Feruloyl Esterase Protocols: Difference between revisions
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===Method=== | ===Method=== | ||
#Grow colonies on agar plates of appropriate media until colonies reach a decent size | #Grow colonies on agar plates of appropriate media until colonies reach a decent size. | ||
#Once the media has | #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. | ||
#Microwave the agar mix until the agar is melted and put in 65°C water bath. | |||
#Once the media has been in the water bath for 15-20 mins: | |||
##Add the 3ml of ethyl ferulate solution and swirl to disperse (NO BUBBLES!). | ##Add the 3ml of ethyl ferulate solution and swirl to disperse (NO BUBBLES!). | ||
##Pour Immediately. | ##Pour Immediately. |
Revision as of 21:27, 8 March 2011
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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
- Grow colonies on agar plates of appropriate media until colonies reach a decent size.
- 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.
- Microwave the agar mix until the agar is melted and put in 65°C water bath.
- Once the media has been in the water bath for 15-20 mins:
- Add the 3ml of ethyl ferulate solution and swirl to disperse (NO BUBBLES!).
- 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
- 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
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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