Feruloyl Esterase Protocols

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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

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.

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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