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Prepared by Law Jeng Yih

CPB 30103 Biochemical Engineering

EXPERIMENT 2:

ENZYME ASSAYS AND FACTORS AFFECTING ENZYME ACTIVITY

OBJECTIVES To comprehend physical characteristic and properties of enzymes To study the factors affecting their activity. INTRODUCTION Enzymes are biological catalysts, functioning to increase the rate of chemical reactions. Most enzymes are proteins, and their catalytic action results from their complex structure. The great diversity of protein structure allows enzyme action to be highly specific. The action of an enzyme in speeding the biochemical conversion of a substance into something else can be simply described as follows:

THEORY Starch makes up a large proportion of our diet, and it needs to be made soluble before it can be absorbed into our bodies. In order to carry out this chemical breakdown, various glands produce digestive juices containing amylases to be mixed with the food. Saliva contains one amylase, and pancreatic juice contains another. Different amylase enzymes are produced by other organisms, including fungi and bacteria, which carry out external digestion. In fact, any organisms that grow on starch must produce an enzyme to break it down. Plant seeds often contain a reserve of rather dry insoluble starch for the embryo plant, and when a seed germinates it must produce an amylase to convert the starch into soluble sugars which can be transported to the growing points at tips of root and shoot of the growing plant. Amylase is a general name for an enzyme which breaks down starch. Amylase hydrolyzes starch (a polysaccharide) into maltose, a sugar (dissacharide). In this investigation, this enzyme will be used to demonstrate the effect of temperature, pH and enzyme concentration on enzyme activity.

Prepared by Law Jeng Yih

CPB 30103 Biochemical Engineering

LABORATORY PROCEDURE Apparatus and Reagent 1. 15-20 test tube 2. Water bath 3. Shaker 4. UV-Vis spectrophotometer 5. Amylase enzyme (commercial) 6. Starch solution (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%; w/v) 7. Glucose standard (0, 20, 40, 60, 80, 100 mg/L) 8. Nelson reagent 9. Copper reagent 10. Distilled water 11. Phosphate buffer pH 5.0, 7.0, 8.0, 9.0, 11.0 Procedure A. Effect of Temperature on Enzyme Activity a) Determine the amylase activity using method shown in Appendix 1. b) Determine the optimum temperature for the enzyme by incubating the reaction mixture at various temperatures: 30C, 40C, 50C, and 60C. c) Determine the amylase activity at all temperature and plot a graph of product formed against temperature. B. Effect of Substrate Concentration on Enzyme Activity a) Prepare a starch solution of concentration 0.5, 1.0, 1.5, 2.0 and 3.0% (w/v) as the substrate. Place 0.5 mL of the substrate of various concentrations into the test tubes. b) Add 0.2 mL enzyme preparation and 0.3 mL, 0.2 M phosphate buffer at pH 7.0 c) Incubate the reaction mixture for 30 min at 30C with shaking of 150 rpm. d) Determine the amylase activity using the method given in Appendix 1. e) Plot the graph of product formed against substrate concentration. C. Effect of pH on Enzyme Activity a) Determine the amylase activity using method shown in Appendix 1. b) Determine the optimum pH for the enzyme by incubating the reaction mixture at various pH: 5.0, 7.0, 8.0, 9.0, 11.0. c) Determine the amylase activity at all pH and plot a graph of product formed against pH.

Prepared by Law Jeng Yih

CPB 30103 Biochemical Engineering

Appendix 1: Amylase Activity Assay a) Enzyme preparation of 0.2 mL is mixed with 0.3 mL, 0.2 M phosphate buffer at pH 7.0 followed by 0.5 mL of 1% (w/v) of starch solution. b) Allow mixture for 30 min at 30C using an incubator with shaking at 150 rpm. c) After 30 min, add 1.0 mL of distilled water followed by 10 mL of copper reagent. d) Add 1.0 mL of the Nelsons reagent followed by 10 mL of distilled water. Allow it to stand for 15 min. e) Determine the optical density at 660nm. Appendix 2: Glucose Standard Curve Preparation a) Prepare glucose solution with concentration in the range of 0-100 mg/L. b) Mix 0.5 mL glucose solution of each concentration with 0.3 mL, 0.2 M phosphate buffer at pH 7.0. c) Determine the optical density by repeating step (b)-(e) in Appendix 1. d) 1U (unit) of amylase activity is defined as the amount of enzyme required to release 1g of reducing sugar per min under the conditions stated. Results/Discussion Describe the predicted/expected results for the investigation of the effect of temperature on enzyme activity. Did you obtain predicted/expected results? If not, what are the predicted/expected results and can you make any reasonable suggestions why you did not get them? What happens to the rate of enzyme reaction when you increase the concentration of an enzyme? What is the effect of a change in pH (away from the preferred pH) on the rate of enzymatically catalyzed reactions? What was the substrate used in this experiment? What was the enzyme used in this experiment?