IN VITRO BIOSYNTHESIS OF HYALURONIC ACID BY RECOMBINANT HYALURONAN SYNTHASE

Background and Aim:Hyaluronic acid (HA) has many applications in the pharmaceutical and cosmetic industries. Hyaluronan synthase (HAS) is the enzyme responsible for the synthesis of HA by coupling of β1-3D-N-acetylglucosamine and β1-4D-glucuronic acid. Since obtaining the fully active recombinant form of enzyme allows a safe and controllable way for HA production, therefore, the aim of the current study is expression and enzymatic assessment of recombinant HAS in E.coli.Methods:has gene was amplified using PCR from S.equisimilis genomic DNA and cloned into pET21a expression vector using NdeI and XhoI restriction enzymes. The recombinant enzyme was expressed in E.coli BL21 strain and analyzed using SDS-PAGE and western blot. The enzyme was purified using nickel affinity chromatography form the host membrane. The polymeric activity of the recombinant enzyme was assessed using Carbazole method according to the USP pharmacopeia.Results:The cloning procedure was confirmed by restriction map analysis and sequencing. 12% gel SDS-PAGE and western blot showed a band around 45 kDa with respect to the recombinant enzyme. The enzyme was isolated from the host membrane with high purity. Activity test showed that the recombinant enzyme has the ability to polymerize monomeric substrates to hyaluronic acid.Conclusion:The results showed successful expression of fully active recombinant HAS in E.coli BL21 strain. This project allows in vitro biosynthesis of HA in a safe manner.

کلیدواژه ها:

Hyaluronan Synthase ، S.equisimilis ، Hyaluronic acid