Feruloyl Esterase Protocols

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Introduction

These are methods to screen for and assay Ferulic-acid Esterase activity. While the screening method outlined here is specific for Lactobacillus 'species' it can be modified for other species by changing the media type to one appropriate for that species.

Plate Screen

Materials

• Ethyl ferulate
• Dimethyl formamide
• MRS agar
  • 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.
• Petri plates

Method

1. Prepare 300ml of MRS media.
2. Autoclave the media for 20 minutes on the liquid cycle.
3. Let the autoclaved media cool in a 65°C water bath for 1 hour.
4. Make ethyl ferulate solution (300mg into 3ml dimethylformamide).
5. 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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