Feruloyl Esterase Protocols

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===Notes===
===Notes===
-
*Donaghy et al. (1998) added the ethyl ferulate solution directly to the media plates at 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
+
*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.
*Agarose instead of agar is better too for top agar.
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===Notes===
===Notes===
==Spectrophotometric Assay==
==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 340nm.
+
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===
===Materials===
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*50mM sodium-phosphate buffer (100ml pH=7, 0.271g monobasic, 0.433g dibasic)   
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*100mM sodium-phosphate buffer (pH = 6.5)   
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*Ethyl-ferulate solution (50mg/mL in dimethyl-formamide)
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*Ethyl-ferulate solution (10mg/mL in dimethyl-formamide)
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*MOPS Buffer
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*Ferulic-acid solution (8.7mg/mL in dimethyl-formamide)
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===Procedure===
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**To be a molar equivalent to the EF solution.
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The amount of substrate buffer is enough to do 10 samples (multiply accordingly).
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1. Make substrate buffer by combining:
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::*5ml MOPS Buffer
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::*3ul ethyl-ferulate solution
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2. Centrifuge 500ul of culture.
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3. Add 100ul of supernatant to 400ul of Sodium phosphate buffer.
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-
4. Add 200ul of buffered supernatant to 400ul substrate solution.
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5. Incubate for 30 mins in 37 degree water bath.
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6. Stop reaction by 3 mins in 99 degree water bath.
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7. Measure absorbance in UV cuvette at 340nm.
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===Notes===
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===Method===
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*Don't forget to include a blank control (no enzyme)  
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:1. Aliquot 800 μL of sodium phosphate buffer into 1.5 mL centrifuge tubes
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*It's also a good idea to include a positive control (no substrate just ferulic acid)
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::*Three tubes for each culture to be assayed (label them)
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::*For each culture there will be a positive (ethyl-ferulate, incubated), negative (ferulic-acid, incubated), and control (ethyl-ferulate, killed).
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:2. Centrifuge 1 mL of each culture to be assayed.<br>
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: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.
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:5. Add 15μL of ferulic acid solution (8.7 mg/mL) to the negative tube.
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:6. Put the control tube in the 99°C degree water bath for 3 minutes.<br>
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:7. Put the positive and negative tubes in the 37°C water bath for 2 hours.<br>
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:8. After two hours stop the reaction by 3 mins in 99 degree water bath.<br>
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:9. Measure absorbance in UV cuvette at 338nm.<br>
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===Method===
 
===Notes===
===Notes===
==References==
==References==
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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>
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*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>
-
 
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*Mastihuba, V., L. Kremnicky, et al. (2002). "A spectrophotometric assay for feruloyl esterases." Analytical Biochemistry 309(1): 96-101.<br>
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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>
-
 
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*Ralet et al.,1994
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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>
+
*Yue et al., 2009
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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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