For many days enzyme experiment, fresh medium of enzyme need to be replaced regularly. At what time interval the medium should be replaced by new to keep activity of enzymes?
That entirely depends on the specific system you are studying, in particular on the stability of your enzyme in the context of your experimental set-up.
With proteases, the main problem is autolysis. For the individual proteases, you might get information about their half-life under assay conditions from the provider. If you use combinations of proteases, you may need to do your own analysis on how fast activity is decaying.
Protease Type XIV is a mixture of at least three caseinolytic activities and one aminopeptidase activity. The caseinolytic enzymes were named as Streptomyces griseus Protease A, Streptomyces griseus Protease B and Streptomyces griseus Trypsin.
Protease Type XIV is highly stable in the pH range 5.0 to 9.0, but fairly unstable below pH 4 and above pH 10.1 The neutral components in the enzyme mixture are stable at pH 5-9 with calcium present; the alkaline components are stable over pH 3-9, with optimal activity at pH 9-10. The aminopeptidase and carboxypeptidase components are stable at pH 5-8, in the presence of calcium ion.2 The optimum activity will be at pH 7-8.2
The product dissolves in 0.01 M sodium acetate with 0.005 M calcium acetate at pH 7.5 at 37°C; at 0.2 mg/mL the clear solution ranges from colorless to light tan. Calcium ion is recommended for protection from autolysis. The activity of a dilute enzyme solution containing 0.01 to 0.1 M calcium ion was stable over 24 hours at neutral pH at 2-8°C. Protease Type XIV is stable at 4°C for at least six months. Stock solutions of 5 to 20 mg/mL in water are usually stored at 20°C.2
(from https://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/protease-type-xiv.html)
Normally, instructions to dissolve the lyophilized powder should be followed, as they have been optimized to efficiently dissolve the powder and to create a stable stock solution for storage, as you usually do not want to start from the lyophilized powder every time you need a new aliquot of the protease to your sample. In addition, some proteins dissolve very poorly neutral pH. E.g. alpha-Chymotrypsin is to be dissolved in dilute HCL (1mM), at a pH at which it is not active, Protease Type XIV in the presence of Ca2+ to inhibit autolysis
Proteinase K
Solubility: 1 mg/mL (aqueous) Storage: Store lyophilized powders at –20 °C. Store solutions at 4 °C. Stability: Proteinase K solutions are stable over a broad pH range (4.0-12.5, optimum pH 8.0) and temperature range (25 to 65 °C). Lyophilized powders are stable for 2 years when stored at –20 °C. pH 8.0 proteinase K solutions are stable for at least 12 months at 4 °C
https://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/proteinase-k.html
The recommended working concentration of Proteinase K is 0.05 to 1 mg/mL. The activity of the enzyme is stimulated by 0.2 to 1% SDS or by 1 to 4 M urea • Ca2+ protects Proteinase K against autolysis, increases the thermal stability, and has a regulatory function for the substrate binding site of Proteinase K
https://www.thermofisher.com/order/catalog/product/EO0491?s_kwcid=AL!3652!3!352479762637!p!!g!!proteinase%20k&ef_id=CjwKCAjw8NfrBRA7EiwAfiVJpaORVH-OF-HLWtsF-AG8aKjNGXkuRyvyx3dmKT-v1eYsuNDppKAnAhoCYNMQAvD_BwE:G:s&s_kwcid=AL!3652!3!352479762637!p!!g!!proteinase%20k&gclid=CjwKCAjw8NfrBRA7EiwAfiVJpaORVH-OF-HLWtsF-AG8aKjNGXkuRyvyx3dmKT-v1eYsuNDppKAnAhoCYNMQAvD_BwE
(20 mg/ml) Purchase as a lyophilized powder and dissolve at a concentration of 20 mg/ml in sterile 50 mM Tris (pH 8.0), 1.5 mM calcium acetate. Divide the stock solution into small aliquots and store at -20°C. Each aliquot can be thawed and refrozen several times but should then be discarded. Unlike much cruder preparations of protease (e.g., pronase), proteinase K need not be self-digested before use.
http://cshprotocols.cshlp.org/content/2006/1/pdb.rec8066.full?text_only=true
In general, to find information
a) read the product sheet
b) use Google or other search engine
c) send an e-mail to the support address of the manufacturer of the product.
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