# Enhanced Hyaluronic Acid Production in Streptococcus equi subsp. Streptococcus zooepidemicus Through Strain Mutagenesis and Optimized Purification

## TL;DR

Scientists improved hyaluronic acid production in a bacteria strain by mutating it and optimizing purification, resulting in higher yields and purity.

## Contribution

A novel strain optimization and purification method for hyaluronic acid production with reduced solvent use and increased yield.

## Key findings

- Mutant strain S. zooepidemicus K12 achieved an 85.7% increase in hyaluronic acid yield.
- Purification method reduced organic solvent consumption by 25-fold while meeting EP standards.
- Hyaluronic acid molecular weight increased from 67,000 Da to 120,000 Da.

## Abstract

Streptococcusequi subsp. S. zooepidemicus is a notable producer of hyaluronic acid (HA), a natural polysaccharide widely utilized in the pharmaceutical, cosmetic, and food industries due to its unique properties.

This study focuses on strain optimization and the purification of HA. The key goals include reducing hyaluronidase activity, which degrades HA, eliminating beta-hemolytic traits, and developing a purification process that significantly minimizes organic solvent consumption to lower costs and reduce environmental impact.

UV-induced physical mutagenesis and chemical mutagenesis utilizing nitrous acid and N-methyl-N'-nitro-N-nitrosoguanidine (NTG) were employed to generate mutant strains with diminished hyaluronidase activity and beta-hemolytic properties. Following strain optimization, an efficient downstream process was implemented, focusing on pre-treatment methods involving pH and temperature adjustments, followed by ultrafiltration and phenol acetate treatment.

The selected mutant, designated as S.
equi subsp. S. zooepidemicus K12, maintained stable mutation characteristics over 15 generations. The HA yield of the selected mutant showed an increase of 85.7%, rising from 0.42 g/L in the wild type to 0.78 g/L. According to gel permeation chromatography, the average molecular weight (Mw) of HA increased from 6.7 × 104 Da to 1.2 × 105 Da. Our purification strategy achieved a recovery rate of 72% with approximately 0.3% protein impurities, meeting European Pharmacopoeia (EP) standards. Additionally, organic solvent consumption was reduced by at least 25-fold compared to conventional methods.

The study presents an integrated approach to HA production, encompassing strain improvement and purification. It addresses key challenges in the manufacturing of streptococcal HA and contributes to the development of more efficient and environmentally friendly purification methods, ultimately resulting in high-purity HA.