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

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作   者：

Cahill, John F.;  Kertesz, Vilmos;  St-Vincent, Patricia;  Valentino, Hannah;  Drufva, Erin;  Thiele, Nikki;  Michener, Joshua K.; 

作者机构：

Oak Ridge Natl Lab; 

关键词：

NYLONS;  MECHANISM;  CHROMATOGRAPHY;  CYCLIC OLIGOMERS; 

期刊名称：

Journal of the American Society for Mass Spectrometry

i s s n：

1044-0305

年卷期：

2023 年 34 卷 7 期

页   码：

1383-1391

页   码：

摘   要：

Enzymatic biodegradation of polymers, such as polyamides(PA),has the potential to cost-effectively reduce plastic waste, but enhancementsin degradation efficiency are needed. Engineering enzymes throughdirected evolution is one pathway toward identification of criticaldomains needed for improving activity. However, screening such enzymaticlibraries (100s-to-1000s of samples) is time-consuming. Here we demonstratethe use of robotic autosampler (PAL) and immediate drop on demandtechnology (I.DOT) liquid handling systems coupled with open-portsampling interface-mass spectrometry (OPSI-MS) to screen for PA6 andPA66 hydrolysis by 6-aminohexanoate-oligomer endo-hydrolase (nylon hydrolase, NylC) in a high-throughput (8-20s/sample) manner. The OPSI-MS technique required minimal sample preparationand was amenable to 96-well plate formats for automated processing.Enzymatic hydrolysis of PA characteristically produced soluble linearoligomer products that could be identified by OPSI-MS. Incubationtemperatures and times were optimized for PA6 (65 degrees C, 24 h) andPA66 (75 degrees C, 24 h) over 108 experiments. In addition, the I.DOT/OPSI-MSquantified production of PA6 linear dimer (8.3 +/- 1.6 mu g/mL)and PA66 linear monomer (13.5 +/- 1.5 mu g/mL) by NylC witha lower limit of detection of 0.029 and 0.032 mu g/mL, respectively.For PA6 and PA66, linear oligomer production corresponded to 0.096 +/- 0.018% and 0.204 +/- 0.028% conversion of dry pellet mass,respectively. The developed methodology is expected to be utilizedto assess enzymatic hydrolysis of engineered enzyme libraries, comprisinghundreds to thousands of individual samples.