Feruloyl Esterase Protocols

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(Method)
(Method)
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#Microwave the agar mix until the agar is melted and put in 60°C water bath.  
#Microwave the agar mix until the agar is melted and put in 60°C water bath.  
#Once the media has been in the water bath for 15-20 mins:
#Once the media has been in the water bath for 15-20 mins:
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##Add the 3ml of ethyl ferulate solution and swirl to disperse (NO BUBBLES!).
+
##Add the 30μL of ethyl ferulate solution (for every ml of top agar) and swirl to disperse (NO BUBBLES!).
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##Pour Immediately.
+
:::*The ethyl ferulate will not really dissolve in the agar but should look cloudy.
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:::*The ethyl ferulate will not really dissolve in the media but will look cloudy.
+
##Pour onto grown colonies immediately.
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:::*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).
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#Incubate for `4 hours
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#If a clear halo forms around the colony in the top agar then it's positive for FAE!!!
==Assay==
==Assay==

Revision as of 00:06, 9 March 2011

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Contents

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. 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 15-20 mins:
    1. Add the 30μL of ethyl ferulate solution (for every ml of top agar) and swirl to disperse (NO BUBBLES!).
  • The ethyl ferulate will not really dissolve in the agar but should look cloudy.
    1. Pour onto grown colonies immediately.
  1. Incubate for `4 hours
  2. If a clear halo forms around the colony in the top agar then it's positive for FAE!!!

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

Notes

  • For the screen Donaghy et al. (1998) added the ethyl ferulate solution directly to the media plates at a concentration of 2mg/ml while Hassan and Pattat (2011) added it to the top agar at a stated concentration of 0.05mg/ml. We've found that the hassan and pattat concentration is too low to make the agar cloudy. But 0.5mg/ml works fine. -- Mike

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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