Preparation of γ – polyglutamic acid and study on its tyrosinase inhibitory activity
Polymers such as homopolymers of amino acids have a wide range of biological functions, biocompatibility, and dispersed molecular weight distribution. At present, the homopolymer amino acids obtained through microorganisms mainly include three types: polyglutamic acid (poly – γ – glutamic acid, γ – PGA), ε – poly lysine (ε – PL), and cyanophycin. Gamma PGA, as one of them, also has its excellent biological characteristics. Since 1913, researchers have successively isolated gamma PGA from the mucus of natto food and fermentation media of Bacillus subtilis, B. licheniformis, B. megaterium, and B. anthracis. The conformational structure of γ – PGA significantly changes with the pH value, concentration, and ionic strength of the solution. The production of γ – PGA by Bacillus subtilis exhibits parallel β – folding structures in acidic solutions, irregular curled structures when approaching neutrality, and random extended structures in alkaline environments.

The finished product of γ – PGA is a white odorless powder with a molecular weight between 100 and 10000 kDa, equivalent to about 50 to 50000 glutamic acid monomers. Gamma PGA and its derivatives have broad research and application prospects in many fields. Such as being used as a food preservative, a moisturizing ingredient in cosmetics, a drug slow-release carrier, a heavy metal adsorbent, and a flocculant for water treatment. In 2013, researchers found that gamma PGA reduces the levels of reactive oxygen species and nitric oxide in B16 cells, increases catalase activity, and inhibits Forsklin induced tyrosinase activity and melanin production. This is the first report on the anti melanogenesis effect of γ – PGA.

Molecular weight is an important characteristic parameter of polymers, and its size determines its application field and effectiveness. Therefore, it is of great significance to prepare different molecular weights of gamma PGA and study the activity differences between different molecular weights in order to expand the application scope of gamma PGA. At present, microbial fermentation is a commonly used method for gamma PGA fermentation. The molecular weight and yield of γ – PGA prepared by this method are affected by the producing bacteria. According to whether glutamic acid is added during the fermentation process, the production strains of γ – PGA are divided into two categories: glutamic acid dependent strains and non glutamic acid dependent strains. Studies have shown that using glutamate dependent strains as producing bacteria results in higher production of gamma PGA. Therefore, this article uses Bacillus subtilis as the fermentation strain to produce γ – PGA through microbial fermentation. Five groups of different molecular weights of γ – PGA were prepared by combining chemical degradation methods, and their intracellular and extracellular inhibition of tyrosinase activity was studied. To lay the foundation for the application of different molecular weights of gamma PGA as whitening agents in the field of cosmetics.

Gamma PGA is an extracellular secreted product. For a long time, extensive research has been conducted on its properties, bacterial improvement and genetic characteristics, fermentation process, and extraction and purification. The molecular weight of γ – PGA directly affects its applications in many fields. With the discovery by researchers that gamma PGA has anti melanin effects, gamma PGA has gradually become a potential whitening agent ingredient.
Reducing the production of melanin is a key step in achieving whitening effects. Accumulated research has shown that tyrosinase is an important rate limiting enzyme involved in the process of melanin production, and its expression level and activity determine the speed and yield of melanin synthesis. Therefore, its inhibitors are usually screened as whitening agents. In 2004, Sasaki et al. discovered that metallothionein can effectively inhibit melanin production stimulated by NO and other melanogens by directly inhibiting tyrosinase activity in melanosomes. In 2013, Jeong et al. discovered that ginsenoside Rh4 aglycone (A-Rh4) inhibits melanin synthesis in B16 melanoma cells through the protein kinase A pathway. An increase in tyrosinase activity was observed in melanin synthesis, and its downregulation can inhibit melanin production. Therefore, the search for natural biomaterials with anti tyrosinase activity has been considered an important strategy for developing new skin pigmentation regulation products.
This article selects glutamic acid dependent Bacillus subtilis as the strain for fermentation preparation of γ – PGA, with a yield of 8g/L and a content of 97.69%. By utilizing its sensitivity to physical and chemical conditions in structure and combining chemical degradation methods, five different molecular weights of γ – PGA with weight average molecular weights of 21, 117, 282, 432, and 501 kDa were prepared. To ensure the biological safety of cells, the safe concentration range that cells can use was first evaluated. Subsequently, the tyrosinase activity inhibition rate was used as an indicator to determine the tyrosinase activity inhibition rates of five different molecular weights of γ – PGA both inside and outside the cell. The comprehensive results of cytotoxicity and tyrosinase inhibition activity showed that all five molecular weights of γ – PGA had tyrosinase inhibition activity, and the extracellular inhibition activity of tyrosinase increased with the increase of molecular weight, providing a theoretical basis for the future application of γ – PGA as a potential whitening ingredient.
The molecular weight of γ – PGA prepared by microbial fermentation method is limited. Jiang et al. constructed a recombinant Bacillus subtilis containing the γ – PGA degrading enzyme gene cluster pgsBAE, which can synthesize low molecular weight (approximately 10kDa) γ – PGA. Qiao Changsheng et al. used a fermentation tank to ferment and prepared 1000kDa γ – PGA by regulating the fermentation process and optimizing the fermentation conditions. Therefore, the next step is to combine genetic engineering and other methods to construct engineering strains by introducing key gene fragments of degradation enzymes or synthesis enzymes to prepare higher or lower molecular weight gamma PGA, in order to further study its whitening activity.
At present, natural ingredients have been added as whitening agents in cosmetic formulas, including quercetin, arbutin, niacinamide, astaxanthin, salicylic acid, fruit acid, etc. Different whitening ingredients have different whitening activities due to their different mechanisms of whitening. The same whitening ingredient also has different effects in cosmetics due to its different concentrations added. Therefore, through the study of the whitening activity of polyglutamic acid with different molecular weights, not only does it provide a potential whitening ingredient for the cosmetics field, but it can also be added to cosmetic formulas to exert different effects based on the differences in whitening activity between different molecular weights, adapting to different populations and meeting their whitening needs. In the future, by improving the extraction technology of natural extracts, qualitative and quantitative analysis of the active ingredients in extracts can be carried out to determine their effects and find the active substances with the best whitening effect in natural organisms.