High extracellular expression of glucose isomerase from Streptomyces murinus in E. coli via OsmY signal peptide System; an integrated experimental and molecular dynamics analysis

Glucose isomerase (GI) is a key enzyme for the industrial conversion of glucose to fructose. A recombinant Escherichia coli strain expressing the GI gene from Streptomyces murinus was engineered, integrating protein design, expression optimization, and structural validation. SDS-PAGE confirmed secretion of the ~43 kDa enzyme, maximized under lactose induction. The OsmY signal peptide enabled efficient extracellular targeting, while an Asp-rich N-terminal extension improved thermostability, increasing T50 from 70 °C to 72 °C. Molecular dynamics revealed reduced loop fluctuations and enhanced electrostatics, supporting stability without loss of activity. Response Surface Methodology produced a predictive cubic model (R² = 0.99), identifying temperature, induction time, carbon and nitrogen sources as key factors. Maximum activity (18.79 U/mL) was achieved at 37 °C, pH 8, glucose, and NH4Cl. Industrial recommendations yielded 5–7 U/mL with improved stability. This integrated approach provides a robust, scalable strategy for thermostable GI production suited to cost-sensitive fructose manufacturing.