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.
- 1. Add the 20μL of ethyl ferulate solution for every ml of top agar (120μL per plate), and swirl lightly to disperse.
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
- 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
- 0.5MKPO4
- Add 20μL protein to 80μL substrate
- 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 = 7.3)
- 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
The amount of substrate buffer is enough to do 5 samples (multiply accordingly).
- 1. Make substrate buffer by combining:
- 5ml 100mM sodium phosphate buffer
- 15ul ethyl-ferulate solution (10mg/ml)
- 2. Make the ferulic acid blanks by combining the following:
- 5ml 100mM sodium phosphate buffer
- 15ul Ferulic Acid solution (8.7mg/ml)
- 3. Aliquot 1ml of these solution into 5 tubes for each solution.
- 4. LABEL THE TUBES. <---don't forget this!!!
- 5. Centrifuge 500ul of each culture to be assayed.
- 6. For each culture, add 200ul of supernatant to each substrate solution.
- This means every culture will have one ferulic acid tube, and one ethyl-ferulate tube.
- 5. Incubate for 1 hour 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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