Enzymatic production of aspartame (a low calorie sweetener) involves the use of
1. amino acylase
2. penicillin acylase
3. lipase
4. thermolysin

Introduction to Aspartame Production

Aspartame is a widely used artificial sweetener known for its low caloric value and high sweetness (approximately 200 times sweeter than sucrose). It is commonly found in sugar-free beverages, chewing gum, and dietary products.

The enzymatic production of aspartame is a highly efficient and environmentally friendly process. Among the various enzymes used, thermolysin plays a critical role in the synthesis of aspartame due to its specific catalytic properties. Understanding the role of thermolysin in aspartame synthesis provides insight into the biotechnological advancements in the food industry.

✅ Key Phrase: Enzymatic production of aspartame

✅ Question and Answer

Question:
Enzymatic production of aspartame (a low-calorie sweetener) involves the use of:

1. Amino acylase
2. Penicillin acylase
3. Lipase
4. Thermolysin

Correct Answer: ✔️ Option 4 – Thermolysin

✅ Explanation of the Correct Answer

🔎 What is Aspartame?

Aspartame is a dipeptide methyl ester composed of two amino acids:

• L-aspartic acid
• L-phenylalanine methyl ester

The chemical structure of aspartame allows it to bind to taste receptors in the human tongue, producing a sweet taste without adding significant calories.

🧬 Enzymatic Synthesis of Aspartame

The production of aspartame involves the enzymatic coupling of:

1. L-aspartic acid
2. L-phenylalanine methyl ester

✅ Role of Thermolysin

Thermolysin is a thermostable metalloprotease enzyme derived from Bacillus thermoproteolyticus. It catalyzes the peptide bond formation between L-aspartic acid and L-phenylalanine methyl ester.

✔️ Thermolysin is preferred for aspartame synthesis due to:

• High specificity for peptide bond formation
• Thermostability (active at high temperatures)
• Low hydrolysis of the product, ensuring high yield

⚙️ Reaction Mechanism of Thermolysin in Aspartame Synthesis

1. Activation:

   • Thermolysin binds to L-aspartic acid and L-phenylalanine methyl ester at the active site.

2. Peptide Bond Formation:

   • The enzyme facilitates nucleophilic attack of the amino group of L-phenylalanine methyl ester on the carboxyl group of L-aspartic acid.

3. Product Release:

   • The dipeptide (aspartame) is released after the formation of a peptide bond.

4. Recycling of Enzyme:

   • Thermolysin remains unchanged after the reaction and can be reused for continuous production.

🌍 Advantages of Enzymatic Synthesis of Aspartame Using Thermolysin

✔️ High Specificity: Thermolysin recognizes specific amino acid sequences, reducing side reactions.
✔️ Eco-Friendly: Enzymatic synthesis reduces the need for chemical reagents, making the process environmentally sustainable.
✔️ Mild Reaction Conditions: Thermolysin operates at moderate pH and temperature, preserving the integrity of the product.
✔️ High Yield: Minimal by-products ensure high purity and yield of aspartame.

✅ Why Thermolysin Over Other Enzymes?

✔️ Thermolysin is highly selective for peptide bonds, making it the enzyme of choice for aspartame production.

✅ Challenges in Aspartame Synthesis

1. Racemization:

   • Maintaining the stereochemistry of L-aspartic acid and L-phenylalanine is critical.
   • Racemization leads to loss of sweetness and potential toxicity.

2. Hydrolysis:

   • Excessive reaction time may lead to hydrolysis of the product, reducing yield.

3. Enzyme Stability:

   • Thermolysin requires optimal temperature and pH conditions to maintain activity.

✅ Biotechnological Importance of Thermolysin

1. Food Industry:

   • Production of low-calorie sweeteners (aspartame)
   • Flavor enhancement and protein modification

2. Pharmaceutical Industry:

   • Peptide drug synthesis
   • Protein hydrolysis for drug formulation

3. Research Applications:

   • Protein sequencing
   • Enzyme-substrate interaction studies

✅ Future Prospects in Aspartame Synthesis

• Enzyme Engineering:

  • Genetic modifications to improve thermolysin’s specificity and stability.
  • Directed evolution to enhance enzyme efficiency.

• Immobilized Enzyme Systems:

  • Using immobilized thermolysin for continuous production.
  • Enhances enzyme recovery and reduces production costs.

✅ Summary of Key Points

 Aspartame is a low-calorie sweetener made of L-aspartic acid and L-phenylalanine methyl ester.
 Thermolysin catalyzes the peptide bond formation in aspartame synthesis.
 High specificity, thermostability, and mild reaction conditions make thermolysin ideal for industrial use.
 The enzymatic process is eco-friendly, scalable, and cost-effective.