INTRODUCTION

Enzymes are proteins that catalyze chemical reactions. In these reactions, the molecules at

the beginning of the process are called substrates, and the enzyme converts them into different

molecules, the products. Almost all processes in a biological cell need enzymes in order to occur

at significant rates. Since enzymes are extremely selective for their substrates and speed up only a

few reactions from among many possibilities, the set of enzymes made in a cell determines which

metabolic pathways occur in that cell. Besides, the microbial amylases meet industrial demands

and a large number of them are available commercially although many microorganism produce

this enzyme. Amylases have attracted the world’s enzyme market because of their wide application

in starch based industries especially food, textile, paper, detergent and baking industries. Most of

the commercially produced amylases are of microbial origin. Therefore enzyme stability and its

stabilization are crucial factors in the application of enzymes.

The physical characteristic and properties of enzyme are first, enzyme generally work very

rapidly. The action and speed of an enzyme is expressed as its turnover number. The turnover

number is the number of substrate molecules which one mole of the enzyme turns into products

per minute. Then, enzymes can work in either direction. This means that the reaction is either to

the left or to the right until an equilibrium is reached between the substrates and the products
formed. Besides that, enzymes are not destroyed or altered by the reaction they catalyzed. Enzymes

can be reused because they are not destroyed by the reactions they catalyze. However, enzymes

cannot be used indefinitely because they are quite unstable as they can be inactivated by certain

parameters. Enzymes are also sensitive to pH changes because they operated at specific pH range

and any alterations can affect their activity and efficiency. Plus, different enzymes have different

specific pH range. Other than that enzymes are specific in their reaction and enzymes cannot

resistant to excessive heat. They are inactivated to excessive heat causing denaturing when

exposed to high temperatures and this explain that only few cell can tolerate with temperature that

exceed than 45oC (O. Tony, 2015).

Furthermore, the activity and stability of enzymes can be affected by some factors

including the concentration of substrate molecule and temperature. As the enzyme concentration

increase, the rate of enzyme activity increases up to a level where it becomes constant. This is

because the more the enzymes are available, the more substrates are broken in less time. It then

becomes constant as the substrate acts as a limiting factor which means that there are not enough

substrate to be broken down compared to the number of enzymes. For temperature, as the

temperature rises, molecular motion causing the collisions between enzyme and substrate speed

up. But as enzyme are proteins, there is a limit beyond which enzyme becomes denatured and

ineffective (M. Abdul Halim, 2016).

 OBJECTIVES

The objectives of this experiment is to study the correlation between enzyme concentration

and activity. Besides, this experiment was also to identify the effect of substrate concentration on

amylase activity.
 METHODOLOGY

The different concentration of starch solution (0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0) were

prepared as the substrate for this experiment. Then, about 0.5 ml of the substrate of each

concentration were added into each test tubes. Besides, an amylase solution is prepared by

dissolving 0.2 ml of amylase, 0.5 ml of starch solution, and 0.3 ml of 0.2M phosphate buffer (pH

7) into every test tubes. The solution then incubated in room temperature for approximately 30

minutes and shaken for every 10 minutes time intervals. Then, about 1 ml of DNS reagent was

added into every solutions. These test tubes then were put into the water bath and boiled for 10

minutes and then cooled down before added in 10 ml of distilled water. The absorbance for these

solution were read at the wavelength of 575 nm. A glucose solution were also prepared for the

standard curve by preparing the pure glucose solution in the range of 0 – 1000 mg/L by repeating

the steps used for the amylase solution preparation. The standard then were used in determining

the unknown glucose concentration for the amylase solution samples. The graph of product formed

against substrate concentration will plotted.

 RESULTS

Table 1.0: Standard curve for glucose

Std. concentration of glucose Absorbance

0 0.000
61.5 0.008
125 0.018
250 0.025
500 0.041
1000 0.064

Std. Concentration of Glucose vs Absorbance

0.07

0.06

0.05
Absorbance

0.04

0.03
y = 0.0122x - 0.0166
0.02
R² = 0.9482
0.01

0
0 61.5 125 250 500 1000
-0.01
Standard concentration of glucose (mg/L)

Figure 1.0: Graph of standard curve of glucose.

 Table 1.1: The data obtained for every percentage of substrate.

Percentage (%) of substrate Absorbance

0 0.000
0.5 0.143
1.0 0.266
1.5 0.318
2.0 0.366
3.0 0.412

Table 1.2: Concentration of glucose calculated (mg/L)

Percentage (%) of substrate Concentration of glucose (mg/L)

0 1.3607
0.5 13.0819
1.0 23.1639
1.5 27.4262
2.0 31.3607
3.0 35.1311
 Product formed vs concnetration of substrate
3.5

Concnetration of substrate (%) 3

2.5

1.5

0.5

0
0 5 10 15 20 25 30 35 40
Product formed (mg/L)

Figure 1.1: A graph of product formed against concentration of substrate.

 DICUSSION

This experiment involved the correlation between the percentages of substrate towards the

concentration of glucose produced. The substrate used in this experiment was starch while the

enzyme used to digest the substrate was amylase. Basically, break down or hydrolyzes of starch

by amylase will produce maltose which is disaccharide that made up of two glucose molecules and

therefore, glucose standard curve was constructed first by checking the absorbance of six different

known concentrations of glucoses. Two-fold series of dilution was applied in order to obtain all

these concentrations ranging from 0 mg/mL to 1000mg/mL and by plotting the standard curve.

In order to study the effect of substrate percentage which is substrate concentration towards

the production of glucoses, six solutions were used with different percentage of substrates starting

from 0%, 0.5%, 1.0%, 1.5%, 2.0% and 3.0%. DNS colorimetric method was applied to determine

the concentration of reducing sugar by adding the DNS reagent before boiled the solution. Based

on the principal, this method will identify the presence of free carbonyl group (C=O) which

represents reducing sugar (J Gou, 2004). Then, the absorbance of every solution was determined

by using spectrophotometer with wavelength of 575nm and the absorbance value obtained can be

used to identify the glucose concentration on the solutions by using glucose standard curve.

According to the results obtained, the concentrations of glucose in the solution were

increased when substrate percentage increased. According to C. J. Fieldeg (1972), in the presence

of a given amount of enzyme, the rate of enzymatic reaction increases as the substrate

concentration increases until a limiting rate is reached. In this case, when starch concentration
increased, there were high amount of starch molecules that bound to the amylase and hydrolyzed

to form glucoses thus increased the rate of reaction. This figure below proved the relationship

between the effects of substrate concentrations towards reaction rate that is catalyzed by fixed

amount of enzyme.

Based on the graph of product formed against concentration of substrate, as shown in

Figure 1.1, the product formed was increasing as the concentration of substrate increased. The

product formed was glucose concentration. As stated by I. Keyser (2011), after a certain

concentration, there will be no effect on the enzyme activity because the enzyme will effectively

become saturated and will be working at their maximum possible even though the substrate

concentrations keep on increasing. From the results obtained, it shows that the amylase were still

active bound to the starch molecules even at 3% concentration because the amount of glucose

concentration was still increased and not yet reached the limiting rate.
 CONCLUSION

The main intention of doing this experiment was to study the correlation between

concentration of enzyme and its activity and also the effect of substrate concentration on the

amylase activity. In this experiment, the enzyme used was amylase while the substrate was starch.

Besides, based on the graph plotted, the product formed increasing as the concentration of the

substrate increased. Hence, by referring to the theory, increasing enzyme concentration will

increased the rate of reaction due to high collision occur between the enzymes and substrates

molecules.
 APPENDIX

Khandpur, S. R. (2006). Colorimeters and Spectrophotometers (Visible-Ultraviolet). Handbook of

Analytical Instruments. 2. 69-70. Tata McGraw-Hill Publishing Company Limited.

M. Abdul Halim. (2016). Enzyme Kinetics Haidhar. [Online]. [Accessed on 27-11-2016].

Available from World Wide Web:

https://www.academia.edu/7347283/Enzyme_Kinetics_Haidhar

M. P. J. Anton. (2000). Microbial Cell Disruption by High-Pressure Homogenization. Chem. Eng.

Sci. 53, 1-16.

O. Tony. (2015). Properties and Characteristic of Enzymes. [Online]. [Accessed on 27-11-2016].

Available from World Wide Web: http://gulpmatrix.com/properties-and-characteristics-of-

enzymes/
St. Rosemary. (2010). Factors Affecting The Activity of Catalase and Amylase Lab Answer.

[Online]. [Accessed on 27-11-2016]. Available from World Wide Web:

http://schoolworkhelper.net/factors-affecting-the-activity-of-catalase-and-amylase-lab-

answers/
 APPENDIXES

To get the concentration of substrate (shown in Table 3) from standard curve of glucose

concentration is; use the gradient equation;

 y = mx + c

 y = 0.0122x – 0.0166

For Absorbance = 0.143, Percentage of substrate = 0.5;

y = 0.0122x – 0.0166

0.143 = 0.0122x – 0.0166

x = 13.0819