the lignin concentration to 0.25g/L. | *Some protocols bump up the lignin concentration to 0.25g/L. | ||
===References=== | ===References=== | ||
*Sundman and Nase. 1974. '''A simple plate test for direct visualization for biological lignin degradation.''' ''Papper och Tra'' 2 | *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 | *Tekere et al. 2001. '''Growth, dye degradation and ligninolytic activity studies on Zimbabwean white rot fungi'''. ''Enzyme and Microbial Technology'' 28 (2001) 420–426 | ||
Back to [[Richard_Lab:protocols | Protocols]] | Back to [[Richard_Lab:protocols | Protocols]] |
Latest revision as of 19:57, 10 October 2011
Back to 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.
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
Bromophenol Blue Plate Screen
Materials
- Agar medium of your choice.
- Bromophenol blue
Procedure
- Supplement the agar medium with 0.2g/L bromophenol blue.
- Autoclave and pour plates.
- Culture microorganism on plates
- Observe clear halos around laccase producting colonies.
Notes
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
Notes
References
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
- 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
- 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
Back to Protocols