Dihydropyrimidinase from Saccharomyces kluyveri can hydrolyse polyamides

Felice Quartinello; Raditya Subagia; Sabine Zitzenbacher; Johanna Reich; Robert Vielnascher; Erik Becher; Mélanie Hall; Doris Ribitsch; Georg M Guebitz

doi:10.3389/fbioe.2023.1158226

Dihydropyrimidinase from Saccharomyces kluyveri can hydrolyse polyamides

Front Bioeng Biotechnol. 2023 Apr 26:11:1158226. doi: 10.3389/fbioe.2023.1158226. eCollection 2023.

Authors

Felice Quartinello^{1

2}, Raditya Subagia¹, Sabine Zitzenbacher¹, Johanna Reich¹, Robert Vielnascher¹, Erik Becher³, Mélanie Hall^{3

4}, Doris Ribitsch^{1

2}, Georg M Guebitz^{1

2}

Affiliations

¹ Austrian Centre of Industrial Biotechnology, Tulln an der Donau, Austria.
² Department of Agrobiotechnology, Institute of Environmental Biotechnology, IFA-Tulln, University of Natural Resources and Life Sciences, Vienna, Austria.
³ Institute of Chemistry, University of Graz, Graz, Austria.
⁴ BioHealth, University of Graz, Graz, Austria.

Abstract

In Saccharomyces kluyveri, 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 E. coli 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_cat/K_m) on 5,6-dihydrouracil (DHU) and para-nitroacetanilide respectively, with 7,229 and 4060 M^-1 s^-1. 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 Nocardia farcinica (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.

Keywords: Saccharomyces kluyveri; dihydropyrimidinase; enzyme; hydrolysis; polyamide; surface functionalization.

Grants and funding

The COMET center: acib: Next-Generation Bioproduction is funded by BMVIT, BMDW, SFG, Standortagentur Tirol, Government of Lower Austria und Vienna Business Agency in the framework of COMET—Competence Centers for Excellent Technologies. The COMET-Funding Program is managed by the Austrian Research Promotion Agency FFG.