Purification and Characterization of Membrane Protein ε-Poly-L-lysine Synthetase from Streptomyces Albulus
摘要
ε-Poly-L-lysine (ε-PL) is a L-lysine homopolymer with wide applications in the fields of food, cosmetics, and pharmaceuticals due to its excellent antimicrobial properties and biodegradability. It is primarily produced through microbial fermentation by Streptomyces species, with its biosynthesis catalyzed by the membrane-bound enzyme ε-PL synthetase (Pls). However, Pls purification from industrial strains has not been achieved, and its enzymatic properties, particularly the effects of metal ions on its activity, remain insufficiently characterized. To address this gap, we successfully expressed, purified, and characterized full-length Pls from the industrial strain Streptomyces albulus GS114 for the first time, achieving milligram-scale yields of high-purity protein. A systematic evaluation of His-tag length and position revealed their impact on recombinant membrane protein expression and purification. To improve purification efficiency, we developed a novel strategy integrating DEAE anion exchange chromatography with Ni-affinity chromatography, enabling efficient Pls isolation. Preliminary enzymatic assays showed that Pls exhibits optimal activity at 20 °C and pH 8.5. Both Mn2+ and Mg2+ effectively support Pls catalytic activity, marking the first report on the impact of metal ions on Pls activity. These findings lay the foundation for further research on enzyme engineering, biosynthetic mechanisms, and potential improvements in ε-PL biomanufacturing.