Hello all, I am working on one of the flavour molecule producing it through biotechnological route. I have two recombinant genes that are involved in the enzymatic reaction that are transformed in E.coli Bl21. While setting the biotransformation reaction with the substrate along with the whole E.coli cells and lysed cells, whole cells are giving me better conversions as compared to lysed cells. My question is when i am using the fresh whole cells its conversion is less as compared to the cells resuspended in phosphate buffer ph7 and then stored at 4C (refrigerator) for one two weeks. why?
Whole cells have reserves that continuously supplement cofactors, and also once you lyse cells everything is out, and some proteins might have conformation change which can reduce the activity. If you are getting good results using whole cells, then it is better to stick with it.
Every strain have viability period. I dont see any reason to keep your cells for long in the 4 C.
If I want to do this experiment, I will take small sample from a week old media and throw it into fresh media in the afternoon and my fresh cells will be ready for an experiment tomorrow morning, and I will also have some cells in media for next
thanks for your reply but my problem is fresh whole cells are not showing better results as compared to whole cells resuspended in buffer for longer periods in 4C. Its like afterone or two week they become viscous (lysed) by seeing their physiology and then they start giving best results. I have to wait for that much time for getting the results are results are not reproducible.
I had the same experience with whole cell vs. cell lysate and I guess it might depend on the stability of your cofactor which might be more destroyed (for example Fe-S-cluster are not very stable) and might also depend on the pH value in the cell. Another possibility is that your substrate is some how being activated which only takes place in the whole cell system.
Are you working in media or in buffer for your fresh cell conversion experiment? Sounded like you work in media which might not have the best conditions for your reaction. Have you checked whether you have the some OD in these experiments to make them comparable or not?
And why are you storing the cells for 1 week at 4 °C? Are ypu already starting the first freezing/defreezing cycle here? (so that you have also kind of a lyse)
Thanks for your reply Anja Himmelstein .
1. My biotransformation reactions are in buffer phosphate buffer pH 7.So after growing E.coli cells, harvested and resuspended in OD50 phosphate bufferph 7 and stored in 4C.
2. Yes everytime i am using constant OD. So If i am using 10OD of fresh E.coli cells they gave me 20% substrate conversion and after keeping cells in 4 C for longer periods even 5 OD E.coli cells give me 100% conversion.
Have you performed a background reaction experiment using an empty vector transformed in your BL21 and perform the same reaction as you would do with your proteins?
What anja suggested is quite important, you should always include negative as well as positive control
Is it possible that after two weeks it is partially lysated?
In this case, it could avoid difusional problems of substrates and products (caused by cell wall), leading to a higher reaction rates (compared to whole cells).
could be Jesús Fernández-Lucas but why then cell lysates/ cell free extrates are not giving better conversions.
Have you measured the final DO at 600-660?
Do you have a bacterial growth curve?
Are you sure you are always using the same amount of cells?
Have you measured the final DO at 600-660?
yes i always take final OD of the culture.
Are you sure you are always using the same amount of cells?
yes i always use same amount of OD.
Hi Archana,
While working some time ago with a P450 for the hydroxylation of complex and hydrophobic compounds, I observed the same effects: Whole cells gave much better results than cell lysates. I think that whole cells are more robust than lysates, and many metabolic pathways will still be functional for certain time in the former case. The problem with lysed cells is that they are not longer alive and many proteins/pathways are activated/desactivated under those conditions. This is why the efficiency is probably better using living cells. It also depends on what you add to your biotransformation. In my case, we would always over-express our target enzymes, wash our cells and directly freeze the samples at minus 20 Celsius. Because we processed many 96-well plates in parallel, we generally froze the plates and the next day or later, we would take out the plates from the freezer, resuspend them in the reaction buffer (phosphate buffer pH 7.4 and NADPH recycling system), followed by a freeze/thaw cycle using liquid nitrogen to semi-permeabilize the membrane to enhance substrate and NADPH transport into the cells (the latter was used in a recycling system with glucose and glucose dehydrogenase). Upon thawing the cells, substrate was added and the reaction started. This was the best protocol we could use to maximize protein activity.
I hope that this helps!
Carlos
hi
i dont know this answer help you or not, but according to my research , the enzymatic depend on concentration of substrate and enzyme , while temperature decreases , because cell is following osmotic pressure , for low temperature the concentration of substrate and enzyme increases , maybe your answer lie down in temperature dependent .
Acevedo-Rocha said something about cell permeability. Did you investigate whether your cells become more permeable after storage than when fresh?