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
- Prepare 300ml of MRS media.
- Autoclave the media for 20 minutes on the liquid cycle.
- Let the autoclaved media cool in a 65°C water bath for 1 hour.
- Add 300μL ethyl ferulate to 2.7ml dimethylformamide (i.e. 10% v/v).
- Once the media has cooled to a reasonable temperature
- Add antibiotic as appropriate (This is if you're using a plasmid).
- Add the 3ml of ethyl ferulate solution and swirl.
- Pour Immediately.
- The ethyl ferulate will nor really dissolve in the media but will look cloudy.
- If it helps you canjust add 150μL of the solution to each plate. When you pour the media and swirl it will disperse the EF solution.
Assay
Materials
Method
Notes
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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