Enzymatic Activity of Salivary Amylase

Enzymatic Activity of Salivary AmylaseArce, Christine Grace S.M.Cua, Lance A.Draheim, Kristella M.Go-Oco, Raquel M.

Introduction

Enzymesconsist of a protein and a non-protein (called thecofactor)Biological catalysts- increase the rate of a reaction- lowers activation energy of reactionshighly selective, and highly sensitive- due to the shapes of the enzyme molecules Globular proteins (with exception of some RNAs)

Figure 1. Comparison of free energy of activation profiles for catalyzed and uncatalyzed reactionsRetrieved from: http://www.columbia.edu/cu/biology/courses/c2005/lectures/lec7_10.html

Figure 2. Maltose is made of two glucose molecules bonded together (1). The maltase enzyme is a protein that is perfectly shaped to accept a maltose molecule and break the bond (2). The two glucose molecules are released (3)Retrieved from: http://science.howstuffworks.com/life/cellular-microscopic/cell2.htm

Factors affecting catalytic activity of enzymesTemperaturepHConcentration of enzyme and substrateInhibition of enzyme activityEffect of timeMetal ion activators

1. TemperatureAs the temperature rises, reacting molecules gain more kinetic energyThere is a certain temperature at which an enzyme's catalytic activity is at its greatest, called the optimal temperature Optimal temperature is usually around human body temperature (37.5oC) for the enzymes in human

Denaturation - above the optimal temperature the enzyme structure begins to break down (denature) since at higher temperatures intra- and intermolecular bonds are broken.Figure 3. Optimal temperature of enzymes found in the human bodyRetrieved from: http://www.rsc.org/Education/Teachers/Resources/cfb/images/07A.jpg

Denaturationof proteins involves the disruption and possible destruction of both the secondary and tertiary structures, but not the primary structure.

2. On pHFigure 4. Optimum pH of different enzymes; Pepsin: 1.5 2, Salivary amylase: 6.8, Arginase: 9 9.5Retrieved from: http://www.rsc.org/Education/Teachers/Resources/cfb/images/07B.jpgOptimal pH: the pH at which the enzyme attains its maximal activity

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3. On Concentration of enzyme and substrateAs the concentration of either is increased, the rate of reaction increases (at constant temperature and pH)The rate of reaction increases with increasing substrate concentration up to a point, above which any further increase in substrate concentration produces no significant change in reaction rate

Figure 5. Two graphs show the effect of increasing the concentration of enzyme and substrate, respectively Retrieved from: http://www.rsc.org/Education/Teachers/Resources/cfb/images/07C.jpg,http://www.rsc.org/Education/Teachers/Resources/cfb/images/07D.jpg

4. Inhibition of enzyme activity

Inhibitors- molecules that reduce or even stop the catalytic activity of enzymes in biochemical reactions. They block or distort the active site.

Types:

Active site-directed(competitive) - inhibitors that occupy the active site and prevent a substrate molecule from binding to the enzyme Non-active site-directed(non competitive) - inhibitors that attach to the enzyme, perhaps distort its shape

5. Effect of time at the beginning, the rate of reaction increases but by time the rate decreases due to:

Depletion of substrateAccumulation of end productsChange in pH of the reaction

6. ActivatorsActivators, which temporarily bind to the active site of the enzyme, switch enzymes from inactive to active state

Chloride ions activate salivary amylase (anionic activation)Calcium ions activate thrombokinase enzyme (cationic activation)

Enzymes

Advantages over chemical catalyst:

enzyme-catalyzed reactions often have much higher ratesEnzymes have greater reaction specifityEnzymes can be regulated

Amylase Alpha Amylase

Salivary Amylase

Amylasedigests starch by catalyzing hydrolysis, which is the breaking of a bond in a molecule by water

Classifications:

-Amylase-Amylase-Amylase

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Saccharification- final stages of depolymerization are mainly the formation of mono-, di-, and tri-saccharides

Alpha amylasemade by the pancreas and salivary glands to hydrolyse alpha bonds of large, alpha-linked polysaccharides, such as starch and glycogen, intodisaccharides and trisaccharides

Activator:Chloride is essential

Salivary AmylaseA digestive enzyme secreted by salivary glandsbegins the chemical process of digestionFormerly known as ptyalinutilizes starch as a substrate and produces sugars as products

Enzymatic hydrolysis of starch breaks large, insoluble starch molecules into dextrins:

1. Amylodextrin intermediate product of hydrolysis of starch, soluble in water. Gives color blue with iodine.2. Eryhtrodextrin gives red color with iodine3. Achrodextrin gives no color with iodine

Hydrolysis is a reaction involving the breaking of a bond in a molecule using water. The reaction mainly occurs between an ion and water molecules and often changes thepHof a solution.

StarchInsoluble in cold water or alcoholstarch molecules are glucose polymers linked together by the alpha-1,4 and alpha-1,6 glucosidic bondsCan be separated into two fractions: amylose and amylopectin

Amylose: 10-20%, unbranched, single chain polymer of 500 to 2000 glucose subunits with only the alpha-1,4 glucosidic bonds (forms colloidal dispersion in hot water); Amylopectin: 80-90%, branched, the presence of alpha-1,6 glucosidic linkages (insoluble)

Amylose in starch is responsible for the formation of a deep blue color in the presence of iodine.

On Starch

Figure 6. Starch molecule in either branched chains (amylopectin) or unbranched chains (amylose) Retrieved from:https://thescienceofnutrition.wordpress.com/2012/04/20/nutrition-101-carbohydrates/

Figure 7.

AmyloseThe structure of amylose consists of long polymer chains of glucose units connected by analpha acetallinkageAll of the monomer units are alpha -D-glucose, and all the alpha acetal links connect C1 of one glucose and to C4 of the next glucose.As a result of the bond angles in theacetal linkage, amylose actually forms a spiral orcoiled springThe iodine molecule slips inside of the amylose coil

On the Iodine Test

Figure 8. Iodine inside amylose coilRetrieved from: http://www.elmhurst.edu/~chm/vchembook/548starchiodine.html

Iodine TestBlue-black color indicates the presence of starchAmylose is responsible for the blue-black color in the presence of iodineIf starch amylose is not present, then the color will stay orange or yellow. Starch amylopectin does not give the color, nor does celluloseThe iodine molecule slips inside of the amylose coilKI Reagent Iodine is not very soluble in water. Reagent is made by dissolving iodine in water in the presence of potassium iodide

Buffers - used as a means of keeping pH at a nearly constant value in a wide variety of chemical applications

Acetic Acid Buffer Solutions (pH 3.75.61)

Phosphate buffer solutions(pH 5.8 8.0) active in unstimulated saliva acid-base pair has a pKa value in the range 6.8-7.2 , which has a maximum buffering capacity that is relatively close to the salivary pH range: 6-8Bicarbonate buffer(pH 9.2 10.8) major buffer in stimulated saliva

Objectives

To determine the optimum enzymatic activity of salivary amylase depending on the different changes of pHTo determine the optimum enzymatic activity of salivary amylase depending on the different changes of temperature

Methodology

Preparing the Solution

1mL saliva

9 mL distilled H2O30 mL 0.5% NaCl40mL Salivary Amylase

Saliva was diluted 40 times.31

Part A: Determining the Optimum Temperature

Incubate for 5 minutes in a 4, 28, 37, 50, and 70Cwater bathMix immediately

Quickly place 2 drops of iodine and 3 drops of solution into well 1 of the spot plate, labeled as 0 timePlace 2 drops of iodine and 3 drops of solution after 1 minute.Repeat for 20 minutes until a yellow - colored solution appearsYellow-Colored SolutionMixture(2mL buffered starch solution + 2 mL enzyme solution)2 mL of enzyme solution2mL of buffered starch solution in a large test tube

Part B: Determining the Optimum pH

Incubate for 5 minutes in a 37C water bathMix immediately

Quickly place 2 drops of iodine and 3 drops of solution into well 1 of the spot plate, labeled as 0 timePlace 2 drops of iodine and 3 drops of solution after 1 minute.Repeat for 20 minutes until a yellow - colored solution appearsYellow-Colored SolutionMixture(2mL buffered starch solution + 2 mL enzyme solution)2 mL of enzyme solution1mL acetate buffer (pH 4, 5, 6.7, 8, 10) + 1mL 2% unbuffered starch in a large test tube

RESULTS

EFFECTS OF TEMPERATURETEAM 1TEMPERATURETIME1/t , min-14oC028oC (room temperature)037oC050oC070oC0

EFFECTS OF TEMPERATURETEAM 2TEMPERATURETIME1/t , min-14oC028oC (room temperature)037oC181/18 = 0.0650oC201/20 = 0.0570oC0

EFFECTS OF pHTEAM 1pHTIME1/t , min-1405161/16 = 0.076.751/5 = 0.208111/11 = 0.09100

EFFECTS OF pHTEAM 2pHTIME1/t , min-1405201/20= 0.056.751/5 = 0.20851/5 = 0.20100

Discussion

A. Effect of TemperatureFor most chemical reactions, the rate of enzyme catalyzed reactions generally increases with temperatureThere is also a temperature range wherein the enzyme is most stable and retains its full activity capacityShape of the graph is SigmoidalRate of most enzymatic reactions approximately doubles for each 10C raised in temperatureVaries per enzyme based on their energy of activation value

The shape of the graph is bell shape which indicates that enzymatic activity has already started at pH 4 but reaches it's optimum level at around 6.7 to 7 pH then steadily decreases in enzymatic activity. The amylase works best at a more neutral pH than in acidic or basic environments.

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B. Effect of pHMost enzymes have a pH value wherein it reaches its optimal conditionsAny pH lower or greater than this would decline the enzymes activityOptimum pH of an enzyme may not be identical with the pH of its normal intracellular surroundingsAverage optimum pH recorded by the group = pH 7The bell shape is the shape of the graph - The enzymatic activity has already started at pH 4 - It then reaches to its optimum level, around pH 6.7 to 7 - Once optimum pH has been reached, the enzymatic activity steadily decreases.Unlike the acidic or basic environments, amylase works best at a more neutral pH.

Conclusion

The optimum temperature of enzymatic activity and specificity of salivary amylase has a value of 37C

The optimum pH level of the enzymatic activity and specificity of salivary amylase has a pH value of 7.

References

Chua-Suba, S. (2011). Laboratory manual in biochemistry. Quezon City: C&E Publishing, Inc.Garret, R., & Grisham, C. (2012). Biochemistry. Boston, Massachusetts: Cengage Learning.Lehninger, A. (1975). Biochemistry. Quezon City: JMC Press, Inc.Mckee, T., & Mckee, J. (2003). Biochemistry: the molecular basis of life. 1221 Avenue of the Americans, New York: McGraw-Hill Company.Gaughan, R. What are the Effects of Boiling & Freezing on Enzyme Activity?. Received from http://education.seattlepi.com/effects-boiling-freezing-enzyme-activity-4327.htmlukessays.com (n.d.). Retrieved February 2015, from http://www.ukessays.com/essays/biology/optimal-temperature-for-enzyme-amylase-biology-essay.php