This experiment was carried out to characterize an enzyme, -amylase by extracting it from the corn. The factors affecting the enzymatic activity of -amylase such as substrate concentration, temperature, pH, ionic strength and inhibitors were inves

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University Degree Biological Sciences

Name: Cheng Chu Yee

Student ID: 22351086

Lab session: Tuesday, 9.00 – 12.00pm

Lab no: A11

Demo: Yan Qi, Yi Lyn

Title: Characterization of an enzyme

Introduction:

Enzyme is a protein molecule that acts as the biological catalyst of chemical reaction where the substrates are converted into products. With the aids of enzyme, the rate of reaction will be increased since the enzyme provides an alternative pathway for reaction by lowering the activation energy needed (Taylor et al., 1997). An enzyme is a tertiary protein with 3 dimensional structure maintained by ionic bonds, hydrogen bonds, hydrophobic interactions and disulphide bridge (Copeland, 2000). It is highly specific as only one type of substrates can bind and catalyze by one type of enzymes. The substrates will bind to a restricted region which is known as the active site which is formed by amino acids with a framework that configures the active site (Campbell & Reece, 2008). For the lock-and-key hypothesis, only substrates which have shapes that are compatible fit to the shape of the active site can be catalyzed by the enzyme while for the induced-fit mechanism, the active site of the enzyme changed its shape to suit the shape of the substrates since the amino acid that determine the shape of the enzyme can mould into a precise shape (Taylor et al., 1997).

When the substrates enter the enzyme’s active site, they are held together to form a enzyme-substrate complex by hydrogen bonds and ionic bonds. The active site then lowers the activation energy to increase the reaction rate by acting as a template to orientate and stress the substrate meanwhile stabilize the transition state of the reaction (Campbell & Reece, 2008). Also, it provides a favorable microenvironment so that the catalysis can be carried out. After that, the substrates will be changed to the products and released from the enzyme which is now free for catalysis since it remains unchanged throughout the reaction.

There are several factors that will affect the enzymatic activity such as pH, temperature, substrate and enzyme concentration, ionic strength, cofactors and inhibitors. Different types of enzymes have different pH range and temperature that allows the activity of enzyme to be carried out optimally. When pH increases, the acidity decreases since the concentration of hydrogen ions decreases. As a result, since the increasing amount of hydroxyl ions compete against the enzymes' ligands for divalent and trivalent cations to form hydroxides and thus affecting the enzymatic activity (Pandey et al., 2006). This applies to low pH value too due to the increase in hydrogen ions as the ionic bonding that maintains the shape of the enzyme will be altered causing the enzyme to be denatured.

Also, higher temperature will increase the rate of reaction since more collisions occur between the molecules. However, extreme temperature will also denature the enzyme as the 3 dimensional structure of enzyme is unfolded due to the breakage of hydrogen bonds and hydrophobic interaction (Taylor et al., 1997). According to Pandey et al. (2006), low temperature will inactivate the enzyme since the enzyme-substrate complex is freeze and little energy is available for collisions to be taken place.  The enzymatic activity will be diminished by the inhibitors. There are two types of inhibitors, the competitive inhibition which can be overcome by increasing the concentration of substrates to reduce the binding of inhibitor at the active site and the non competitive inhibition which permanently denature the enzyme since the inhibitors bind to the separate site of the enzyme (Taylor et al., 1997).

Besides that, increasing ionic strength will inhibit enzymatic activity since it will lower the binding chance of substrates to the enzyme as the enzyme and substrate are held together by ionic bonds and hydrogen bonds (Campbell & Reece, 2008). Low ionic strength will also denature the enzyme since the interaction of ions in the enzyme increases in low level of ionic strength. However, as stated by Scopes (2002), different enzymes will function optimally at different ionic strength since some may need substantial level or low level of ionic strength. Moreover, higher amount of substrate will increase the enzymatic activity. However, when all the enzymes are saturated with substrate, the rate of reaction will not be further increase even more substrates are added (Taylor et al., 1997).

In this experiment, α-amylase extracted from corn was used. It is an enzyme that  long-chained carbohydrate such as starch and glycogen to  from amylose, glucose and limit dextrin fron amylopectin by breaking the α-1, 4 –glycosidic bond. It can be found in animals as a digestive enzyme, fungi, plants and bacteria. In human, α-amylase is present in saliva and pancreas By breaking down the starch or simple sugars obtaining from daily diet for animals or through photosynthesis by plants, energy can be produced and used for metabolism purposes. Furthermore, α-amylase is used in food industries to manufacture food such as glucose syrup and corn syrup and for bread making. Arora and Bhatnagar (2005) also claimed that it is also used in ethanol production to break down starch into sugars. Some detergents also contain α-amylase. Another use of this enzyme is that it can remove adhesive in textile as ageing starch will damage the textile since it becomes acidic and hydrolyze the fiber of textile (Arora & Bhatnagar, 2005).

Objectives:

This experiment was carried out to characterize an enzyme, α-amylase by extracting it from the corn. The factors affecting the enzymatic activity of α-amylase such as substrate concentration, temperature, pH, ionic strength and inhibitors were investigated. Besides that, the percentage of recovery of the enzyme from the crude extract was also determined after a series of purification to purify the enzyme.

Results:

  1. Protein determination of α-amylase

Table 1: The absorbance and mass of protein in standard BSA and α-amylase crude extract at wavelength of 560nm

Figure 1: The graph of absorbance at 560nm against the mass of protein in solutions, µg

Table 2: The absorbance and mass of protein in standard BSA and α-amylase after 1st dialysis at wavelength of 560nm

Figure 2: The graph of absorbance at 560nm against the mass of protein, µg after first dialysis

Calculations:

Mass of standard BSA

The concentration of standard BSA used = 500µg/ml

Volume of standard BSA used = 0.30ml

Mass of standard BSA = (concentration of standard BSA)(volume of standard BSA)

= (500)(0.30)

= 150µg

Mass of α-amylase crude extract 

From figure 1, it can be shown that the relationship between the absorbance at 560nm and the mass of protein in solutions can be illustrated using the equation, y = 0.0019x where y is the absorbance and x is the mass of protein in solutions.

y = 0.0019x

0.044 = 0.0019x

X = 23.16µg

Since the solution was diluted ten times,

Mass of protein in α-amylase crude extract before dilution = 23.16 x10

= 231.60µg

It was shown that mass of protein in 0.3ml of α-amylase crude extract used in FC method

= 231.60µg, therefore the mass of protein in 30ml of α-amylase crude extract can be determined since 30ml of α-amylase  was used for the investigation in this experiment.

The mass of protein in 30ml of α-amylase =

 = 23160µg

Total mass of α-amylase after first dialysis

From figure 2, it can be shown that the relationship between the absorbance at 560nm and the mass of protein in solutions can be illustrated using the equation, y = 0.0026x where y is the absorbance and x is the mass of protein.

y = 0.0026x

0.110 = 0.0026x

Mass of α-amylase dialyzed = 42.31µg

It was shown that mass of protein in 0.3ml of α-amylase extract dialyzed in FC method

 = 42.31µg, therefore the mass of protein can be determined for 5ml of the extract since 5ml of α-amylase was collected after the first dialysis.

The total mass of protein in 5ml of α-amylase extract dialyzed =

= 705.17µg

  1. Enzyme assay (α-amylase)

Table 3: The absorbance of starch with and without the enzymatic reaction of α-amylase at wavelength of 620nm

Figure 3: The graph of absorbance at 620nm against the mass of starch in solutions, mg

Calculation:

The concentration of starch

The starch used = 0.15%

 = 0.15g/100ml

  = 150mg/100ml

  = 1.50mg/ml

Mass of starch

The concentration of starch used = 1.50mg/ml

Volume of starch used = 1.00ml

Mass of starch = (concentration of starch)(volume of starch used)

= (1.50)(1.00)

 = 1.50mg

Mass of starch with α-amylase

From figure 3, it can be shown that the relationship between the absorbance at 620nm and the mass of protein in solutions can be illustrated using the equation, y = 0.2540x where y is the absorbance and x is the mass of starch in solutions.

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y = 0.2540x

0.078 =0.2540x

Mass of starch with α-amylase = 0.31 mg

Mass of starch hydrolysed by α-amylase

= Mass of starch – Mass of starch with α-amylase

= 1.50–0.31

= 1.19mg

Mass of α-amylase used

It was shown that mass of protein in 0.3ml of α-amylase extract dialyzed in FC method

 = 42.31µg, therefore the mass of protein can be determined for 1ml of the extract since 1ml of α-amylase was used.

The mass of protein in 1ml of α-amylase extract dialyzed =

= 141.03µg        

Enzyme activity of α-amylase

Enzyme activity of α-amylase =

= ...

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This experiment was carried out to characterize an enzyme, -amylase by extracting it from the corn. The factors affecting the enzymatic activity of -amylase such as substrate concentration, temperature, pH, ionic strength and inhibitors were inves
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