Feruloyl Esterase Protocols

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Introduction
These are methods to screen for and assay Ferulic-acid Esterase activity.

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!!!

Materials

 * Protein desalting columns
 * HEPES
 * sodium azide
 * Dnase
 * 4-nitrophenyl ferulic acid

Method

 * 1) Make Protein buffer
 * 2) 100mM hepes
 * 3) 10μg/mL sodium Azide
 * 4) 5μL/mL Dnase
 * 5) Concentrate cellular proteins from 1mL culture into 100μL buffer
 * 6) Make Substrate buffer
 * 7) 2.5mM 4-nitrophenyl ferulic acid
 * 8) 0.5MKPO4
 * 9) Add 20μL protein to 80μL substrate
 * 10) Incubate for 30 mins at 37°C