Development of Streptococcus equisimilis Group G Mutant Strains with Ability to Produce Low Polydisperse and Low-Molecular-Weight Hyaluronic Acid

Authors
Bahareh Jafari
Malihe Keramati
Reza Ahangari Cohan
Seyed Mohammad Atyabi
Sara Ali Hosseinzadeh
Department of Nano-Biotechnology, New Technologies Research Group, Pasteur Institute of Iran, Tehran, Iran
10.52547/ibj.3789
Abstract
Background: Hyaluronic acid (HA), a natural polymer with wide applications in biomedicine and cosmetics, is mainly produced by Streptococcal fermentation at industrial scale. In the present study, chemical random mutagenesis was used for development of Streptococcus equisimilis group G mutant strains with high HA productivity.

Methods: The optimum of the pH of culture condition and cultivation time for HA production by wild strain group G were assessed. At first, two rounds of mutation at different concentrations of NTG was used for mutagenesis. Then, the nonhemolytic and hyaluronidase-negative mutants were screened on the blood and HA agar. HA productivity and molecular weight were determined by carbazole assay, agarose gel electrophoresis and specific staining. Moreover, stability of the high producer mutants was evaluated within 10 generations.

Results: The results showed that the wild-type strain produced 1241 ± 2.1 µg/ml of HA at pH 5.5 and 4 hours of cultivation, while the screened mutants showed a 16.1-45.5% increase in HA production. Two mutant strains, named Gm2-120-21-3 (2470 ± 8.1 µg/ml) and Gm2-120-21-4 (2856 ± 4.2 µg/ml), indicated the highest titer and a consistent production. The molecular weight (Mw) of HA for the mutants was less than 160 kDa, considering as a low Mw HA.

Conclusion: The mutant strains producing a low polydisperse, as well as low Mw of HA with high titer might be regarded as potential industrial strains for HA production after further safety investigations.
Keywords
Hyaluronic acid
Microbial production
Molecular weight
Mutation
Streptococcus equisimilis