Development of Streptococcus equisimilis group G mutant strains produce low polydisperse and low molecular weight Hyaluronic Acid.

Abstract

Hyaluronic acid (HA), a natural polymer with unique properties and wide application, is mainly produced by Streptococcal fermentation at industrial scale. In the present work, two rounds of chemical random mutagenesis were applied on Streptococcus equisimilis group G to obtain high-producing strains using 1-methyl-3-nitro-1-nitrosoguanidine. The non-hemolytic and hyaluronidase-negative mutants were screened on blood agar and HA agar, respectively. HA productivity and molecular weight (Mw) was determined by carbazole assay and agarose gel electrophoresis, respectively. Moreover, the stability of strains was assessed within 10 generations in term of HA productivity. The findings showed that the wild type strain produced 1241 ± 2.1µg/ml HA at pH 5.5 and 4 hours of cultivation, while the screened mutants showed a 16.1 - 45.5 % increase in the production. Two mutant strains, named Gm2-120-21-3 (2470± 8.1µg/ml) and Gm2-120-21-4 (2856± 4.2µg/ml), showed the highest titer and a consistent production. The molecular weight of HA for the mutants was less than 160 kDa which considered a low Mw. The mutant strains producing a low polydisperse and low Mw HA at high titer can be considered potential industrial strains for HA production after further safety investigations.