Dihydropyrimidinase from Saccharomyces kluyveri can hydrolyse polyamides
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) wit...
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2023-04-01
|
| Series: | Frontiers in Bioengineering and Biotechnology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1158226/full |
| _version_ | 1797839105061552128 |
|---|---|
| author | Felice Quartinello Felice Quartinello Raditya Subagia Sabine Zitzenbacher Johanna Reich Robert Vielnascher Erik Becher Mélanie Hall Mélanie Hall Doris Ribitsch Doris Ribitsch Georg M. Guebitz Georg M. Guebitz |
| author_facet | Felice Quartinello Felice Quartinello Raditya Subagia Sabine Zitzenbacher Johanna Reich Robert Vielnascher Erik Becher Mélanie Hall Mélanie Hall Doris Ribitsch Doris Ribitsch Georg M. Guebitz Georg M. Guebitz |
| author_sort | Felice Quartinello |
| collection | DOAJ |
| description | 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 (Kcat/Km) 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. |
| first_indexed | 2024-04-09T15:51:50Z |
| format | Article |
| id | doaj.art-43bb705ef75f44a5a6156ce252e62b69 |
| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-04-09T15:51:50Z |
| publishDate | 2023-04-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-43bb705ef75f44a5a6156ce252e62b692023-04-26T05:54:24ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852023-04-011110.3389/fbioe.2023.11582261158226Dihydropyrimidinase from Saccharomyces kluyveri can hydrolyse polyamidesFelice Quartinello0Felice Quartinello1Raditya Subagia2Sabine Zitzenbacher3Johanna Reich4Robert Vielnascher5Erik Becher6Mélanie Hall7Mélanie Hall8Doris Ribitsch9Doris Ribitsch10Georg M. Guebitz11Georg M. Guebitz12Austrian Centre of Industrial Biotechnology, Tulln an der Donau, AustriaDepartment of Agrobiotechnology, Institute of Environmental Biotechnology, IFA-Tulln, University of Natural Resources and Life Sciences, Vienna, AustriaAustrian Centre of Industrial Biotechnology, Tulln an der Donau, AustriaAustrian Centre of Industrial Biotechnology, Tulln an der Donau, AustriaAustrian Centre of Industrial Biotechnology, Tulln an der Donau, AustriaAustrian Centre of Industrial Biotechnology, Tulln an der Donau, AustriaInstitute of Chemistry, University of Graz, Graz, AustriaInstitute of Chemistry, University of Graz, Graz, AustriaBioHealth, University of Graz, Graz, AustriaAustrian Centre of Industrial Biotechnology, Tulln an der Donau, AustriaDepartment of Agrobiotechnology, Institute of Environmental Biotechnology, IFA-Tulln, University of Natural Resources and Life Sciences, Vienna, AustriaAustrian Centre of Industrial Biotechnology, Tulln an der Donau, AustriaDepartment of Agrobiotechnology, Institute of Environmental Biotechnology, IFA-Tulln, University of Natural Resources and Life Sciences, Vienna, AustriaIn 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 (Kcat/Km) 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.https://www.frontiersin.org/articles/10.3389/fbioe.2023.1158226/fullpolyamidehydrolysisenzymesurface functionalizationSaccharomyces kluyveridihydropyrimidinase |
| spellingShingle | Felice Quartinello Felice Quartinello Raditya Subagia Sabine Zitzenbacher Johanna Reich Robert Vielnascher Erik Becher Mélanie Hall Mélanie Hall Doris Ribitsch Doris Ribitsch Georg M. Guebitz Georg M. Guebitz Dihydropyrimidinase from Saccharomyces kluyveri can hydrolyse polyamides Frontiers in Bioengineering and Biotechnology polyamide hydrolysis enzyme surface functionalization Saccharomyces kluyveri dihydropyrimidinase |
| title | Dihydropyrimidinase from Saccharomyces kluyveri can hydrolyse polyamides |
| title_full | Dihydropyrimidinase from Saccharomyces kluyveri can hydrolyse polyamides |
| title_fullStr | Dihydropyrimidinase from Saccharomyces kluyveri can hydrolyse polyamides |
| title_full_unstemmed | Dihydropyrimidinase from Saccharomyces kluyveri can hydrolyse polyamides |
| title_short | Dihydropyrimidinase from Saccharomyces kluyveri can hydrolyse polyamides |
| title_sort | dihydropyrimidinase from saccharomyces kluyveri can hydrolyse polyamides |
| topic | polyamide hydrolysis enzyme surface functionalization Saccharomyces kluyveri dihydropyrimidinase |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1158226/full |
| work_keys_str_mv | AT felicequartinello dihydropyrimidinasefromsaccharomyceskluyvericanhydrolysepolyamides AT felicequartinello dihydropyrimidinasefromsaccharomyceskluyvericanhydrolysepolyamides AT radityasubagia dihydropyrimidinasefromsaccharomyceskluyvericanhydrolysepolyamides AT sabinezitzenbacher dihydropyrimidinasefromsaccharomyceskluyvericanhydrolysepolyamides AT johannareich dihydropyrimidinasefromsaccharomyceskluyvericanhydrolysepolyamides AT robertvielnascher dihydropyrimidinasefromsaccharomyceskluyvericanhydrolysepolyamides AT erikbecher dihydropyrimidinasefromsaccharomyceskluyvericanhydrolysepolyamides AT melaniehall dihydropyrimidinasefromsaccharomyceskluyvericanhydrolysepolyamides AT melaniehall dihydropyrimidinasefromsaccharomyceskluyvericanhydrolysepolyamides AT dorisribitsch dihydropyrimidinasefromsaccharomyceskluyvericanhydrolysepolyamides AT dorisribitsch dihydropyrimidinasefromsaccharomyceskluyvericanhydrolysepolyamides AT georgmguebitz dihydropyrimidinasefromsaccharomyceskluyvericanhydrolysepolyamides AT georgmguebitz dihydropyrimidinasefromsaccharomyceskluyvericanhydrolysepolyamides |