03.11 Practical: Enzymes

Objective

• Purpose: To immobilise the enzyme lactase in alginate beads for converting lactose in milk to lactose-free milk. This method ensures enzyme reusability and product purity, as the enzyme does not mix with the final product.

Materials

• Sodium alginate solution
• Lactase enzyme solution
• Calcium chloride solution (for forming beads)
• Milk (substrate for lactase)
• Dropper or syringe
• Beaker for calcium chloride solution
• Column or tube (to hold and test immobilised beads)
• Stirring rod and spatula

Method

1. Prepare the Enzyme Mixture:

• Mix equal parts of sodium alginate solution and lactase enzyme in a small container.
• Stir gently to ensure even distribution of the enzyme within the alginate.

1. Form Alginate Beads:

• Fill a beaker with calcium chloride solution.
• Using a dropper or syringe, add small droplets of the enzyme-alginate mixture into the calcium chloride.
• Observe the reaction: As each drop enters, it forms a gel-like bead due to an instant reaction between sodium alginate and calcium chloride, creating a jelly-like shell that immobilises the enzyme.

1. Rinse and Collect Beads:

• Allow the beads to set for a few minutes, ensuring they fully solidify.
• Rinse the beads gently in distilled water to remove any excess calcium chloride, preventing contamination in the reaction.

1. Pack Beads into a Column:

• Place the beads carefully into a vertical column or tube, avoiding compacting them too tightly (to ensure good flow of substrate).
• The beads should be loosely packed so that milk can flow smoothly through and around them.

1. Run Substrate Through the Column:

• Pour milk (containing lactose) into the column, allowing it to flow slowly over the beads.
• As milk passes over the beads, lactase immobilised in the beads hydrolyses lactose into glucose and galactose.
• The lactose-free milk trickles out from the bottom of the column.

1. Collect and Test Product:

• Collect the milk that exits the column. This milk should be free of both lactose and lactase enzyme.
• You may test the milk for glucose presence to confirm lactose breakdown (using glucose test strips).

Why This Method is Effective

1. Cost Efficiency:

• Enzyme Reusability: The lactase remains immobilised in the beads and can be reused for multiple batches, reducing the cost of enzyme replenishment.

1. Product Purity:

• No Contamination: Lactase does not mix with the milk, so the final product is free from enzyme contamination, simplifying purification.

1. Enhanced Stability:

• Protection from pH and Temperature Changes: Enzyme molecules are held in place by the alginate gel, providing a more stable environment that makes them less susceptible to denaturation.

Tips and Considerations

• Bead Size: Ensure droplets are of consistent size to maintain uniform flow rate.
• Flow Rate: The milk should trickle slowly; a rapid flow may reduce contact time between substrate and enzyme, leading to incomplete lactose conversion.
• Column Packing: Beads should not be packed too tightly to avoid blockages, which can limit substrate contact with the enzyme.

Summary

This immobilisation method is widely used in food technology and medicine for its efficiency, stability, and cost-saving benefits, making it ideal for processes like producing lactose-free dairy products.

Overview of a Colorimeter

• Colorimeter: Instrument that quantitatively measures the color intensity of a solution by detecting light absorption at specific wavelengths.
• Measures how much light of a particular wavelength passes through a solution to determine concentration of the colored substance.
• Key Components:
  • Light source: Provides light of a known wavelength.
  • Filter: Selects specific wavelengths of light for the measurement.
  • Cuvette: Holds the sample solution.
  • Light meter: Measures the light that passes through the sample and displays the absorbance or transmission reading on the readout.

Application in Enzyme Reactions – Amylase-Starch Experiment

• Purpose: To monitor the breakdown of starch by amylase by measuring the color intensity change in an iodine test.
• Iodine Test: Iodine solution turns blue-black in the presence of starch.
• Reaction Observation: As amylase breaks down starch into maltose, the blue-black color fades, indicating decreasing starch concentration.

Steps for Using a Colorimeter in Enzyme Experiments

1. Prepare the Reaction Mixture:

• Mix starch solution, iodine solution, and amylase enzyme in a test tube.
• The reaction starts once amylase is added, so prepare the colorimeter beforehand to take immediate readings.

1. Calibrate the Colorimeter:

• Place a blank cuvette (usually containing water or iodine solution without starch) into the colorimeter and set the absorbance to zero. This ensures all subsequent readings are relative to a baseline.

1. Insert the Reaction Sample:

• Pour a small amount of the reaction mixture into a clean cuvette.
• Wipe the cuvette with a lint-free cloth to avoid fingerprints, which can interfere with the reading.
• Place the cuvette into the colorimeter’s sample holder, ensuring it’s aligned correctly.

1. Measure Absorbance or Transmission:

• Select the wavelength filter that matches the color change (often a red or orange filter for blue-black iodine-starch complexes).
• Start timing and take an initial absorbance reading.
• Record absorbance or transmission readings at regular intervals (e.g., every 30 seconds or 1 minute) to track changes over time.

1. Record Observations Over Time:

• As starch concentration decreases (due to enzyme action), the blue-black color fades to brown, pale brown, and eventually colorless.
• The decreasing absorbance values (or increasing transmission) correspond to the breakdown of starch.

Plotting Results and Calculating Reaction Rates

• Graphing:
  • Plot ‘Absorbance’ (or ‘Starch remaining’) vs. ‘Time’ to produce a reaction curve.
  • The curve typically starts steep (high rate of starch breakdown) and gradually levels off as starch is consumed.
• Calculating Initial Rate of Reaction:
  • Use the initial slope of the curve at the start of the reaction for the initial rate.
  • Alternatively, calculate the change in absorbance over the first 30 seconds to determine the initial reaction rate, which represents the enzyme’s efficiency when substrate concentration is highest.

Practical Notes and Tips

Sample Collection:

• For a more accurate result, take regular samples at set intervals and test each one’s absorbance immediately after sampling.

Avoid Iodine Interference:

• Adding iodine directly to the reaction mixture can slow down the reaction; consider testing separate samples instead of mixing iodine into the main reaction tube.

1. Introduction to Amylase

• Definition: Amylase is an enzyme that catalyzes the hydrolysis of starch into sugars.
• Sources: Commonly found in saliva (salivary amylase) and the pancreas (pancreatic amylase) of mammals.
• Function: Initiates the digestion of dietary starches in the mouth and continues in the small intestine.

2. Enzyme Activity Assay Using Starch-Agar Plates

• Starch-Agar Composition:
  • A jelly-like medium containing starch, which serves as the substrate for amylase.
• Procedure:
  1. Preparation of Wells:
     • Small holes (wells) are cut into the starch-agar using a cork borer.
  2. Application of Enzyme Samples:
     • Equal volumes of amylase samples are placed into each well.
  3. Diffusion and Starch Digestion:
     • Enzyme molecules diffuse outward from the well, breaking down starch in their path.
  4. Iodine Staining:
     • After incubation, iodine in potassium iodide solution is poured over the plate.
     • Reaction: Iodine binds to remaining starch, turning the agar blue-black.
     • Halo Formation: Areas where starch has been digested remain clear, forming a halo around each well.
• Interpretation:
  • Halo Size: Indicates enzyme activity.
    • Larger Halo: Higher amylase activity (more starch digestion).
    • Smaller Halo: Lower amylase activity (less starch digestion).

3. Experimental Design: Assessing Denaturation of Amylase at 60 °C

• Objective: To investigate the rate at which mammalian amylase is denatured when exposed to 60 °C.
• Methodology:
  1. Sample Preparation:
     • Multiple enzyme samples are prepared for different heating durations.
  2. Heat Treatment:
     • Samples are heated in a water bath at 60 °C for varying times: 0, 1, 5, 10, and 30 minutes.
  3. Cooling:
     • Heated samples are allowed to cool to room temperature to halt the denaturation process.
  4. Application to Starch-Agar Plates:
     • Equal volumes of each cooled sample are placed into separate wells on five starch-agar plates.
  5. Incubation:
     • Plates are incubated in an oven at 40 °C for 24 hours to allow enzyme diffusion and starch digestion.
• Expected Outcomes:
  • 0 Minutes (Control): Maximum enzyme activity, largest halo.
  • Increasing Heating Times: Progressive denaturation, resulting in smaller halos.

4. Data Interpretation

• Halo Size vs. Heating Time:
  • Graphical Representation: Typically, a graph plotting halo diameter against heating time would show a decreasing trend.
• Denaturation Kinetics:
  • Initial Phase: Rapid loss of activity as the enzyme begins to unfold.
  • Later Phases: Slower rate of denaturation as the remaining active enzymes stabilize or aggregate.
• Rate of Denaturation:
  • Calculation: Measure halo diameters at each heating time and plot to determine the rate constants.
• Implications:
  • Understanding thermal stability of amylase is crucial for applications in food industry, biotechnology, and understanding physiological processes.

5. Controlled Variables and Considerations

• Controlled Variables:
  • Enzyme Concentration: Equal volumes ensure consistent enzyme amounts across samples.
  • Starch-Agar Composition: Uniform agar ensures consistent substrate availability.
  • Incubation Conditions: Same temperature and time for all plates to ensure comparability.
• Potential Sources of Error:
  • Inconsistent Well Sizes: May affect diffusion rates.
  • Temperature Fluctuations: In water bath or oven could lead to variable denaturation.
  • Measurement Accuracy: Precision in measuring halo diameters is crucial.

6. Conclusions

• Denaturation Effect: Prolonged exposure to 60 °C leads to progressive denaturation of amylase, reducing its enzymatic activity as evidenced by smaller halo sizes.
• Thermal Stability Insights: The experiment elucidates the thermal limits of amylase activity, providing valuable data for its application under varying temperature conditions.
• Further Research: Additional studies could explore denaturation at different temperatures, the reversibility of denaturation, or the effect of stabilizing agents.

7. Diagram Reference

• Starch-Agar Plate Diagram:
  • Illustrates wells with surrounding halos.
  • Visual comparison of halo sizes corresponding to different heating times.
• Real Halo Sizes:
  • Actual measurements provide quantitative data for analysis.