Experiment 3: Enzyme

EXPERIMENT 3: ENZYME

Objectives:

• ·         To study the reactions of enzyme in different environment.
• ·         To investigate the properties of the environment that may influence the rate of an enzyme to react.
• ·         To determine the effect of such reaction to amylase.

Introduction

Enzymes are types of protein act as catalyst. Catalysts will speed up the chemical reactions. Biochemical reactions that take place will react too slowly without enzymes to keep up with the metabolic needs and the life functions of organisms. Enzymes have a three-dimensional structure that is very complex. This three-dimensional structure consists of one or more polypeptide chains. These polypeptide chains form an active site, an area into which the substrate will fit. There are four factors that will have an effect on the structure of an enzyme’s active site, the activity of the enzyme, and the rate of the reaction in which the enzyme is involved. The four factors that can affect the activity of an enzyme include temperature, pH, enzyme concentration, and substrate concentration.

When the enzyme’s active site binds with the substrate like a key fitting a lock, an enzyme-substrate complex will form. The shape of the enzyme must match with the shape of the substrate. Enzymes are very specific. This is because they will only function correctly if the shape of the substrate matches the active site. After the reaction take place, the products are released and the enzyme returns to its normal shape. The enzyme remains unchanged and does not form chemical bonds with the substrate. As a result, the enzyme molecule can be reused. Only a small amount of enzyme is needed because they can be used repeatedly.

                                                                           

       

Procedure

The effect of temperature

The experiment were prepared as the table below

Test tube	8 ml starch of x mg/ml	Water (ml)	Amylase (ml)	Each sample were incubated at 15, 28, 36.6 and 40 ºC for 10 minutes	Iodine (ml)	All test tube were placed in an ice bath for 10 minutes. The absorbance were measured at 590nm
1	0	8	1		1
2	0.01	0	1		1
3	0.025	0	1		1
4	0.05	0	1		1
5	0.10	0	1		1
6	0.30	0	1		1
7	0.50	0	1		1
8	0.70	0	1		1
9	1.00	0	1		1

The Lineweaver-Burke line for the results were plot and compared.

The values of V_max ang K_m were calculated.

 The effect of pH

The experiment were prepared as the table below

Test tube	Starch 0.5 mg/ml (ml)	2ml buffer of pH x	Amylase (ml)	Each sample were incubated 36.6 for 10 minutes	Iodine (ml)	All test tube were placed in an ice bath for 10 minutes. The absorbance were measured at 590nm
1	5	4	1		1
2	5	5	1		1
3	5	6	1		1
4	5	7	1		1
5	5	8	1		1
6	5	9	1		1
7	5	10	1		1
Blank	5	3 ml of H2O			1

The values of V for all pH were calculated.

The velocity for each of the pH test were compared.

Result

A. standard reference

Tubes	Starch concentration (M)	Amylase (ml)	Absorbance (590 nm)
1	0.000	1	0.018
2	0.010	1	0.060
3	0.025	1	0.148
4	0.055	1	0.279
5	0.100	1	0.472
6	0.300	1	1.245
7	0.500	1	1.912
8	0.700	1	2.471
9	1.000	1	3.324

i)     Graph of Standard Reference

B. The effect of substrate concentration 

Tubes	Starch concentration (M)	Amylase (ml)	Absorbance (590 nm)	Absorbance (mg/ml) / Sf
1	0.000	1	0.064	0.015
2	0.010	1	0.061	0.013
3	0.025	1	0.055	0.010
4	0.050	1	0.058	0.011
5	0.100	1	0.063	0.014
6	0.300	1	0.061	0.013
7	0.500	1	0.062	0.014
8	0.700	1	0.064	0.015
9	1.000	1	0.065	0.016

ii) Graph for the effect of substrate concentration

C. The effect of temperature (40 ˚C, 35 ˚C and 10 ˚C)

Tubes	Starch concentration /S◦	Amylase (ml)	Absorbance (590 nm) / 40˚C	Absorbance (mg/ml) / Sf
1	0.000	1	0.082	0.020
2	0.010	1	0.107	0.025
3	0.025	1	0.110	0.028
4	0.050	1	0.093	0.021
5	0.100	1	0.100	0.029
6	0.300	1	0.088	0.018
7	0.500	1	0.091	0.020
8	0.700	1	0.074	0.012
9	1.000	1	0.184	0.043

Tubes	Starch concentration (M)	Amylase (ml)	Absorbance (590 nm) / 35˚C	Absorbance (mg/ml) / Sf
1	0.000	1	0.064	0.015
2	0.010	1	0.061	0.013
3	0.025	1	0.055	0.010
4	0.050	1	0.058	0.011
5	0.100	1	0.063	0.014
6	0.300	1	0.061	0.013
7	0.500	1	0.062	0.014
8	0.700	1	0.064	0.015
9	1.000	1	0.065	0.016

Tubes	Starch concentration	Amylase (ml)	Absorbance (590 nm) / 10˚C	Absorbance (mg/ml) / Sf
1	0.000	1	0.013	0.000
2	0.010	1	0.057	0.004
3	0.025	1	0.101	0.022
4	0.050	1	0.274	0.070
5	0.100	1	0.548	0.148
6	0.300	1	1.542	0.432
7	0.500	1	2.241	0.639
8	0.700	1	3.153	0.885
9	1.000	1	4.441	-

Temperature     1/S◦ (M)	10 ˚C	35 ˚C	40 ˚C
20.00	1379	1025	920
10.00	963	465	660
3.33	141	120	98
2.00	83	83	83
1.43	58	58	58
1.00	41	40	40

(data used to plot LB plot graph)

ii) Graph for the effect of temperature

D. the effect of pH

Discussion 

A standard curve is a type of graph used as a quantitative research technique. Multiple samples with known properties are measured and graphed, which then allows the same properties to be determined for unknown samples by interpolation on the graph. The samples with known properties are the standards, and the graph is the standard curve. Standard curves are most commonly used to determine the concentration of a substance in this experiment, we want to know the concentration of starch present in each test tubes. The spectrophotometer reading will give us readings in unit nm, thus, standard curve will be plotted (absorbance nm vs absorbance in unit mg/ml). The reading of results from spectrophotometer in this experiment, will be converted to unit mg/ml by referring to this standard curve.

         From the experiment conducted and the data obtained, the rate of digestion of the reaction for each sample can be calculated by referring to the formula given. The formula is:

                       

                                         V = ∆ S/ ∆ t = (S₀ - S_F) / 40 minutes

The result for the effect of substrate concentration was tabulated in a table form. Then, from the table-containing data, a graph of a Michaelis-Menten (MM plot) was plotted. The graph shown that the V_max value is 0.025 V and the K_m value is 0.520 M. The value of K_m can be marked on the graph by dividing the V_max value with 2 (V_max/2), and the x-horizontal shown the reading of K_m.

            Noted that the concentration of starch increases (differ concentration in each tube) and the volume of amylase added is constant (1 ml for each tube). In tube 1 only amylase is present since no starch was added. In tube 2 which contain least concentration of starch, when amylase is added, most of the starch has been broken down into maltose. This occurred as the substrate (starch) combine with the active site of enzyme (amylase) before being broken down into maltose. As a result, once tested with iodine, none or less amount of starch concentration is detected. In contrast, in the test tube 9 that has the highest concentration of starch, not all starch was succeed to be broken down into maltose since the amount of amylase provided is not enough. In the other word, the active site of the enzyme became very saturated and no more substrate can be broken down. Hence after tested with iodine, large amount of starch left in the tube was detected.

           For an experiment on the effect of temperature towards enzyme (amylase), different temperature of 40 ˚C, 35 ˚C and 10 ˚C were prepared to observe the reaction rate of the amylase. The rate of reaction can be seen from the Lineweaver-Burke graph plotted, where different temperature performed different K_m but have same V_max cut at the y-axis. The value of V_max for all temperature should be the same since the amount of active site of enzyme is the same. By analyzing the graph, temperature that closer to the most effective temperature for the amylase to break down the starch will have smallest K_m, and in this experiment the most desired temperature for the reaction to be occurred is 40 ˚C followed 35 ˚C and 10 ˚C. this means that reaction at 40 ˚C has the higher affinity for the reaction to occur while reaction at 10 ˚C has the lowest affinity for the reaction to occur. Thus, the values of K_m at 40 ˚C, 35 ˚C and 10 ˚C are 0.22 M, 0.29 M and 1.43 M. The value of V_max is 0.032 V and same to all temperature provided. The value of 1/V_max and 1/K_m can be marked on the LB plot, there the actual value of  V_max and K_m can be calculated.

         α-amylases are found in saliva, pancreatic juice, human breast milk, serum and certain tissues such as the liver, beta amylase in plant product and gamma amylase also is found in human liver, but it is exist in weak acidic condition. This enzyme catalyzes the hydrolysis of α (1-4) linkages in starch by breaking it down to maltose and some glucose. As the starch is broken down, the coiled structure of α-amylase is unfolded. Therefore, iodine will no longer be able to form the blue complex with the α-amylase. Starch will be converted to maltose when amylase is added. The addition of iodine to starch will turn the solution into blue black when amylase are added, the colour turns to colorless indicate the presence of maltose and no presence of starch. It can be assumed that the decrease in color (absorbance) is proportional to the product formed. In this experiment, the absorbance of the starch-iodine complex wil be measured at 590 nm in 1 cm cuvettes using single beam spectrophotometers. The intensity of the emerging beam will be decreased because the solution absorbs some of the radiation. In this experiment, results shows that the low absorbance in neutral condition (pH 7), and thus, we predict that this is the alpha amylase. The fact is,  there is an optimum pH for every enzyme. pH is capable of altering the structure of the active center in the enzyme causing denaturation. The optimum pH is not a solid value. Usually they work quite well within a ph range (note that there are those that will work properly in any pH value) Out of this range they will act slowly tending to no inactivity (cause of denaturation). 

Conclusion:
Enzymes are biological or organic catalysts which speeds up a reaction without undergoing any permanent change themselves. The enzyme that we used was amylase. Amylase is an enzyme that catalyses the breakdown of starch into sugars that can be found in saliva. As the conclusion for this experiments, our objective was failed because our results does not follow the theory.

References:

Campbell, Reece, Urry, Cain, Wasserman, Minosky, Jackson (2008). Biology 8^th edition. Pearson International Edition, 1301  Sansome St., San Francisco.

Bah Hock Guan, Tor Siong Hoon, Sri Nasariya (2009). Complete reference matriculation biology 1 (2^nd edition). Oriental Academic Publication, Kuala Lumpur.

(2010, 02). The Effect of Temperature and Ph on Α-Amylase Enzyme Activity. StudyMode.com.Retrieved May 1, 2012, from http://www.studymode.com/essays/The-Effect-Of-Temperature-And-Ph-286442.html