Purification and Characterization of Membrane Protein ε-Poly-L-lysine Synthetase from Streptomyces Albulus
Tingting He1, Daojun Zhu1, Hao Yang1
1Key Laboratory of Industrial Biotechnology, School of Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.
Abstract:
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.
Insights
Researchers purified and characterized ε-Poly-L-lysine synthetase (Pls) from Streptomyces albulus. This enzyme is crucial for producing ε-Poly-L-lysine (ε-PL), a biopolymer with broad industrial applications.
Area of Science:
- Biotechnology
- Enzyme Engineering
- Microbial Fermentation
Background:
- ε-Poly-L-lysine (ε-PL) is a biodegradable antimicrobial biopolymer with applications in food, cosmetics, and pharmaceuticals.
- Its production relies on ε-PL synthetase (Pls), a membrane-bound enzyme from Streptomyces species, which has been difficult to purify and characterize.
Purpose of the Study:
- To achieve the first successful expression, purification, and characterization of full-length Pls from the industrial strain Streptomyces albulus GS114.
- To investigate the effects of metal ions on Pls activity and optimize purification strategies.
Main Methods:
- Recombinant expression and purification of Pls using optimized His-tag strategies.
- Development of a novel purification strategy combining DEAE anion exchange and Ni-affinity chromatography.
- Enzymatic assays to determine optimal conditions (temperature, pH) and metal ion dependency.
Main Results:
- Milligram-scale yields of high-purity Pls were obtained.
- A purification strategy integrating DEAE anion exchange and Ni-affinity chromatography proved efficient for Pls isolation.
- Pls showed optimal activity at 20°C and pH 8.5, with Mn2+ and Mg2+ found to support its catalytic activity.
Conclusions:
- The successful purification and characterization of Pls provide a foundation for enzyme engineering and improving ε-PL biomanufacturing.
- This study offers the first report on the impact of metal ions on Pls activity, highlighting the roles of Mn2+ and Mg2+.
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