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BMI_Lab6PhysicalProp - Enzyme Physical Properties Lab 6...

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Unformatted text preview: Enzyme Physical Properties Lab 6 Objective To study the effect of pH and temperature on amylase enzyme To understand the enzymesubstrate specificity Enzyme activity Depends on Substrate specific pH Temperature Substrate concentration or Substrate:enzyme ratio (last week) How enzymes work An enzyme has an active or catalytic site into which a substrate molecule can fit, much like a key fits into a lock. Only substrate with a particular structure will fit into the active site Substrate Substrates must possess two features: 1. a specific chemical bond or linkage that can be attacked by the enzyme. 2. the substrate molecule must properly fit to the active site Substrate specificity Amylase and Acid phosphatase both catalyze the same chemical reaction (hydrolysis) Enzyme is specific for substrate Amylases (salivary, pancreatic, bacterial) Starch ~ neutral pH and at range of temperatures Acid phosphatase Phosphate bonds (PNPP, ATP) in acidic environment Effects of Temperature Mammalian enzymes have optimum temperatures in the range of 35 45 C Thermophilic and Psychrophilic enzymes found in bacteria are useful in biotechnology Temperature accelerate the rate of enzymatic DNA polymerases reactions. High temperatures also accelerate the rate of The initial velocity increases with temperature. enzyme denaturation. Enzyme is inactivated. Effect of pH pH affects the efficiency of an enzyme Enzyme active at a range of pH Optimal pH provides maximum activity Very high or very low pH can also denature enzymes Cellular pH changes help regulate the activity of enzymes pH is an important factor for cellular enzyme regulation Enzyme activity Purity and the source of enzyme varies Activity varies with batch and source Standardized by expressing in units/mg Unit The amount of product formed in unit time at specified pH and temperature Today's Experiment Hydrolysis of starch Starch (the substrate) Amylase (the enzyme) Amylases hydrolyzes the alpha1,4 and alpha1,6 glucosidic bonds in starch. Breaks down starch polymer to smaller maltose units Today's Experiment The enzyme activity can be detected with iodine http://www.elmhurst.edu/~chm/vche mbook/548starchiodine.html Starch + Iodine Starch Iodine complex (violet to blue color) Starch + Amylase Maltose + maltotriose (2 glucose units) (3 glucose units) Maltose+maltotriose + Iodine No blue color Slight variations in color maybe observed depending on the bonds cleaved and fragments formed Effects of temperature and pH on amylase activity Test amylase at 4C (set A) and 37 C (set B) Three different buffers 1 tube with and 1 tube without amylase for each Acetate at pH 4 2 tubes for set A and 2 tubes for set B Phosphate pH 7.2 2 tubes for set A and 2 tubes for set B Carbonate pH 10 2 tubes for set A and 2 tubes for set B temperature and pH Negative controls have no amylase No hydrolysis Blue color Effects of temperature and pH on amylase activity Bring test solution to temperature Incubate for 15mins at temperature Then add amylase or water (negative control) Incubate again to perform the reaction Add iodine to test whether the reaction has occurred Compare with those containing amylase Add an additional drop of iodine if necessary Table 1: set A at 4 C and set B at 37 C Tub e# Starc h Acetat e buffer PO4 CO3 buffer Buff er --------- 1 2 3 4 5 6 0.1ml 0.9ml 0.1ml 0.9ml 0.1ml --0.1ml --0.1ml --0.1ml --- 0.9ml --0.9ml ------0.9m l 0.9m l I N C U 0.1ml B A --T E 0.1ml 1 --5 m 0.1ml in s --- Amylas e Distill ed water --0.1ml --0.1ml --0.1ml Iodine I drops Color Set A 4 C Set B 37 C N C U 2 drops B A T E 2 drops 1 5 m i 2 drops n s Substrate specificity To test if starch is hydrolyzed by acid phosphatase Use the optimal conditions determined as in Table 1 Use a positive control Positive control has amylase hydrolysis No Blue color (yellow color!) Table 2: Substrate specificity Tube # Starch Optimal buffer 1 0.1ml 0.9ml IN Amylase Acid IN Iodine C enzyme phosphatase C enzyme U UB BA AT --TE 0.1ml E 2 drops 15 min s --0.1ml 15 min s 2 drops Color 3 0.1ml 0.9ml NEXT WEEK MIDTERM EXAMS # of Questions Types of QUESTIONS Total points 53 ( 35 for midterm + 18 for Quiz, Short Answers Fill in the blanks Multiple choice lab reports & techniques) On average students who do not get >50% (26 points/53) in the midterm usually fail the finals Study and come on time No Make ups ...
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