High-Throughput Characterization and Optimization of Polyamide Hydrolase Activity Using Open Port Sampling Interface...

Area of Science:

• Biotechnology
• Polymer Science
• Analytical Chemistry

Background:

• Plastic waste, particularly polyamides (PA), poses environmental challenges.
• Enzymatic biodegradation offers a sustainable solution for plastic waste reduction.
• Directed evolution of enzymes requires efficient screening methods for improved degradation activity.

Purpose of the Study:

• To develop and validate a high-throughput screening method for enzymatic hydrolysis of polyamides.
• To assess the efficiency of nylon hydrolase (NylC) in degrading PA6 and PA66.
• To enable rapid screening of engineered enzyme libraries for enhanced plastic degradation.

Main Methods:

• Utilized robotic autosampler (PAL) and I.DOT liquid handling systems.
• Employed open-port sampling interface-mass spectrometry (OPSI-MS) for rapid analysis.
• Screened for PA6 and PA66 hydrolysis by NylC in a high-throughput manner (8-20 s/sample).

Main Results:

• Optimized incubation conditions for PA6 (65 °C, 24 h) and PA66 (75 °C, 24 h).
• Quantified PA6 dimer (8.3 ± 1.6 μg/mL) and PA66 monomer (13.5 ± 1.5 μg/mL) production.
• Achieved low limits of detection (0.029-0.032 μg/mL) for hydrolysis products.

Conclusions:

• The developed I.DOT/OPSI-MS methodology enables rapid, automated screening of enzymatic polyamide degradation.
• This approach significantly accelerates the assessment of engineered enzymes for plastic waste management.
• The method is scalable for screening large libraries of enzymes.