CHE506 Lab 8
Lab Manual
Edited Feb 2017
Laboratory 5: Investigation Investigation on enzyme activit activit y and kinetics
Objectives •
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Determination Determinat ion of the effects of temperature temperat ure on the enzymatic activity and changes in enzyme concentration of an enzyme-catalysed enzyme-catalysed reaction. Describe the relationship relationshi p between substrate substrat e concentration concentrat ion and the maximum velocity of an enzyme. Estimation of Michaelis-Men Michaelis-Menten ten parameters, parameters, effect effect of pH and and temperature on enzyme enzyme activity and kinetics of inhibition.
1. Introduction Amylases are a family of enzymes that degrade starch (polymers (polymers of glucose) glucose) into smaller disaccharides disaccharides (maltose). A molecule of water is also split during this reaction and the OHand H+ ions bind to the exposed ends of the broken starch polymer. This type of reaction is called hydrolysis (water splitting).Hydrolysis is a common mechanism used by enzymes to break chemical bonds.
The hydrolysis of starch can be measured through the use of an enzyme test or assay. assay. An enzyme enzyme assay assay will will test for the simple simple presence of enzyme enzyme activity activity but but can also be used used to measure the reaction rate of an enzyme-catalyzed reaction. The assay can measure either the appearance of one of the products or the disappearance of one of the substrates over time. To measure your amylase amyla se activity, you will monitor the disappearance of amylase’s substrate, starch. Starch reacts with iodine (which is yellow) to form a blue compound (Amax 620 nm). This reaction is the basis of a colorimetric assay for amylase activity. Broth culture supernatant (which contains the secreted amylase) will be incubated with starch. After the incubation period, a portion (an aliquot) aliquot ) of this mixture is combined with acidic iodine. The acid stops the enzymatic reaction and the iodine reacts with the starch to produce the blue color. Any starch that has not yet been hydrolyzed by the amylase will turn blue, with the intensity of the blue color being proportional to the amount of starch remaining. The intensity of the blue color can be quantified spectrophotomet spectrophotometrically rically by measuring its absorbance at 620 nm. The greater the change in absorbance between a
CHE506 Lab 8
Lab Manual
Edited Feb 2017
sample containing the initial amount of starch (without enzyme) and the hydrolyzed mixture containing the enzyme, the greater the amount of st arch degraded by the enzyme, therefore the greater the activity of the enzyme being measured. Enzyme activity (reaction rates) is dependent upon the environmental conditions either in nature or in the laboratory (e.g. temperature, pH, etc.). This is because these conditions can alter the amino acid side chains in a protein, affecting protein structure and folding and sometimes the enzyme's active site. The effects of some of those conditions will be explored in this exercise. Just as in any chemical reaction, the concentration of reactants (substrates) will affect enzymatic reaction rates. In regards to substrate concentration, enzyme kinetics follows the Michaelis-Menton Model:
2. Apparatus and Reagent 1. 2. 3. 4. 5. 6. 7. 8.
Alpha Amylase enzyme Starch pH buffer solution (pH 4-9) DNSA Reagent Beaker Measuring cylinder Cuvette Falcon tube rack
9. Falcon tube 10. Micropipet and tips 11. Label sticker 12. Schott bottle 13. Vortex mixer 14. Water bath 15. Spectrophotometer 16. Hotplate
CHE506 Lab 8
Lab Manual
Edited Feb 2017
3. Experimental Procedures i. Preparatio n of 2% Starch Soluti on a) Mix 4 g of soluble starch in approximately 50 ml of cold water. b) While stirring, add the slurry to approx. 100 ml of gently boiling water in a large beaker. c) Then top up to final volume of 200ml and mix well.
ii. Effect of pH on the activit y and stabili ty of amylase enzyme. a) Label five test tubes with pH 5, 6, 7, 8 and 9. In each tube, place 1 mL of 2% starch solution. Add 1 mL of the appropriate buffer (at corresponding pH) to each tube. b) Get five additional clean test tubes and put 2 mL of amylase solution in each tube. c) Place all 10 tubes in the 37°C water bath for about 5 minutes to allow the temperature to equilibrate. d) Pour the content of each amylase test tube into each starch test tube and mix them on vortex mixer. e) Return the tubes to the 37°C water bath. f) Let the hydrolysis reaction proceed for exactly 10 minutes. g) Determine the amylase activity using the method given in Appendix 1. h) Plot a graph of pH vs. enzyme activity.
iii. Effect of temperature on the activity and stabili ty of amylase enzyme. a) Label one test tube with 30ºC. In the tube, place 1 mL of 2% starch solution and 1 mL of pH=7 buffer to the tubes. b) Get additional clean test tub and put 2 mL of amylase solution in the tube. c) Place both tubes in the 30°C water bath for about 5 minutes to allow the temperature to equilibrate. d) Pour the contents of the amylase test tube into starch test tube and mix them on vortex mixer. e) Return the tubes to the 30°C water bath. f) Let the hydrolysis reaction proceed for exactly 10 minutes. g) Determine the amylase activity using the method given in Appendix 1. h) Repeat step a-g at 4 different temperatures ranging from 30-70 ºC. i) Plot a graph of temperature vs. amylase activity.
CHE506 Lab 8
Lab Manual
Edited Feb 2017
iv. Effect of subs trate concentration on the activity of amylase enzyme. a) Prepare starch solutions of varying concentration (0.5, 1.5, 2.0, 2.5, and 3.0% w/v) as the substrate. b) Label each tube with starch concentration and place 1ml of each starch solution into the test tubes. c) Add 1 mL of pH=7 buffer to the tubes. d) Get five additional clean test tubes and put 2 mL of amylase solution in each tube. e) Place all tubes in the 37°C water bath for about 5 minutes to allow the temperature to equilibrate. f) Pour the content of each amylase test tube into starch test tube and mix them on vortex mixer. g) Return the tubes to the 37°C water bath. h) Let the hydrolysis reaction proceed for exactly 10 minutes. i) Determine the amylase activity using the method given in Appendix 1. j) Plot the graph of starch concentration against amylase activity.
Appen di x 1 (Dem on st rat io n o f En zyme Ac ti vi ty ) a. b. c. d. e.
After 10 minutes (the time of hydrolysis reaction), stop the reaction by adding 4 ml of DNS reagent. Boil for 10 minutes and then left to cool to room temperature. Measure the absorbance of the samples at λ=540 nm. Compare the absorbance value with glucose standard curve prepared to obtain the glucose concentration. Calculate the enzyme activity. Note: Enzyme activity is the amount of glucose formed in reaction mixture per unit time.
Appen di x 2 (Gl uc os e Standar d Curv e Prep aratio n) a. b. c. d. e. f.
Prepare standard solutions of glucose at five different concentrations ranging from 01000mg/L by serial dilution. Put 1 ml of each glucose solution in test tubes. Add 1 ml of DNS reagent in each tube and mix for few seconds on vortex mixer. Place the test tubes in water bath (T=100°C) for 10 min and then left to cool at room temperature. The absorbance of the samples is measured at λ=540 nm. Draw the standard curve of absorbance vs glucose concentration. Note: The graph is in straight line for absorbance less than 0.7.
CHE506 Lab 8
Lab Manual
Edited Feb 2017
4. Report : (100 marks) Plagiarism is highly prohibited.
Abstract/Summary
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Introduction
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Aims/Objective
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Theory
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Apparatus
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Methodology/Procedure
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Results
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Calculations
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Discussion
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Conclusion
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Recommendation
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Reference/Appendix
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