pectinase precipitation - how to precipitate (Feb/04/2008 )
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hi!
I would be happy if there is somebody help me. I just do my experiment, it's about pectinase purification from bacteria Anoxybacillus isolated from hot spring. This bacteria can produce pectinase with activity 1.1 U/ml. But, i got some problems when i precipitated this enzyme. I already tried using 3 precipitation methods, first, i tried using ammonium sulphate and divided become 4 fraction, and the result, i just got 20% recovery from all fractions. Second, i tried using ethanol precipitation, and it showed similar result. third, i tried using ultrafiltration with MWCO 10kDa, but the enzyme was leak and pass the membrane. I tried to use the membrane with MWCO 5 kDa. Finally i know that my enzyme's MW is between 5-10kDa. but the problem, i dont have membrane with MWCO 5 kDa for loading my crude enzyme in big scale. Could you please give me some suggestion how to precipitate/purify my enzyme? Thanks before.
hi!
I would be happy if there is somebody help me. I just do my experiment, it's about pectinase purification from bacteria Anoxybacillus isolated from hot spring. This bacteria can produce pectinase with activity 1.1 U/ml. But, i got some problems when i precipitated this enzyme. I already tried using 3 precipitation methods, first, i tried using ammonium sulphate and divided become 4 fraction, and the result, i just got 20% recovery from all fractions. Second, i tried using ethanol precipitation, and it showed similar result. third, i tried using ultrafiltration with MWCO 10kDa, but the enzyme was leak and pass the membrane. I tried to use the membrane with MWCO 5 kDa. Finally i know that my enzyme's MW is between 5-10kDa. but the problem, i dont have membrane with MWCO 5 kDa for loading my crude enzyme in big scale. Could you please give me some suggestion how to precipitate/purify my enzyme? Thanks before.
You could try size-exclusion chromatography... collect the fractions, test them and then concentrate, for example by freeze-drying and then dissolving the protein in a smaller volume...
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Thanks for your suggestion. But i still confused how to reduce the volume of my crude enzyme (CE)? because as i know, to load the CE in size exclusion chromatography, i need small amount of CE. Tomorow, i'll try using ethanol to precipitate and prolonge the equilibrate time after adding cold ethanol.