Hie can any one help me out with the step by step protocol for fractionating the polygalacturonase and amylase enzymes using ammonium sulphate present in my fermentation broth. My fermenting organism is a yeast belonging to Geotrichum species. I tried it once I found good amount of protein present at 80% fraction but it did not show any enzyme activity. My control OD value was more than my test enzyme value.
Hi, there
First, I think if you study the chapter recommended by Carlos, you can get good information about ammonium sulphate precipitation method.
Second, you can try solid precipitation instead of liquid sedimentation.
Try to increase the saturation in step manner for example 20, 40, 60, 80, and 95%. Use the Bradford method for measuring the protein content both in the precipitate and the supernatant. Try to dissolve the precipitate in the least amount of buffer.
Then you will find out which concentration is good for your work.
Good luck
hi, if your enzyme concentration is very low, you can have trouble to measure in ammonium sulfate precipitation. to alter this problem i think you should use Centrifugal Filter Units (Amicon by MerckMilipore). this can concentrate and you may easily measure the activity.
the other point is your activity procedure may have deficiencies if you precipitated but did not measure the acitivity. good luck with that.
Thank You Carlos and Karim. I'll try this.
Hie Berna Can I concentrate my fermented broth using vaccum evaporator ??
In my opinion first you have to concentrate the crude enzyme than you can try gradient precipitation using Ammonium sulphate.
In second way you can use Ultra filtration Technique using different cutoff membrane.
I agree with Dr. Berna Genç, it is better to use molecular cut-off membranes to concentrate the proteins/enzymes and based on pI, use chromatography resin/s to partially or completely purify the enzyme. Use of ammonium sulphate is OK, but in large scale, it is difficult to dispose or treat ammonium sulphate in the effluent treatment plant. With simple steps, you can purify the enzymes.
I also agree with Shah Ali Ul Qader on his suggestions on using ultrafiltration technique. In my opinion ultrafiltration (5 KD to 10 KD) would only concentrate the proteins and you can get rid of salts; remove the biomass from fermented broth either by centrifugation or by micro-filtration, concentrate the protein, and purify through chromatography resin. Use of ammonium sulphate is a tedious process, cannot be scaled up because of ETP problem.
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