Currently I'm doing research on production of hydrolytic enzymes via SFF. Does anyone know the best way to store crude enzyme while maintaining its freshness in term of enzyme activity. And how long can it maintain?
First point: it is impossible to generalise and you need to play around with conditions to find out what your enzyme likes and does not like!
Second point, although it is a bit boring, that initial exploration will give rich rewards and could save you much grief. People often persist with conditions in which their enzyme is terribly unstable when a few experiments could eliminate the problem.
Third, you haven't told us what kind of hydrolytic enzyme. If it is a proteinase then this is the toughest challenge, because, given enough time a proteinase will digest itself. Even if your enzyme is not a proteinase, a crude extract is bound to contain protepytic activity and so a wise precaution is to use a cocktail of proteinase inhibitors. There are cocktails commercially available to take account of the fact that a proteinase might be a serine proteinase, a cysteine proteinase, a metalloproteinase, an aspartate proteinase.
Fourth you should try some simple experiments to explore pH, temperature and choice of buffer substance in terms of how they affect stability over time. Sample and assay e.g. every hour and you will soon find out:
if it is a good idea to keep on ice (some enzymes are actually cold-labile!);
if the enzyme is perhaps stable in Tris but not in phosphate, or vice versa;
if the enzyme is really happy at pH6 but terribly unhappy at pH8;
Finally, if you find that after these preliminary basic experiments you still have a problem, there a number of well-tried additives to explore - glycerol, bovine serum albumin, sucrose, trehalose, PEG, salt etc.
Above all, remember that each protein is unique and what works for one protein may be quite different from what works for the next.
TQ Dr. Engel for the information. My research focus on the production of xylanase, protease and pectinase. Currently I'm thinking of store these enzyme in -20 degree Celsius which I believe its the common practice of crude enzyme storage.
anyone have experience on this?
I agree, as Dr. Engel said pH, Temperature, storage conditions and all need to be characterized for your crude enzymes.
Dear,
I think it is better to freeze-dry the enzyme for storage.
Freeze-drying causes enzyme get dehydrated, and you would be able to keep it into the room condition.
As Alireza says, freeze drying is usually a very good way of maintaining activity in the long term. But of course it is not as simple as just freezing - not everyone has access to a good freeze drier. As Nizzal says, for long-term storage, freezing at -20C is the commonest procedure. You can very easily test this by taking a small sample, freezing overnight and next day thawing and assaying. Any loss of activity that occurs will almost certainly happen during the actual freezing and thawing process and not once the solution is securely frozen. Once again, each protein is individual and so it is much better to test rather than assuming that because the method is OK for most proteins it is bound to be OK for yours! The same is true of another favoured procedure, storing at 4C a suspension of protein precipitated in ammonium sulphate. Again OK for many proteins but still not every protein likes ammonium sulphate.
I'm working about protease extraction from ecologic process and my question is: what is the best method (economic, quality and keeping best enzymatic activity) to store and stabilize the crude extract (crude ou partially purified) : zeodratation, atomisation, microencapsulation, freeze-drying, what else ? Thanks a lot
Hello Nizzal Syafiq. Yes i kept my crude enzyme (cold active endoglucanase) extracted by using citrate buffer 5.1pH at -20 degree for 2 months and after that I ran that for SDS PAGE and I got good results and also have above 95-98% relative activity.
we are generally keeping in incubator at temp 20degree alone to avoid infection
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