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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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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. ...read more.


1.50 0.381 0.000 Figure 10: The graph of enzymatic activity of ?-amylase, mg/min/ �g against the temperature, �C Calculations: Initial concentration of starch The concentration of starch used = 0.15% = 0.15g/100ml = 150mg/100ml = 1.50mg/ml Initial 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 This calculation was repeated for all the starch solutions with temperature 4, 37, 50 and 70 to obtain the mass of starch. The results were tabulated in table 9. Mass of starch remaining 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. y = 0.2540x** 0.189 = 0.2540 x Mass of starch remaining solution at 4�C = 0.744mg Mass of starch hydrolysed by ?-amylase Mass of starch hydrolysed in starch solution at 4�C = Initial mass of starch - Mass of starch remaining = 1.50 -0.744 = 0.756mg This calculation was repeated for all the starch solutions with temperature 4, 37, 50 and 70 to obtain the mass of starch hydrolysed by ?-amylase so that enzymatic activity can be determined. Concentration of ?-amylase Mass of ?-amylase extracted from DEAE purification (values taken from week 3) = 142.94�g Volume of ?-amylase extract: 5 � 1.40 = 7.00ml Concentration of ?-amylase extracted = = = 20.42�g/ml This calculation was repeated for all the starch solutions with temperature 4, 37, 50 and 70 to obtain the concentration of ?-amylase so that enzymatic activity can be determined. Mass of ?-amylase Since the volume of ?-amylase extract added to each of the solution = 0.20ml Mass of ?-amylase extract used in assay = concentration x volume = 20.42 x 0.2 = 4.084�g This calculation was repeated for all the starch solutions with temperature 4, 37, 50 and 70 to obtain the mass of ?-amylase so that enzymatic activity can be determined. ...read more.


According to Hsieh et al. (2008), EDTA inhibits completely on ?-amylase while urea only inhibit ?-amylase partially. This was because since calcium ions are present in the enzyme, EDTA which is a strong chelating agent will diminish the enzymatic activity by forming complex molecules with the ions (Satyanarayana & Johri, 2005). Thus, the enzyme loss its ability to hydrolyze the starch and can be considered as denatured since EDTA is a non-competitive inhibitors. It can be seen that the enzymatic activity. On the other hand, urea will inhibit enzyme by replacing the enzyme to form hydrogen bonds with the substrate as it attached to the carboxyl group of amino acids of the enzyme (Choudhary, 1996). This causes the enzyme to be not able to bind with starch as the active site which is moulded by amino acid. Thus the enzymatic activity of ?-amylase in the presence of urea should be higher than that of EDTA since urea is a competitive inhibitor. However, the results showed that the enzymatic activity in the presence of urea is lower than that of the EDTA. This may due to the reason that the concentration of urea added was 8M which was higher as compared to that of EDTA which was only 3mM. As shown by the results, ?-amylase can carry out its function to break down starch at pH 7, temperature of 37�C, 0.5M of buffer and without any inhibitors since the enzymatic activity with these conditions were high. This was because the enzyme is not denatured at this point y extreme condition such as low or high pH, temperature and ionic strength. Conclusion: In conclusion, ?-amylase has a maximum enzymatic activity (Vmax) of 0.087 mg of starch /min/�g of ?-amylase and the concentration of starch needed for ?-amylase to reach half of the maximum enzymatic activity (Km) was 4.79mg/ml. The optimum conditions for the enzyme to have optimum enzymatic activity were pH 7, temperature of 37�C, 0.5M of buffer and without any inhibitors since the enzymatic activities with these conditions were high which were 0.022, 0.024, 0.032, 0.018 mg of starch /min/�g of ?-amylase respectively. ...read more.

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