AUTHOR=Quartinello Felice , Subagia Raditya , Zitzenbacher Sabine , Reich Johanna , Vielnascher Robert , Becher Erik , Hall Mélanie , Ribitsch Doris , Guebitz Georg M. 

TITLE=Dihydropyrimidinase from Saccharomyces kluyveri can hydrolyse polyamides

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=11

YEAR=2023

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2023.1158226

DOI=10.3389/fbioe.2023.1158226

ISSN=2296-4185

ABSTRACT=<p>In <italic>Saccharomyces kluyveri,</italic> dihydropyrimidinase (DHPaseSK) is involved in the pyrimidine degradation pathway, which includes the reversible ring cleavage between nitrogen 3 and carbon 4 of 5,6-dihydrouracil. In this study, DPHaseSK was successfully cloned and expressed in <italic>E. coli</italic> BL-21 Gold (DE3) with and without affinity tags. Thereby, the Strep-tag enabled fastest purification and highest specific activity (9.5 ± 0.5 U/mg). The biochemically characterized DHPaseSK_Strep had similar kinetic parameters (K<sub>cat</sub>/K<sub>m</sub>) on 5,6-dihydrouracil (DHU) and <italic>para</italic>-nitroacetanilide respectively, with 7,229 and 4060 M<sup>−1</sup> s<sup>−1</sup>. The hydrolytic ability of DHPaseSK_Strep to polyamides (PA) was tested on PA consisting of monomers with different chain length (PA-6, PA-6,6, PA-4,6, PA-4,10 and PA-12). According to LC-MS/TOF analysis, DHPaseSK_Strep showed a preference for films containing the shorter chain monomers (e.g., PA-4,6). In contrast, an amidase from <italic>Nocardia farcinica</italic> (NFpolyA) showed some preference for PA consisting of longer chain monomers. In conclusion, in this work DHPaseSK_Strep was demonstrated to be able to cleave amide bonds in synthetic polymers, which can be an important basis for development of functionalization and recycling processes for polyamide containing materials.</p>