The effect of pH on the Invertase enzyme

Summary

The video explains an experiment investigating how pH affects the activity of the enzyme invertase, which breaks down sucrose into glucose and fructose. It details the equipment needed, safety precautions, and step-by-step procedures involving different pH buffers, sucrose solutions, and a spectrophotometer. The experiment uses DNS reagent to measure enzyme activity at 540 nm absorbance, indicating sugar reduction. After analyzing the results, the video concludes that the optimal enzyme activity occurs at pH 5. Safety guidelines like using gloves, goggles, and a fume hood are emphasized throughout the process.

Takeaways

• 🌐 The experiment focuses on studying invertase, an enzyme that breaks down sucrose into fructose and glucose.
• 🍬 Invertase is commonly obtained from yeast and is used in the production of certain candies like cherry cordials.
• 🔍 The experiment aims to determine how pH affects the activity of invertase.
• 🧪 Essential equipment includes a spectrophotometer, water baths, test tubes, glass cuvettes, and a 0.3% sucrose solution.
• 🧴 Safety gear such as gloves, goggles, lab coats, fume hoods, and hot mitts are required.
• ⏱️ The spectrophotometer needs to warm up for at least 30 minutes before use.
• 🧪 Test tubes are labeled and filled with sucrose solution and pH buffers to test different pH levels.
• 🌡️ Test tubes are incubated in a 50-degree water bath for 15 minutes to allow the enzyme to act.
• 🔴 DNS (dinitro salicylic acid) is used to react with glucose, and the reduced DNS is measured using a spectrophotometer.
• 🌡️ After the reaction, test tubes are placed in a 100-degree water bath for 10 minutes to stop the reaction.
• 📊 The absorbance at 540 nanometers is measured, which indicates the amount of glucose produced and thus enzyme activity.
• 📊 The results show that optimal enzyme activity occurs at pH 5.

Q & A

What is the purpose of studying invertase in this experiment?

The purpose is to study how pH affects the activity of invertase, an enzyme that breaks down sucrose into fructose and glucose.

What is the source of invertase used in the experiment?

Invertase is usually obtained from yeast.

What candies are invertase enzymes commonly used in?

Invertase enzymes are commonly used in making candies such as cherry cordials.

What equipment is necessary for the experiment and why?

The necessary equipment includes a spectrophotometer, water baths at 50-100 degrees, test tubes, test tube racks, glass cuvettes, kim wipes, transfer pipettes, and various buffers. These are used to measure enzyme activity under different pH conditions.

What safety equipment is recommended for this experiment?

Gloves, goggles, a lab coat, a fume hood, and hot mitts are recommended to ensure safety while handling corrosive solutions and working with hot water baths.

Why is it important to warm up the spectrophotometer before starting the experiment?

Warming up the spectrophotometer ensures it is functioning correctly and provides accurate readings, as it needs time to stabilize.

What is the role of the sucrose solution in the experiment?

The sucrose solution serves as the substrate for the invertase enzyme, which breaks it down into monosaccharides.

How does the addition of pH buffers and inverted suspension to the test tubes relate to the experiment's objective?

The addition of pH buffers and inverted suspension allows for the observation of invertase activity under various pH conditions, which is crucial for understanding how pH affects enzyme activity.

Why is the test tube incubated in a 50-degree water bath?

Incubating the test tube in a 50-degree water bath provides the optimal temperature for the enzyme to function and break down sucrose.

What is the purpose of adding DNS (dinitro salicylic acid) to the test tubes?

DNS is added to reduce glucose, and the reduced DNS is then measurable on a spectrophotometer, allowing for the quantification of glucose produced by the enzyme's action.

How does the spectrophotometer measure the enzyme activity?

The spectrophotometer measures the absorbance at 540 nanometers, which is the optimal wavelength for detecting sugars reduced by DNS, thus indicating the amount of glucose produced and the enzyme's activity.

What is the significance of the graph showing optimal enzyme activity at pH 5?

The graph indicates that invertase enzyme activity is highest at a pH of 5, which is important for understanding the enzyme's efficiency in different environments.