Determination, Preparation and Purification of Enzymes
All enzymes are proteins. The activities of enzymes enable all cells (plants and animals) to function. These activities result in constant changes in cells which are regulated by catalytic reactions and these reactions are also regulated by enzymes.
An important property of enzymes is that they act as catalysts to a reaction and that they affect only the reaction rate. A good knowledge of the activities of enzymes will help define the properties of cells. It is therefore necessary to study enzymes in vitro after extraction from cells.
Properties of Enzymes
Like all proteins, the enzymes are characterized by peculiar properties. They include the following;
1. Enzymes only act as catalysts to a reaction and they affect only the reaction rate and do not change the chemical composition of the reaction.
2. Enzymes have differing solubility in salt solutions.
3. Minute changes in the temperature of a reaction will significantly alter the reaction rate.
4. Extremely high temperatures can denature the enzymes.
5. Enzymes are highly sensitive to changes in pH.
6. Salts cause the denaturation of enzymes, especially heavy metal salts and irreversibly alter the structure of the enzymes except for the effects of ammonium sulfate which may be reversible.
Mode of Action of Enzymes
1. An enzyme works by binding to a given substrate such that the substrate undergoes the reaction more rapidly.
2. This reaction is a perfect fit. It means that there is a specific part of the enzyme structure that binds in a lock and key mode to a substrate.
3. The enzyme makes the substrate in an accurate alignment for it to react spontaneously or together with other substance(s).
4. Every reaction goes on usually by a random kinetic action of molecules running into each other and whenever these molecules align, they react faster.
5. In ideal conditions (when the pH, and temperature are optimal) and with the right concentration of enzyme, the reaction will proceed fastest provided the substrate concentration is adequate.
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Preparation of Enzymes
The success of the purification of enzymes begins at the extraction stage. Each stage in the extraction procedure should be closely monitored for enzyme activity.
Monitoring during the extraction process can be achieved in several manners which usually entail a measurement of the decrease in substrate, or the increase in product specific to the enzyme.
The product of the process of enzyme extraction is not a pure form of the target enzyme but a cocktail of the enzyme and numerous other proteins.
Extraction of Enzymes
The protocol for the extraction of the enzyme is as follows;
Choose the tissue from which the enzyme will be extracted.
Homogenize the tissue in a buffer (for example phosphate buffer) that will preserve the enzyme in its soluble state.
Centrifuge and collect the supernatant, this process will allow the enzyme to be separated from all insoluble proteins.
Repeat the step c (combining the supernatant and cold buffer).
Use the supernatant from step d as the source of the enzyme. The enzyme will be impure in the supernatant because no other proteins are present in the solution.
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Purification of Enzymes
Pure enzymes can be obtained from the impure enzyme extract. This is by the purification of the impure extract by salt precipitation. The sequential addition of varied salt concentrations such as ammonium sulfate solution to the extract will cause individual proteins to precipitate depending on their solubility.
However, the precipitate containing the target enzyme can be subjected to addition processing if absolute purity of the enzyme is required. This is in the form of dialysis, gel filtration, electrophoresis and column chromatography, and ion exchange chromatography to obtain the enzyme in the purest form possible. The pure enzyme could then be utilized for further analysis.
Protocol for Purification of Enzymes
Material required:
1. Ice bath.
2. Ammonium sulfate (finely ground).
3. Weighing scale.
4. Centrifuge.
Buffer (Potassium phosphate) Protocol:
1. Weigh the ammonium sulfate powder.
2. Add the ammonium sulfate powder slowly and carefully to the impure extract, constant stirring to ensure complete solubility.
3. Centrifuge the solution at the required speed (in rpm) for some time (in minutes) at a cold temperature (°C).
4. Different precipitation steps will need to be carried out for different enzymes by varying the salt concentration (45–80%) and precipitates will be collected.
An example is the purification of tyrosinase enzyme as stated below;
1. Weigh the finely grounded ammonium sulfate powder.
2. Add the ammonium sulfate powder slowly and carefully to the impure extract, constant stirring to ensure complete solubility.
3. Centrifuge the solution at 5000 rpm for 30 minutes at 4°C.
4. Vary the salt concentration between 45-80 percent and collect precipitate.
5. Dialyze the precipitate against 100 mM potassium phosphate buffer (pH 7.0) for 24 hours by changing the buffer thrice.
6. The enzyme is now ready for use for further studies.
In conclusion, the properties of enzymes allow them to be utilized in fast-tracking a reaction under ideal conditions. The ability of the enzymes to speed up the reaction rate can only be possible if the right concentration of enzyme is in the mixture, and ideal temperature and pH are maintained.
A successful enzyme recovery process depends on proper monitoring of the process at the extraction stage. It is also worth noting that the product of the enzyme extraction process is not a pure form of the target enzyme but a cocktail of enzymes and other proteins.
A successful enzyme recovery process depends on proper monitoring of the process at the extraction stage. It is also worth noting that the product of the enzyme extraction process is not a pure form of the target enzyme but a cocktail of enzymes and other proteins.
Enzymes are proteins, they influence the rate of reaction and not the final product of the reaction Enzymes are highly sensitive to pH, temperature, and salt concentration.
Reactions proceed more rapidly if the right concentration of enzyme is present in the reaction mixture and optimum temperature, and pH is maintained for the specific reaction.
Purification of the enzyme is the next step in the process after extracting the impure enzyme. Varying the salt concentration added to the impure extract will cause different proteins in the cocktail to precipitate.
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