How is polyglutamic acid obtained?

The polyglutamic acid is a molecule derived from the polymerisation of an amino acid, glutamic acid. Discovered a few decades ago, it has been utilised in various sectors, such as the food industry, due to its interesting nutritional properties, medicine, particularly to support the healing process, and the wastewater treatment industry. Today, the polyglutamic acid is also used to formulate cosmetic care products, mainly with a hydrating purpose. Indeed, this active ingredient works through several mechanisms to maintain skin hydration, notably by forming a film on its surface and stimulating the synthesis of certain molecules belonging to the natural moisturising factor, naturally present in the stratum corneum.

Polyglutamic acid is traditionally obtained through biosynthesis from L-glutamic acid using a Gram-positive bacterium from the Bacilli class. At Typology, we utilise the bacterium Bacillus subtilis.

The production of polyglutamic acid is carried out exclusively by Gram-positive microorganisms belonging to the class of Bacilli. Among the main ones used are Bacillus subtilis, Bacillus licheniformis, and Bacillus anthracis. The synthesis of polyglutamic acid occurs through a non-ribosomal process, catalysed solely by enzymes, as opposed to so-called ribosomal peptides, which are synthesised by ribosomes. As a reminder, ribosomes are complexes that ensure the translation of mRNA and enable protein synthesis. The non-ribosomal process is a unique synthesis mechanism, found only in bacteria and fungi. The synthesis of polyglutamic acid takes place in two main stages:

• Production of the amino acid L-glutamic: L-glutamic acid is produced from 2-oxoglutarate, an intermediate of the Krebs cycle, through the action of glutamate synthase and glutamate dehydrogenase. In certain strains, such asB. licheniformis, these enzymes are not inhibited by the end product, allowing for a high accumulation of L-glutamate.

• Glutamate polymerisation: A portion of L-glutamate is racemised into D-glutamate via a glutamate racemase. The two forms are then co-polymerised into polyglutamic acid via a specific enzymatic pathway. The L-glutamate is first activated by ATP to form a γ-glutamyl-AMP intermediate. This activated group is then transferred to a carrier enzyme containing a thiol group before being converted into D-glutamate. This activated glutamate is then transferred to a growing chain of polyglutamic acid via a γ-amide bond. This mechanism allows for a gradual growth of the polymer, with precise control of the D/L ratio depending on the bacterial strains and cofactors, such as Mn²⁺ ions, which regulate racemase activity.

It's also worth noting that the synthesis of polyglutamic acid is controlled by a set of genes organised into operons, the expression of which depends on the biological role the polymer plays in the bacteria: either as a protective capsule or in a free extracellular form, as is the case for industrial and cosmetic applications. In the latter case, the synthesis of polyglutamic acid is directed by an operon named pgsBCA or pgsBCAAE depending on the strains, composed of the genes pgsB, pgsC, pgsAA and sometimes pgsE. These genes can be located on the bacterial chromosome or on a plasmid.

In practical terms, the industrial production of polyglutamic acid relies on a controlled fermentation process using specific bacterial strains. These bacteria are cultivated in large-scale fermenters, in an environment rich in carbon and nitrogen substrates, in the presence of mineral salts and cofactors such as Mn²⁺. The temperature, pH, aeration, and agitation are finely regulated to optimise both bacterial growth and polymer synthesis. After several tens of hours of fermentation, the polyglutamic acid is excreted by the bacteria into the extracellular culture medium, which facilitates its extraction. The supernatant is then collected, and the polyglutamic acid is precipitated, often with the help of ethanol, purified, dried, and sometimes fractionated, depending on the desired molecular weight.