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 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
• 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 340nm.

Materials

• 50mM sodium-phosphate buffer (use 0.135g monobasic and 0.217g dibasic to make 50ml of buffer pH = 7)
• Ethyl-ferulate solution (50mg/mL in dimethyl-formamide)
• 100mM MOPS Buffer (use 1g MOPS to make 50ml of buffer pH=6)

Method

The amount of substrate buffer is enough to do 10 samples (multiply accordingly).

1. Make substrate buffer by combining:

• 5ml MOPS Buffer
• 3ul ethyl-ferulate solution

2. Centrifuge 500ul of culture.

3. Add 100ul of supernatant to 400ul of Sodium phosphate buffer.

4. Add 200ul of buffered supernatant to 400ul substrate solution.

5. Incubate for 30 mins in 37 degree water bath.

6. Stop reaction by 3 mins in 99 degree water bath.

7. Measure absorbance in UV cuvette at 340nm.

Notes

• Don't forget to include a blank control (no enzyme)
• It's also a good idea to include a positive control (no substrate just ferulic acid)

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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