Utilisation of Amino Acids | Microbial Biotechnology

The following points highlight the two main tests conducted for utilisation of amino acids:- 1. Decarboxylase Test 2. Phenylalanine Deaminase Test.

1. Decarboxylase Test:

This test helps in differentiating Gram-negative organisms based on their ability to degrade amino acid substrates enzymatically. All bio­logically active proteins are composed of 20 es­sential amino acids. Amino acids have an alpha carbon (-C-) an amino group (—NH₂), a carboxylgroup (-COOH) and a hydrogen atom (-H). Attached to the alpha carbon, a side group, or an atom designated by an (-R) is there which differs in each amino acid.

Decarboxylase enzymes are adaptive en­zymes and they remove the carboxyl (-COOH) group to yield end products like an amine or diamine plus carbon dioxide. This process is known as decarboxylation. Such decarboxylated amino acids play an essential role in cellular metabolism, since the amines pro­duced may serve as end products for the syn­thesis of other molecules that are required by the cell.

This adaptive enzyme is produced in presence of specific amino acid substrates on which they act. However, these amino acid substrates must possess at least one chemi­cal group other than an amine (-NH₂) or a carboxyl (-COOH) group. Here the organ­isms are grown in an acid environment and the presence of a specific substrate. The end produces products (amines) will shift to alkaline pH.

Requirements:

1. 24 hr. Nutrient broth culture of Proteus vulgaris, E.coli and Citrobacter freundii.

2. Medium.

3. 3 tubes with Moeller’s decarboxylase broth supplemented with L-lysine 10 g/1 (labelled LD⁺).

4. 3 tubes with Moeller’s decarboxylase broth without L-lysine (LD^–).

5. Bunsen flame.

6. Inoculating loop.

7. Sterile Pasteur pipettes.

8. Sterile mineral oil.

9. Test tube rack.

Procedure:

1. Label three tubes with LD⁺ medium and name of organism to be inoculated.

2. Label three tubes with LD^– medium and name of organism to be inoculated (control medium). These tubes should re­main yellow indicating that only glu­cose was oxidised.

3. Inoculate each organism into the tubes as per label and add 1 ml of sterile min­eral oil on top with Pasteur pipettes, by holding the tubes in a slanting position and without the tip of the pipette touch­ing either the medium or sides of the tubes. (Decarboxylation reaction occurs under anaerobic conditions).

4. Incubate at 37°C for 24-48 hours. Acid end products will change bromocresol purple from purple to yellow which shows that decarboxylase enzymes have been activated. This leads to production of an alkalinising diamine, cadaverine, and CO₂ leading to a final colour change from yellow back to purple. This indicates that L-lysine has been decarboxylated.

2. Phenylalanine Deaminase Test:

Some organisms remove the amino group (- NH₂) from amino acids and other NH₂ con­taining compounds.

Principle:

The amino acid when acted upon by deaminase will produce ketoacids and NH₃ as end products.

Requirements:

1. 24 hr. Nutrient broth culture of Esch­erichia coli and Proteus vulgaris.

2. Two phenylalanine slants:

3. 10-12% ferric chloride solution.

4. Bunsen flame.

5. Pasteur pipettes.

6. Test tube rack.

7. Inoculating loop.

Procedure:

1. Streak inoculate each organism into the labelled tubes.

2. Incubate at 37°C for 24-48 hours.

Phenyl pyruvic acid produced can be detected by adding 10-12% ferric chlo­ride solution to the surface of the me­dium. If substrate deamination has taken place, green colour develops.