Feruloyl Esterase Protocols
Back to Protocols
These are methods to screen for and assay Ferulic-acid Esterase activity.
- 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.
- Grow colonies on agar plates of appropriate media until colonies reach a decent size.
- 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).
- Microwave the agar mix until the agar is melted and put in 60°C water bath.
- Once the media has been in the water bath for 15-20 mins:
- Add the 30μL of ethyl ferulate solution (for every ml of top agar) and swirl to disperse (NO BUBBLES!).
- The ethyl ferulate will not really dissolve in the agar but should look cloudy.
- Pour onto grown colonies immediately.
- Incubate for `4 hours
- If a clear halo forms around the colony in the top agar then it's positive for FAE!!!
- Protein desalting columns
- sodium azide
- 4-nitrophenyl ferulic acid
- Make Protein buffer
- 100mM hepes
- 10μg/mL sodium Azide
- 5μL/mL Dnase
- Concentrate cellular proteins from 1mL culture into 100μL buffer
- Make Substrate buffer
- 2.5mM 4-nitrophenyl ferulic acid
- Add 20μL protein to 80μL substrate
- Incubate for 30 mins at 37°C
- 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
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.
Back to Protocols