Enzyme Lab

Enzyme Activity and Invertase Overview

  • Enzymes: Biological catalysts that speed up chemical reactions.

  • Experimental Focus: The laboratory will illustrate the relationship between enzyme activity and four key parameters:

    • 1) Substrate amount

    • 2) Enzyme amount

    • 3) pH

    • 4) Temperature

Invertase Enzyme

  • Enzyme Specification: The experiment will utilize the enzyme invertase, sourced from Saccharomyces cerevisiae, commonly known as baker's or brewer's yeast.

  • Function of Invertase: Invertase catalyzes the hydrolysis of sucrose, which is a disaccharide, into two products:

    • Glucose

    • Fructose

Measuring Enzyme Activity

  • Challenge: The enzymes, substrate, and products involved in the experiments are colorless, which raises the question of how to track enzyme activity.

  • Solution: The Benedict's Test will be employed to detect enzyme activity.

Benedict's Test Details

  • Purpose: Detects the presence of simple reducing sugars, which includes monosaccharides containing free aldehyde groups in a solution.

  • Benedict's Solution: This blue solution undergoes a color change upon boiling with simple sugars, leading to the formation of precipitates ranging in color from light green to brick red.

  • Qualitative Measure of Enzyme Activity: The extent of the color change can be used to estimate the amount of sucrose that has been hydrolyzed by the enzyme invertase.

  • Absence of Sugar Indicator: No colored precipitate is formed in the absence of simple reducing sugars, indicating no enzyme activity.

Table 9.1: Color Change Indication of Benedict’s Test

  • Enzyme Activity/Reaction Strength: Based on the amount of simple sugar indicated by the color change in the solution.

Dispensing Solutions Using a Burette

  • Equipment: Students will dispense solutions using a burette, which is a glass tube designed to hold 50 ml of liquid and features gradations of 1 ml and 0.1 ml.

  • Functionality: Precise volumes can be dispensed through a stopcock located at the bottom of the burette.

  • Preparation: Familiarize yourself with using the burette prior to starting the experiment. Refer to the accompanying handout titled "Using a Burette" from the BLC.

Important Burette Handling Instructions

  • Stopcock Operation: Do not push down while turning the stopcock; simply turn it to dispense liquid.

Safety Precautions

  • Eye Protection: Mandatory when working with acids and bases.

  • Skin Contact Response: In case of contact with pH buffer or Benedict's solution, immediately wash hands generously with water.

  • Handling Hot Water Baths: Caution is advised when working with high-temperature water baths, particularly those at 100°C (boiling), due to the risk of burns from steam and hot water.