Feruloyl Esterase Protocols

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(Method)
 
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**This means that you will have to make this media yourself and can't buy a premix.
**This means that you will have to make this media yourself and can't buy a premix.
*Water
*Water
-
*Agar
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*Agar or Agarose (agarose is preferred)
===Method===
===Method===
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#Grow colonies on agar plates of appropriate media until colonies reach a decent size.
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1. Grow colonies on agar plates of appropriate media until colonies reach a decent size.<br>
-
#For each plate to be screened, add 25-30mg agar to 6ml of water (i.e. if your screening 3 plates thats 80mg agar to 18ml water).
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2. For each plate to be screened, add 25-30mg agar to 6ml of water (i.e. if your screening 3 plates thats 80mg agar to 18ml water).<br>
-
#Microwave the agar mix until the agar is melted and put in 65°C water bath.  
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3. Microwave the agar mix until the agar is melted and put in 60°C water bath.<br>
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#Once the media has been in the water bath for 15-20 mins:
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4. Once the media has been in the water bath for 10 mins:
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##Add the 3ml of ethyl ferulate solution and swirl to disperse (NO BUBBLES!).
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::1. Add the 20μL of ethyl ferulate solution for every ml of top agar (120μL per plate), and swirl lightly to disperse.
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##Pour Immediately.
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:::*You want the ethyl ferulate to look cloudy in the agar so don't swirl too hard.
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:::*The ethyl ferulate will not really dissolve in the media but will look cloudy.
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:::*Bubbles = Enemy
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:::*If it helps you can just add 150μL of the EF solution to each plate.  When you pour the media and swirl, it will disperse the EF solution (this is based on using 15ml per plate).
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::2. Pour onto grown colonies immediately.
 +
5. Incubate for ~4 hours.<br>
 +
6. If a clear halo forms around the colony in the top agar then it's positive for FAE!!!<br>
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==Assay==
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===Notes===
 +
*Donaghy et al. (1998) added the ethyl ferulate solution directly to the media immediately before pouring the plates, and used a final concentration of 2mg/mL while Hassan and Pattat (2011) added it to the top agar at a stated concentration of 0.05mg/ml.  We've found that the hassan and pattat concentration is way too low to make the agar cloudy but 1mg/ml can work well in a pinch. -- Mike
 +
*Agarose instead of agar is better too for top agar.
 +
 
 +
==Nitrophenyl Ferulic Acid Assay==
===Materials===
===Materials===
*Protein desalting columns
*Protein desalting columns
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#Incubate for 30 mins at 37°C
#Incubate for 30 mins at 37°C
-
==Notes==
+
===Notes===
-
*For the screen Donaghy et al. (1998) added the ethyl ferulate solution directly to the media plates at a concentration of 2mg/ml while Hassan and Pattat (2011) added it to the top agar at a stated concentration of 0.05mg/ml.  We've found that the hassan and pattat concentration is too low to make the agar cloudy. But 0.5mg/ml works fine. -- Mike
+
==Spectrophotometric Assay==
 +
This method quantifies the release of free ferulic-acid from ethyl-ferulate or methyl-ferulate. It is dependent on their absorbant divergence at 338nm.
 +
===Materials===
 +
*100mM sodium-phosphate buffer (pH = 6.5)
 +
*Ethyl-ferulate solution (10mg/mL in dimethyl-formamide)
 +
*Ferulic-acid solution (8.7mg/mL in dimethyl-formamide)
 +
**To be a molar equivalent to the EF solution.
-
==References==
+
===Method===
-
Donaghy, J., P. F. Kelly, et al. (1998). "Detection of ferulic acid esterase production by Bacillus spp. and lactobacilli." Applied Microbiology and Biotechnology 50(2): 257-260.<br>
+
:1. Aliquot 800 μL of sodium phosphate buffer into 1.5 mL centrifuge tubes
 +
::*Three tubes for each culture to be assayed (label them)
 +
::*For each culture there will be a positive (ethyl-ferulate, incubated), negative (ferulic-acid, incubated), and control (ethyl-ferulate, killed).
 +
:2. Centrifuge 1 mL of each culture to be assayed.<br>
 +
:3. Add 200ul of the appropriate culture supernatant to the tubes containing 800 μL phosphate buffer.<br>
 +
:4. Add 15μL of ethyl ferulate solution (10 mg/mL) to the positive and control tubes.
 +
:5. Add 15μL of ferulic acid solution (8.7 mg/mL) to the negative tube.
 +
:6. Put the control tube in the 99°C degree water bath for 3 minutes.<br>
 +
:7. Put the positive and negative tubes in the 37°C water bath for 2 hours.<br>
 +
:8. After two hours stop the reaction by 3 mins in 99 degree water bath.<br>
 +
:9. Measure absorbance in UV cuvette at 338nm.<br>
-
Mastihuba, V., L. Kremnicky, et al. (2002). "A spectrophotometric assay for feruloyl esterases." Analytical Biochemistry 309(1): 96-101.<br>
+
===Notes===
-
Nsereko, V. L., B. K. Smiley, et al. (2008). "Influence of inoculating forage with lactic acid bacterial strains that produce ferulate esterase on ensilage and ruminal degradation of fiber." Animal Feed Science and Technology 145(1-4): 122-135.<br>
+
==References==
 +
*Donaghy, J., P. F. Kelly, et al. (1998). "Detection of ferulic acid esterase production by Bacillus spp. and lactobacilli." Applied Microbiology and Biotechnology 50(2): 257-260.<br>
 +
*Mastihuba, V., L. Kremnicky, et al. (2002). "A spectrophotometric assay for feruloyl esterases." Analytical Biochemistry 309(1): 96-101.<br>
 +
*Nsereko, V. L., B. K. Smiley, et al. (2008). "Influence of inoculating forage with lactic acid bacterial strains that produce ferulate esterase on ensilage and ruminal degradation of fiber." Animal Feed Science and Technology 145(1-4): 122-135.<br>
 +
*Ralet et al.,1994
 +
*Yue et al., 2009
Back to [[Richard_Lab:protocols | Protocols]]
Back to [[Richard_Lab:protocols | Protocols]]
 +
 +
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[[Category:Protocol]]
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[[Category:In vitro]]
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[[Category:Protein]]

Current revision

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Contents

Introduction

These are methods to screen for and assay Ferulic-acid Esterase activity.

Plate Screen

Materials

  • Ethyl ferulate solution (100mg/ml in dimethylformamide).
  • Agar plates of media appropriate to your microorganism.
    • If screening natural strains some find it helpful to eliminate glucose from the media to drive FAE secretion.
    • This means that you will have to make this media yourself and can't buy a premix.
  • Water
  • Agar or Agarose (agarose is preferred)

Method

1. Grow colonies on agar plates of appropriate media until colonies reach a decent size.
2. For each plate to be screened, add 25-30mg agar to 6ml of water (i.e. if your screening 3 plates thats 80mg agar to 18ml water).
3. Microwave the agar mix until the agar is melted and put in 60°C water bath.
4. Once the media has been in the water bath for 10 mins:

1. Add the 20μL of ethyl ferulate solution for every ml of top agar (120μL per plate), and swirl lightly to disperse.
  • You want the ethyl ferulate to look cloudy in the agar so don't swirl too hard.
  • Bubbles = Enemy
2. Pour onto grown colonies immediately.

5. Incubate for ~4 hours.
6. If a clear halo forms around the colony in the top agar then it's positive for FAE!!!

Notes

  • Donaghy et al. (1998) added the ethyl ferulate solution directly to the media immediately before pouring the plates, and used a final concentration of 2mg/mL while Hassan and Pattat (2011) added it to the top agar at a stated concentration of 0.05mg/ml. We've found that the hassan and pattat concentration is way too low to make the agar cloudy but 1mg/ml can work well in a pinch. -- Mike
  • Agarose instead of agar is better too for top agar.

Nitrophenyl Ferulic Acid Assay

Materials

  • Protein desalting columns
  • HEPES
  • sodium azide
  • Dnase
  • 4-nitrophenyl ferulic acid

Method

  1. Make Protein buffer
    1. 100mM hepes
    2. 10μg/mL sodium Azide
    3. 5μL/mL Dnase
  2. Concentrate cellular proteins from 1mL culture into 100μL buffer
  3. Make Substrate buffer
    1. 2.5mM 4-nitrophenyl ferulic acid
    2. 0.5MKPO4
  4. Add 20μL protein to 80μL substrate
  5. Incubate for 30 mins at 37°C

Notes

Spectrophotometric Assay

This method quantifies the release of free ferulic-acid from ethyl-ferulate or methyl-ferulate. It is dependent on their absorbant divergence at 338nm.

Materials

  • 100mM sodium-phosphate buffer (pH = 6.5)
  • Ethyl-ferulate solution (10mg/mL in dimethyl-formamide)
  • Ferulic-acid solution (8.7mg/mL in dimethyl-formamide)
    • To be a molar equivalent to the EF solution.

Method

1. Aliquot 800 μL of sodium phosphate buffer into 1.5 mL centrifuge tubes
  • Three tubes for each culture to be assayed (label them)
  • For each culture there will be a positive (ethyl-ferulate, incubated), negative (ferulic-acid, incubated), and control (ethyl-ferulate, killed).
2. Centrifuge 1 mL of each culture to be assayed.
3. Add 200ul of the appropriate culture supernatant to the tubes containing 800 μL phosphate buffer.
4. Add 15μL of ethyl ferulate solution (10 mg/mL) to the positive and control tubes.
5. Add 15μL of ferulic acid solution (8.7 mg/mL) to the negative tube.
6. Put the control tube in the 99°C degree water bath for 3 minutes.
7. Put the positive and negative tubes in the 37°C water bath for 2 hours.
8. After two hours stop the reaction by 3 mins in 99 degree water bath.
9. Measure absorbance in UV cuvette at 338nm.

Notes

References

  • Donaghy, J., P. F. Kelly, et al. (1998). "Detection of ferulic acid esterase production by Bacillus spp. and lactobacilli." Applied Microbiology and Biotechnology 50(2): 257-260.
  • Mastihuba, V., L. Kremnicky, et al. (2002). "A spectrophotometric assay for feruloyl esterases." Analytical Biochemistry 309(1): 96-101.
  • Nsereko, V. L., B. K. Smiley, et al. (2008). "Influence of inoculating forage with lactic acid bacterial strains that produce ferulate esterase on ensilage and ruminal degradation of fiber." Animal Feed Science and Technology 145(1-4): 122-135.
  • Ralet et al.,1994
  • Yue et al., 2009

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