In this experiment we investigated the effect of temperature on enzyme activity and protein content. After the calculations we plotted two graphs these were protein content versus temperature and total activity versus temperature. According to the graphs we can say that the total activity increases up to a point then it starts to fall down. Initially this increase is caused by the increase in temperature up to the optimum temperature for reaction to gain sufficient energy to reach the activation energy. Then the activity decreases since further increase in temperature cause denaturation of protein. According to the graph the optimum temperature is about 400C. Also at that temperature the purity of the enzyme is highest. In contrast to total activity, protein content decreases down to a point then it starts to increase.

QUESTIONS

1. State the temperature at which the activity is highest.

– Activity is highest at about 400C

2. State the temperature at which the purity is highest.

– The highest the specific activity, the highest the purity. So at 400C purity is highest.

3. What is the use of purification? Does it always necessary?

– Purification involves removing any contaminants that are present in the mixture; these may be other enzymes or proteins, carbohydrates and ash. For the some determinations it is necessary to purify enzyme from other substances in the case of determination amino acid composition, molecular weight, tertiary and quaternary structure, substrate specificity and kinetic

parameters like Km, Vmax, kcat.

4. Under which conditions a purified enzyme is required for activity determination?

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In this experiment, Major affect of enzyme activity and stability is temperature. Since enzymes are biochemical catalysts, made up at least partially of protein, they are sensitive in varying degrees to heat. Raising temperatures of the environment generally multiplies the degree of activity by the enzyme. The most dissolved protein is denatured by heat when the temperature higher than about 50oC. Heat denaturation results in protein precipitation as a result of destruction of the secondary structure and formation of random aggregates. Because all proteins are not stable when heated in aqueous solution. The precipitate enzyme is recovered by filtration or centrifugation and dried in atm. or vacuum driers. For most commercial application cost is most important than high purity and is usually unnecessary.

When the presence of contaminating enzymes or other substance will adversely affect the product and activity. If the turbidity is observed in taken tube from ice bath, it is centrifuged. Because protein is denatured due to heat treatment and so sedimentation will occur. Therefore the sedimentation part must be removed. When we look at our results it is obviously seen that increase in temperature increases the activity of enzyme up to a level. According to our result this temperature is 40oC. In 60oC the protein content is more than in 40oC but specific activity of 60oC is less. On the other hand when we compare the velocities we can say the same thing. In 30oC velocity reaches the highest value. Finally we can say that because of protein denaturation in high temperatures, enzyme activity increases up to a level with increase in temperature.

Answers of The Questions:

1.At room temperature is highest than other temperature. Because the temperature is increased as the activity is decreased. The activity is affected for the temperature. The protein structure (interaction bond) is changed.

2.State the temp. At which the purity is highest. At 40 C the purity is highest.

3.Purification helps to elimination the contaminants, which is effect the purification. The foreign material that is not wanted in product for the pure product to be best product is changed the purification. But this is more expensive, so it is usually unnecessary for most commercial application.

4.The factor that, will affect the purification. The process condition is important for purification. If the purification is not best, the enzyme activity is not best. The unpurification enzyme is not work in process. This reason is important for working and activation enzymes.

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