Effect of temperature on enzyme activity

The most substrates require an input of energy to get the reaction going, (the reaction is not spontaneous). The energy required to initiate the reaction is called the activation energy. When the substrate(s) react, they need to form a complex called the transition state before the reaction actually occurs. This transition state has a higher energy level than either the substrates or the product. Outside the body, high temperatures often supply the energy required for a reaction. We have enzymes that provide an alternative way with a different transition state and lower activation energy. The rate of the reaction without any external means of providing the activation energy continues at a much faster rate with an appropriate enzyme than without it. The maximum rate that any reaction can proceed at will depend, among other things, upon the number of enzyme molecules and therefore the number of active sits available. There are four factors which can effect the enzyme controlled reaction i.e. enzyme concentration, substrate concentration, temperature, and pH.

AIM

Study the effect of temperature on enzyme activity.

METHOD

APPARATUS:

Boiling tubes

Rack

00cm³ measuring cylinder

, 5 and 10 cm³ pipettes and pi-pumps to fit

Stop clock

Colorimeter

Ice and water baths

CHEMICALS:

0.5% starch buffered to pH 6.7

% NaCl solution

Fearons reagent (methylamine hydrochloride)

20% NaOH solution

pH 6.7 buffer

Distilled water

Amylase solution

It was made sure that all apparatus and chemicals handled carefully. Sodium hydroxide solution NaOH is dangerous for skin therefore hands were protected by wearing gloves.

> 7 tubes of starch were prepared and labelled S1 - S7. Each tube contained 5 cm³ of buffered starch, 2cm³ of pH 6.7 buffer, and 1cm³ of 1% NaCl.

> Salivary amylase solution were prepared by washing out the mouth with 50cm³ of distilled water and discarded into the beaker. This process was repeated twice to get 100cm³ of salivary amylase. 7 separate tubes were prepared and labelled E1 - E7. Each tube contained 5cm³ of salivary amylase.

> Then the tubes were distributed as follows

S1 + E1 › beaker of ice

S2 + E2 › at room temperature

S3 + E3 › into water bath at 30ºC

S4 + E4 › into water bath at 40ºC

The remaining tubes were paired into water baths at 50ºC, 60ºC, and 70ºC.

> All the tubes were allowed to equilibrate at the chosen temperature for 5 minutes.

> At zero time on the stop clock the contents of tube E1 and S1 were mixed thoroughly. The mixture was placed into the ice.

> After exact 30 second the mixing procedure was repeated with tubes E2 and S2. The mixing process continued at 30 second intervals with the remaining tubes until all the reactants were mixed and were at their correct temperatures.

> After exactly 5 minutes on the stop clock 1 cm³ of 20% NaOH were added into tube 1 and contents were mixed thoroughly. The addition of NaOH stopped any further reactions and the tube was removed from the ice.

> The above step was repeated after exactly 30 second intervals with the rest of the tubes. It was ensured that each mixture had exactly 5 minutes reaction time at the chosen temperature.

> Boiling water bath was prepared by boiling water into beaker over Bunsen burner.

> 0.5 cm³ of Fearons reagent was added to each tube and mixed thoroughly. The tubes were then heated in boiling water for 2 minutes. Then the tubes were removed and cooled at room temperature.

> The colorimeter was used to make quantitative colour comparison for each tube. The colorimeter was adjusted correctly to get precise readings. 430 filter and water was used as a reference. The absorbance for each solution was taken. The redder colour in the solution the higher the concentration of maltose in a solution.

RESULTS

Tubes

Temperature

Absorbance value (nm)

S1 + E1

Ice

0.17

S2 + E2

Room temperature

0.24

S3 + E3

30ºC

0.40

S4 + E4

40 ºC

0.80

S5 +E5

50 ºC

0.35

S6 +E6

60 ºC

0.20

S7 +E7

70 ºC

0.18

Table 1

Graph A

DISCUSSION

The above experiment was performed to evaluate the effect of temperature on enzyme activity. In this experiment amylase is used as an enzyme and starch is a substrate. Amylase catalyses the breakdown of starch. When amylase reacted with starch, it cut off the disaccharide maltose. As the reaction progressed, more starch converted into sugar. As amylase broke down starch, the colour of the solution changed from light to bright red. The activity of amylase was observed by colorimeter.

The data recorded in (table 1) was plotted on a graph to get precise conclusion. The (graph A) clearly explains the effect of temperature on enzyme activity. The x-axis is representing increase in temperature and y-axis is representing absorbance value (rate of reaction).

We can see from the graph, as temperature increases the rate of reaction increases as well. This is due to the increase in kinetic energy of molecules. Enzymes work best at an optimum temperature. Below this, an increase in temperature provides more kinetic energy to the molecules involved. The numbers of collisions between enzyme and substrate will increase so the rate will too as shown in graph 1.

The optimum temperature in this case was 40 ºC. Above optimum temperature enzyme get denatured. This was because bonds holding the structure together were broken and the active site lost its shape and no longer worked. As temperature increased, molecules moved faster (kinetic theory) the rate of reaction decreased. As the temperature continued to raise shown in graph, the hydrogen and ionic bonds, which hold the enzyme molecules in shape, were broken.

If the molecular structure is disrupted, the enzyme ceases to function, as the active site no longer accommodates the substrate. The enzyme is denatured.

References

. Biology for advanced level 4th edition Glenn & Susan Toole

2. http://scool.co.uk/default.asp