When I store the laccase enzyme in freezing, i always have less activity than at the beginning, I would like to know how I can preserve it to not loss enzymatic activity . I would appreciate it if you could suggest how to keep it active for later determinations.
Hi there,
Freezing/thawing is denaturing for most of proteins therefore if you want to preserve enzyme activity you have to consider including a cryoprotectant like glycerol (at least 10%), flash freeze in liquid nitrogen in order to make the transition liquid to solid the shortest and storage at -80°C. One more parameter to look at is the protein concentration which has not to be too low.
Hello Maryen,
An enzyme is a biocatalyst -life stuff at the end, and it cannot escape to the second law of thermodynamics. Therefore, an enzyme will always face its unidirectional fate (loosing activity is one of the many tragic consequences). This is worsened by any type of processing (including natural ones). Freezing and thawing also aggravate the situation.
For preservation: refer to the bio MSDS
All the best
Thank you very much for yours suggestions, I will keep it in mind for upcoming conservation.
Hi, Maryen!
For lower enzyme degradation rate while freezing you could:
1) store you protein as several aliquotes to prevent freeze-thaw cycles.
2) do not dilute your protein too much - it'll degrade faster.
3) if possible, add glycerol (e.g. solutions with 40% glycerol remain liquid at -20C).
4) sometimes addition of BSA can help, especially when you work with low concentrations of your own protein, but it is less convinient than addition of glycerol.
5) addition of enzyme cofactors and other ligands could significantly increase its lifetime, but they may interfere with your assays.
Thank you vasily, I will try to have minor aliquots to not affect the enzyme after thawing, and i will add glycerol, which i didnt´proved before, I purified the laccase of liquid cultures of ligninolitic mushrooms, but I thought about not freezing the enzyme to see how it behaved at 4ºC and at the next day my enzyme preparation was gelled, but even this way, it had a high activity, do dou think that the preparation was very concentrated then or does that happen with many protein samples?
@Maryen Alberto It is interesting to know that after gelling the enzyme prepartion has higher activity. Please make sure that you have used same protein concentration.
Best
Dear Nadda, when I store the enzyme preparation at 4 degrees for several days, it often gels, its activity is not lost by full, what I wanted to say was that even in this way has great activity but not as much as my initial purified preparation, I just do not know why this happens, because the concentration is not as high as in other cases that gels for this reason.
Thank you very much for your appreciations.
Regards
Lyophilised protein usually has a good stability at -20°C, so if you have the possibility it could be worth a test.
Dear Anders:
I want to do the test to see how it turns out, because I had not done it before because I thought that its activity decreases a lot as freeze and freeze drying takes a pre-freezing step, that's why I had not done it, I'll try it a lot of thanks and as soon as I have results I will comment with you. regards
Dear Maryen,
Proteins are complex systems and their response to different factors can be difficult to predict. Generalizations can be made for several factors, but there are exceptions almost always. All the above suggestions are things that have been used to stabilize proteins when frozen and constitute good thing to try.
Now, freezing changes water structure and modifies the balance of forces in the hydrophobic effect. Those proteins with tendency to aggregate due to the sticking of hydrophobic patches, may do so faster at low temperatures. In addition, low temperatures slow down some chemical and physical processes, but solubility of gases in water increases at low temperature, and soluble oxygen with Copper from the enzyme may produce free radicals even at low temperature. An antioxidant is recommended, such as 2-mercaptoetanol.
Cryoprotectants and other additives as mentioned above may help:
Glycerol or BSA may help (mentioned by Vasily), the first, if used in high concentration stops the solution to freeze at -20, so this may help. Other common additives are sucrose, trehalose, ethyleneglycol, fructans (like achicoria soluble fructans) , or polyethyleneglycol.
Some enzymes tolerate low temperatures as ammonium sulphate suspensions and can be stored as such for long periods. I.e., you precipitate your enzyme centrifugate, resuspend the pellet in a small amount of the supernatant to prevent the pellet to dry, then store as suspension in the refrigerator, you may not have to use a freezer.
Some enzymes may require a particular pH, and this is usually very important; not necessarily the optimum for activity is optimum for stability. Using a pH close to the pI usually favours aggregation and leads to activity loss (not always though).
Best wishes,
Rogelio
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