Production of High Molecular Weight Hyaluronic Acid via Recombinant Bacillus subtilis 1A752

Author(s): Nouhan Doumbouya, Alper Akkaya

This study aimed to synthesize hyaluronic acid (HA) using a non-pathogenic recombinant bacterial host, Bacillus subtilis 1A752. The HA synthase (hasA) gene, originally isolated from Streptococcus equi subsp. zooepidemicus, was introduced into B. subtilis 1A752. This host strain naturally contains functional analogs of the Streptococcus genes hasB, hasC, and hasD, namely tuaD, gtaB, and gcaD. The hasA gene (or operon) was amplified from S. zooepidemicus genomic DNA using polymerase chain reaction (PCR) and subsequently cloned into B. subtilis 1A752 through recombinant DNA technology. The engineered strain was then employed for HA production, followed by deproteinization of the product using the Sevag method and quantification via the Carbazole assay. The purified HA was characterized further through chromatographic techniques, with its molecular structure verified using Nuclear Magnetic Resonance (NMR) and Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy. The viscosimetric analysis determined the intrinsic viscosity (ηi) and molecular weight, ranging from 1.7 to 2.7 MDa. The synthesized HA exhibits promising potential for pharmaceutical, biomedical, and cosmetic applications with its high molecular weight and non-pathogenic production platform.