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An Investigation into the activity of A Amylase

Alpha amylase is an enzyme. It specific binds with water and starch . It hydrolyses starch and glycogen to give glucose and maltose. It acts on the alpha bonds of polysaccharides. Because of the specificity of the enzymes activity the structure of the enzyme must be precise. Any factor which will cause denaturation of the enzyme will restrict its rate of activity. Two of the most important factors effecting Alpha amylase activity are temperature and pH. The following experiment is designed to investigate the effect of different environmental temperatures and pH on the activity rate of Barley amylase.

Materials and Methods

As per schedule, No procedures were changed from original schedule.

Results:

The results are recorded as the time when colour change indicated that all the starch had been hydrolysed. A dark blue-black colour signified the presence of starch. When this colour is lost and an amber-yellow colour develops indicating that all the starch is hydrolysed, the time is recorded.

Experiment 1:

Investigating the effect of environmental pH on the activity of Barley Amylase

Figure 1 Effect of pH on the activity of Amylase, increasing pH appears to increase amylase activity

Time (minutes)

pH

G1

G2

G3

G4

G5

Average

1/Average

4.0

>30

>30

>30

>30

>30

30

0.033

5.5

23

28

28

28

20

24.5

0.039

6.0

28

25

25

28

19

25

0.040

6.4

17

29

26

30

16

23.6

0.042

7.0

17

18

24

30

30

23.8

0.042

Table 2: Experimental results recorded by Groups 1 to 5, on the effect of pH on amylase activity.

Experiment 2:

Investigating the effect of environmental Temperature on the activity of Barley Amylase

Figure 2 effect of temperature on Amylase activity; Amylase appears to be active at lower temperatures and inactive at higher temperatures

Time (minutes)

Temp ° C

G1

G2

G3

G4

G5

Average

1/Average

5

>30

>30

>30

>30

>30

30

0.033

22

20

25

22

20

25

22.4

0.045

30

21

27

25

22

30

25

0.04

40

25

16

27

25

28

24.2

0.041

80

>30

>30

>30

>30

>30

30

0.033

Table 2: Experimental results recorded by Groups 1 to 5, on the effect of temperature on amylase activity.

The results indicate that the activity of alpha amylase increases with decreasing acidity and is highest at pH7. The trend in effect of temperature on amylase activity is that it increases in the middle range but is inactive at extreme temperatures. The results do not agree with the expected results of previous similar experiments and repeats of the same experiment within the class do not concur with others

Discussion

The hypothesis being tested is that enzymatic reactions are effected by a number of external factors. Temperature and pH are thought to be the most important extrinsic factors. The objective was to examine the activity of the enzyme α-amylase under the effect of increasing environmental temperatures and increasing pH levels and to determine the optimal temperature and pH for Alpha amylase activity.

The starch medium selected was Barley. This is a good choice due to its ready availability, the ease of preparation and the body of work available on understanding its germination process due to its importance in the brewing industry. Barley is composed of 53% to 65% dry weight starch (Fox et al (2003), MacGregor 1978, Sanford et al 2003). Barley produces its own amylases during the germination period. The changes in the levels of α- amylase detected in barley during germination are outlined by McGregor et al (1984). The breakdown of starch in barley involves two types of amylase α-amylase and β-amylase. The former works by hydrolysing the 1-4 bonds within the glucose chain exposing non-reducing ends for the beta amylase to split (fig 3.) (Keusch 2003). Prior to germination there is no amylase detected in Barley, a trace was found after 24 hours germination, but after that it was found to increase rapidly (MacGregor 1978).

figure 3. www.uni-regensburg.de/Fakultaeten/nat_Fak_IV/Organische_Chemie/Didaktik/Keusch/Grafik/sucrose.gif&imgrefurl

The optimal temperature and pH for α-amylase extracted from barley is well studied. Fox et al (2003) state an optimal temperature of 65°C and a pH of 5.5. O’Rourke (2002) gives optimal values of 67°C and pH 5.2 (table 3) while lower temperature values of 55°C for optimum activity of alpha amylase are given in other papers (Al-Bar 2009 and MacGregor 1978). The first two papers are written from an industrial and brewing viewpoint whereas the latter are written from a pure scientific evaluation of the characteristics of barley, this may have some bearing on the different temperatures cited keeping in mind that 60°C is the temperature used in mashing.

ENZYME

ACTION

OPTIMUM T°C

OPTIMUM pH

α-AMYLASE

Random hydrolyses of starch

67

5.2

β-AMYLASE

Hydrolysis of parts of sugars from the non reducing end

62

5.5

Table 3. Optimal Temperature and pH for Enzyme activity in germination of barley (adapted from O’Rourke 2002)

The optimal temperature for amylase activity differs for different sources, Azuki beans , finger millet and wheat have optimum temperatures of 70°C, 45°C and 55°C respectively (Al-Bar 2009).  In mammals the temp & ph of the body fluids are kept constant at homeostatic condition. If the body temp & ph varies from the optimum body temp & ph, the enzymes activity decreases and the cellular respiration process which produces ATP energy cell mover would be affected in the body. Less ATP energy the body cells cannot perform the work which they need. The results of the present experiment did not comply with these expected results. From the literature the theory was that Amylase activity would increase with increasing temperature until a maximum level would be reached around 55°C and there after it would decrease as it was denatured at higher temperatures, ultimately showing no activity at 80°C. Likewise the effect of pH was anticipated to find increasing amylase activity with increased pH level to optimal between 5-6 and a decrease reaching neutral pH. While the average results were basically compliant there was a great variation in individual group results.

Amylases are important across the spectrum of living organisms; they are required for the breakdown of carbohydrates, which is one of the four essential food groups and the main source of energy to living cells. Amylases are enzymes that increase the activity of a reaction without being consumed in the reaction. The reaction they are involved in is hydrolysis of carbohydrate which is the cleaving of bonds and the addition of water (Hogg 2005). Two main forms exist, alpha amylase and beta amylase, both hydrolyse carbohydrates but in different way. Alpha amylase is the faster acting as it can act on any part of the carbohydrate chain but beta amylase can only act on the non reducing ends which are produced in increasing amounts after the activity of alpha amylase. In plants amylases are largely involved in the germination of seed; temperature may be the trigger for onset of this process. In animals amylases are found in association with the digestive system, ptyalin in the mouth and from the pancreas. The amylase enzymes hydrolysis the disaccharides  sugar and maltose into monosaccharides glucose & galactose which are the smaller molecules of starch that are suitable for absorption in the small intestine for ultimate body use. As an enzyme it binds the substrates together, promotes the hydrolysis and frees the products (fig 4.), and if denatured at high temperatures this cannot happen

Figure 4. http://kvhs.nbed.nb.ca/gallant/biology/biology.html

From the results obtained and the discussion hitherto the indications are that the results of the present experiment are in error. The anticipated optimal temperature and pH based on previous experiments were Temperature 55°C and pH 5. The optimal results from the present experiment were 22°C and pH 6.4. This is a well practiced experiment with well recognised results. looking at the results where some conform to the expected and others have results that are un expected it is most likely that the deviation is due to human error, as in not mixing solutions correctly, not maintaining the test tubes at a constant temperature, mixing up samples and not monitoring the time of reaction correctly. The observation of colour changes is also highly subjective, the precise time of change being difficult to detect. Improvements could include using:

a spectrophotometer to measure change of amount of starch remaining and then it must be blanked correctly before use.

shaking equipment to ensure correct mixing

using the same amylase as preparation by different groups might not have been adequate

Concentration of substrate, exactness of measurement.

Lack of closer attention by the operator can lead to error. Reaction rates, solutions and indicator need to be observed or calculated correctly to avoid errors.

In conclusion, to improve on the experiment in future, proper attention and observation is very important.


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