Can you give me some more details:
Which class of enzymes are you using (just the family if you would like to maintain confidentiality)?
What is the nature of your substrate(s) and product(s)?
How do you follow your reaction?
What do you want to measure, Km, Vmax, Kcat, efficiency? or some other parameters?
Is your protein pure or do you use a crude extract?
Thanks dear Amit but I think your protocol for the purified enzyme or known enzyme concentration but l will work with enzyme detection in the sample with known substrate so l have to make the standard curve with the product or the substrate most articles work with the product but I can not understand the calculation of the enzyme activity
Thanks dear Islam for your concern l will work with proteinases and peptidases enzymes from lactic acid producing bacteria ,l want to know their activity aganist chromogenic and flourogenic substrates ,so some articles did not mention the method for standard curve also some mention incubation for certain period other with time interval,when I made general search I found using para nitroaniline as product for the standard curve but the calculation of the enzyme activity still unclear for me especially the specific activity
I found this protocol but did not mention standard curve ??????
Chromogenic Method
In this method, the amount of p-nitroanilide (pNA) released from the
chromogenic peptide by the action of LAB proteinase is measured at 410 nm.
The method is as follows:
1. Grow the microorganism in 100 mL MRS–Ca at 30°C (mesophilic bacteria) or 37°C
(thermophilic bacteria) at the optical density at 600 nm of 1.5 (see Notes 3 and 6).
2. Harvest cells by centrifugation at 10,000g for 10 min at 4°C (see Note 7).
3. Wash twice with CaCl2–saline solution (see Note 8).
4. Resusped in 5 mL Tris buffer (50 mM, pH 7.8).
5. To 200 μL resuspended cells (enzyme solution), add 287.5 μL phosphate buffer
(0.2M, pH 7.0), 225 μL 5M NaCl, and 37.5 μL S-Ala (see Notes 9–11).
6. Mix gently and incubate at 30°C (mesophilic bacteria) or 37°C (thermophilic
bacteria) for 30 min.
7. Stop the reaction by the addition of 175 μL of 80% (v/v) acetic acid.
8. Centrifuge for 5 min at 13,000g.
9. Measure the release of pNA at 410 nm.
10. The concentration of pNA released can be calculated from the derived value of
molar absorption coefficient ( = 8.800/M/cm):
μM pNA = A410 F ×103
where A410 is the experimentally observed change of absorbance at 410 nm
using a 1-cm light path and F is the dilution factor corresponding to the assay
procedure.
One unit of enzyme is defined as the amount of enzyme required to release 1 μmol
pNA per minute under the conditions of the assay
Thanks dear Amit,
I already mentioned my enzymes it would be proteolytic enzymes,so if I understand you correctly I will make my standard curve with the reaction product (p nitroaniline) as I will use chromogenic peptides ,but the curve will make relation between the different concentrations of the product and absorbance or the concentration and time and how I will calculate the enzyme activity
Thanks too muck for you cooperation
It depends on what your study/ aim is ; in the present study you want to determine the enzyme activity it is better to observe the product formation so that the kinetic parameters will be very specific to substrate to product formation ratio corresponding to time., optimum transformation and peak production time. .
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