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

Method

1. Grow colonies on agar plates of appropriate media until colonies reach a decent size
2. Once the media has cooled to a reasonable temperature:
   1. Add antibiotic as appropriate (This is if you're using a plasmid).
   2. Add the 3ml of ethyl ferulate solution and swirl to disperse (NO BUBBLES!).
   3. Pour Immediately.

• The ethyl ferulate will not really dissolve in the media but will look cloudy.
• If it helps you can just add 150μL of the EF solution to each plate. When you pour the media and swirl, it will disperse the EF solution (this is based on using 15ml per plate).

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

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.

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