Hi everybody, somebody knows how can I obtain a protein extract of macerated fruit, maintaining the enzyme activity of some proteases present in the solution?
Do you mean preserving the enzyme activity ? By working on ice/water (0-4 C°) and studying a buffer composition containing salts (Ca++, Mg++ or others) possibly involved in protease activity ; 1° without EDTA
Hey a general precaution....don't add protease inhibitor cocktail or PMSF while following purification protocols.
u have to make sure your condition during extraction to be at 4degree celcius and below. Which included your extraction buffer. so, keep the buffer at cold room@refrigerated temperature a day before extraction.
and the most important think u cannot keep your enzyme at room temperature for long time
If you do not want to spend your time at 4°C , I suggest to work in a box with ice/water bath, it is
Thanks for all. I will try work in ice throughout the process, when i have news i tell everyone.
Dr Paola I'm in Brazil and here is also very hot ... I would say the whole year! i love it!
What you need here is to protect your protease from the action of protease inhibitors, that may cause a loss in activity of enzyme of your interest. To do that you need to do the following:
- Maintain the experiment on ice to avoid temperature above 4°C in all steps. This is foremost requirement as inhibitors are also a type of enzymes. Low temperature prevents the degrading action of these inhibitors.
- Use suitable buffer to maintain enzyme activity
- Make sure that you do not add any protease inhibitors to your extraction buffer.
- If you have preliminary idea about the type of protease you expect, incorporate suitable co-factors which stabilize the protease structure (and hence, the activity) in solution. Presence of co-factors improves enzyme activity.
Dhananjay i pretend obtain cysteine protease of fruit, as pineapple. You know same co-factor to stabilize the protease structure? I thank the help.
The activity of all cysteine proteases depends on a catalytic dyad consisting of cysteine and histidine. If you specifically talk of Bromelein activity, it is inhibited by Hg++, Ag+, Cu++, a-1-antitrypsin, estatin A&B, Iodoacetate, and activated by bisulfite salts, NaCN, H2S, Na2S, and benzoate.
Thank so much Dhananjay! i will do a fisrt assay on next week and after i´ll post my experience here.
My intence is work with Bromelein and Papain.
If you intent purify bromelain talk with prof. Hamilton Cabral from USP Ribeirao Preto (Website: www.fcfrp.usp.br/dcf/hcabral) I worke with him studing specificity of papain and bromelain on FRET peptides Abz-peptidyl-EDDnp. He purified bromelein from fruit and stem. Good luck.
Oi Wagner, não conhecia o trabalho do Dr. Hamilton vou olhar com a atenção. Obrigado pela dica!
How do you extract bromelein from Pineapple in stable form.What is the protocol to be followed and the conditions to be maintained.
I recomend you make small aliquotes of your enzyme to use during your experiment and keep only one aliquote on ice and the others in -20 ou less. Papain is very hard exemple of cysteine protease that the activity is not the same in the begining and the and of experiment if it take several hours. If the aliquote is high concentration you can take autoproteolisys e at low concentration you can take unfolding. I recomend too use a 20% of glycerol in the enzyme stock to stabilize them. You can put glycerol in the reactio0n buffer too (10%). If you verify the enzyme activity is down during experiment (not a linear curve but a hyperbolic one - either the substrate is ending or the enzyme is dying - check how long your experiment are linear) you can add triton-x100 or brij 35 in the reaction buffer (if you get a hyperbolic curve and the substrate is not the limiting factor), not in the aliquotes. But you will nedd check if this additives change your results. Make your experiment with and without the additives in the buffer first.
Cisteino proteases principalmente a papaína perde facilmente a atividade durante seu tempo de uso no período experimental. Se vc medir a atividade de sua enzima mantida mesmo no gelo muito provavelmente haverá redução de atividade, que pode ser devido à diluição ou autoproteólise, perda de estrutura ou agregação. Para aumentar sua estabilidade vc pode utilizar tamponamento com 20% de glicerol e/ou bridj 0,01 ou 0,02%. Além disso, como as cisteíno proteases frequentemente trabalham em pH mais ácido, um tampão menos ácido poderá minimizar o processo de autólise. Lotes velhos de proteases também podem tornar-se instáveis. Minimizo esses fatores nas minhas cisteíno proteases de tripanossomatídeos e catepsinas usando glicerol 20%. No tampão de ensaio, frequentemente acetato de sodio 100mM, pH 5,5, uso 100mM de NaCl (estabilização da força iônica que ajuda na estabilidade da velocidade) glicerol 20% com 0,02% de bridj obtendo maior tempo de leitura linear de velocidade. Mas recomendo fazer testes para suas proteases.
- Badges
- Science method