Enzymatic-Activity-of-Salivary-Amylase.pptx

Powerpoint about our lab experiment on enzymatic activity of salivary amylase

1. Experiment 4: Enzymatic Activity of Salivary Amylase Jalipa , Jomari Mesa, Hannah Pichon, Jasmine San Pedro, Anna Villasenor , Maiko

2. Introduction

3. Introduction What are ENZYMES? Most diverse group of proteins Acts as biological catalysts cause or speed up chemical reactions by lowering the activation energy ( E a ) Important biological reactions catalyzed by enzymes: Metabolism DNA synthesis RNA synthesis Protein synthesis Digestion

4. Introduction What are ENZYMES? Active site pocket or cleft contains amino acid side chains that participate in substrate binding and catalysis Substrate Reactant molecules

5. Introduction Properties of Enzymes Large protein molecules Reusable (example: 2 H 2 O 2 [catalase] → 2 H 2 O + 1 O 2 ) Remain unchanged VERY specific Operate at very high speeds Rate of reaction is dependent upon temperature, pH, [E], and [S] Usually work best with 60°C Denaturation occurs at very high temperatures

6. Introduction BINDING MODELS: LOCK & KEY MODEL Emil Fischer Enzyme specificity Substrate has a complimentary shape to the active site Substrate: key :: Enzyme: lock Wrong key = no reaction

7. a.k.a. “hand and glove model” substrate induces a slight change in the shape of the active site so that they both compliments each other’s shape. BINDING MODELS: INDUCED FIT MODEL

8. HOW DO ENZYMES WORK?

9. SALIVARY AMYLASE Digestive enzyme responsible for catalyzing starch molecules into simpler sugars (i.e. maltose and isomaltose ) Secreted by the salivary glands OPTIMUM TEMPERATURE: 37ᴼC OPTIMUM pH: 5.6 – 6.7

10. STARCH Mixture of two polysaccharides: amylose and amylopectin White, tasteless, solid carbohydrate Converted to glucose via hydrolysis Detected by iodine (blue-black color)

11. THE BASIC REACTION: SUBSTRATE enzyme PRODUCT

12. OBJECTIVES: to observe the effect of different temperatures and pH on the enzymatic activity and specificity of the salivary amylase to determine the optimal temperature and pH values of the salivary amylase

13. Materials FOR A. EFFECT OF TEMPERATURE: Enzyme solution (1 mL saliva + 9 mL distilled H2O+ 30 mL 0.5% NaCl Buffered starch (1% starch in phosphate buffer pH 6.7) 0.001 M Iodine solution 2 Spot plates Test tubes Medicine droppers Constant Temperature bath (4, room temp, 37, 50, 60 and 70 °C)

14. Materials FOR B. EFFECT OF PH : Enzyme solution (1 mL saliva + 9 mL distilled H2O+ 30 mL 0.5% NaCl 2% Unbuffered starch 0.001 M Iodine solution Acetate buffer solutions (pH 4 and 5) Phosphate buffer solutions (pH 6.7 and 8) Bicarbonate buffer (pH 10) 2 Spot plates Test tubes Medicine droppers Water bath set at 37°C

15. Methodology A. Effect of Temperature 2 mL enzyme solution In separate test tube, 2 mL of buffer starch solution Incubate in 4°C ice bath for 10 minutes Immediately mix 2 mL enzyme solution to 2 mL buffer starch solution. Enzyme- starch solution Immediately take 3 drops and put in spot plate well. Add 2 drops Iodine solution.

16. Continue incubation for 1 minute Q uickly take 3 drops of enzyme solution and 2 drops of iodine and add it to the second spot plate well. Record color Label as “1 minute” Continue incubation for 1 minute until a light yellow- colored solution is observed. Light yellow colored solution Repeat procedures for other temperatures (RMT, 37, 50, 60 and 70°C) *Use hot water for high temperatures Plot the reciprocal of time (1.t, min -1) versus the temperature (T). Determine optimum temp. of Amylase Blue-Violet colored solution Note time (t)

17. B. Effect of PH 1 mL 2% u nbuffered starch With 1 mL acetate buffer (pH4) 2 mL enzyme solution Incubate for 10 minutes in 37° C Immediately take 3 drops and put in spot plate well. Add 2 drops Iodine solution. Label as “zero minute” Record color

18. Blue-Violet colored solution Light yellow colored solution Continue incubation for 1 minute Q uickly take 3 drops of enzyme solution and 2 drops of iodine and add it to the second spot plate well. Record color Label as “1 minute” Continue incubation for 1 minute until a light yellow- colored solution is observed. Note time (t) Repeat procedures for the other pH (5, 6.7, 8, and 10) using the appropriate buffer. Plot the reciprocal of time (1/t, min -1) versus the buffer pH (pH). Determine optimum pH of Amylase

19. Results

20. Effect of Temperature Temperature Time (min.) Rate (1/time) 4 12 0.083 32.8 1 1 37 1 1 60 5 0.20 70 ∞

21. Rate (1/min) Temperature 4 32.8 37 60 70

22. Effect of pH pH Time (min.) Rate (1/time) 4 ∞ 5 12 0.0833 6.7 3 0.33 8 4 0.25 10 14 0.07

23. pH Rate (1/min)

24. Discussion

25. Salivary Amylase Alpha amylase Form produced by your salivary gland and pancreas Hydrolyzes alpha bonds of large, alpha linked oligosaccharides and polysaccharides Acts on linear alpha(1,4) glycosidic linkages Starch + amylase  maltose + salivary amylase + dextrin

26. Salivary Amylase Chloride-dependent enzyme AMY1A gene encodes human salivary amylase Influence salivary amylase concentration and enzymatic activity

27. Effect of Temperature Enzymes work best at a certain temperature (optimum temperature) where the reaction rate is at maximum Optimum temperature: 37 °C (body temperature) Lower than 37°C = slower reaction At 40 °C and above, enzymes start to denature - Destruction of 2° and 3° structures

28. Effect of pH pH affects the detailed structure of the active site of the enzyme Requires certain level of acidity and alkalinity Too much or too little H+ interferes with electric charges and disrupts H-bonds Extremely high or low pH will result in the complete loss of enzymatic activity

29. Effect of pH Optimum pH: pH 6.7 – 7 pH 10 is when most enzymes are denatured

30. Conclusion

31. Conclusion: Effect of Temperature on Enzymatic Activity Enzymes work best at a certain temperature (optimum temperature) where the reaction rate is at maximum Optimum temperature = 37 °C

32. Conclusion: Effect of pH on Enzymatic Activity Affects the ionization of acidic or basic amino groups Affects the shape of the substrate Optimum pH for salivary amylase = pH 6.7 -7