Comparison of Four Immobilization Methods for Different Transaminases
Biocatalytic syntheses often require unfavorable conditions, which can adversely affect enzyme stability. Consequently, improving the stability of biocatalysts is needed, and this is often achieved by immobilization. In this study, we aimed to compare the stability of soluble and immobilized transam...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-01-01
|
| Series: | Catalysts |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2073-4344/13/2/300 |
| _version_ | 1797621770783555584 |
|---|---|
| author | Tobias Heinks Nicolai Montua Michelle Teune Jan Liedtke Matthias Höhne Uwe T. Bornscheuer Gabriele Fischer von Mollard |
| author_facet | Tobias Heinks Nicolai Montua Michelle Teune Jan Liedtke Matthias Höhne Uwe T. Bornscheuer Gabriele Fischer von Mollard |
| author_sort | Tobias Heinks |
| collection | DOAJ |
| description | Biocatalytic syntheses often require unfavorable conditions, which can adversely affect enzyme stability. Consequently, improving the stability of biocatalysts is needed, and this is often achieved by immobilization. In this study, we aimed to compare the stability of soluble and immobilized transaminases from different species. A cysteine in a consensus sequence was converted to a single aldehyde by the formylglycine-generating enzyme for directed single-point attachment to amine beads. This immobilization was compared to cross-linked enzyme aggregates (CLEAs) and multipoint attachments to glutaraldehyde-functionalized amine- and epoxy-beads. Subsequently, the reactivity and stability (i.e., thermal, storage, and solvent stability) of all soluble and immobilized transaminases were analyzed and compared under different conditions. The effect of immobilization was highly dependent on the type of enzyme, the immobilization strategy, and the application itself, with no superior immobilization technique identified. Immobilization of HA<sub>GA</sub>-beads often resulted in the highest activities of up to 62 U/g beads, and amine beads were best for the hexameric transaminase from <i>Luminiphilus syltensis</i>. Furthermore, the immobilization of transaminases enabled its reusability for at least 10 cycles, while maintaining full or high activity. Upscaled kinetic resolutions (partially performed in a SpinChem<sup>TM</sup> reactor) resulted in a high conversion, maintained enantioselectivity, and high product yields, demonstrating their applicability. |
| first_indexed | 2024-03-11T09:01:41Z |
| format | Article |
| id | doaj.art-2540904060c64b508a65d459e9d918db |
| institution | Directory Open Access Journal |
| issn | 2073-4344 |
| language | English |
| last_indexed | 2024-03-11T09:01:41Z |
| publishDate | 2023-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Catalysts |
| spelling | doaj.art-2540904060c64b508a65d459e9d918db2023-11-16T19:41:12ZengMDPI AGCatalysts2073-43442023-01-0113230010.3390/catal13020300Comparison of Four Immobilization Methods for Different TransaminasesTobias Heinks0Nicolai Montua1Michelle Teune2Jan Liedtke3Matthias Höhne4Uwe T. Bornscheuer5Gabriele Fischer von Mollard6Faculty of Chemistry, Biochemistry, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, GermanyFaculty of Chemistry, Organic and Bioorganic Chemistry, Bielefeld University, Universitätsstraße 25, 33615 Bielefeld, GermanyFaculty of Chemistry, Biochemistry, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, GermanyFaculty of Chemistry, Biochemistry, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, GermanyProtein Biochemistry, Institute of Biochemistry, University of Greifswald, Felix Hausdorff-Str. 4, 17487 Greifswald, GermanyDepartment of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, Felix Hausdorff-Str. 4, 17487 Greifswald, GermanyFaculty of Chemistry, Biochemistry, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, GermanyBiocatalytic syntheses often require unfavorable conditions, which can adversely affect enzyme stability. Consequently, improving the stability of biocatalysts is needed, and this is often achieved by immobilization. In this study, we aimed to compare the stability of soluble and immobilized transaminases from different species. A cysteine in a consensus sequence was converted to a single aldehyde by the formylglycine-generating enzyme for directed single-point attachment to amine beads. This immobilization was compared to cross-linked enzyme aggregates (CLEAs) and multipoint attachments to glutaraldehyde-functionalized amine- and epoxy-beads. Subsequently, the reactivity and stability (i.e., thermal, storage, and solvent stability) of all soluble and immobilized transaminases were analyzed and compared under different conditions. The effect of immobilization was highly dependent on the type of enzyme, the immobilization strategy, and the application itself, with no superior immobilization technique identified. Immobilization of HA<sub>GA</sub>-beads often resulted in the highest activities of up to 62 U/g beads, and amine beads were best for the hexameric transaminase from <i>Luminiphilus syltensis</i>. Furthermore, the immobilization of transaminases enabled its reusability for at least 10 cycles, while maintaining full or high activity. Upscaled kinetic resolutions (partially performed in a SpinChem<sup>TM</sup> reactor) resulted in a high conversion, maintained enantioselectivity, and high product yields, demonstrating their applicability.https://www.mdpi.com/2073-4344/13/2/300amine transaminaseenzyme stabilityenzyme immobilizationsite-selective immobilizationreusabilitystorage stability |
| spellingShingle | Tobias Heinks Nicolai Montua Michelle Teune Jan Liedtke Matthias Höhne Uwe T. Bornscheuer Gabriele Fischer von Mollard Comparison of Four Immobilization Methods for Different Transaminases Catalysts amine transaminase enzyme stability enzyme immobilization site-selective immobilization reusability storage stability |
| title | Comparison of Four Immobilization Methods for Different Transaminases |
| title_full | Comparison of Four Immobilization Methods for Different Transaminases |
| title_fullStr | Comparison of Four Immobilization Methods for Different Transaminases |
| title_full_unstemmed | Comparison of Four Immobilization Methods for Different Transaminases |
| title_short | Comparison of Four Immobilization Methods for Different Transaminases |
| title_sort | comparison of four immobilization methods for different transaminases |
| topic | amine transaminase enzyme stability enzyme immobilization site-selective immobilization reusability storage stability |
| url | https://www.mdpi.com/2073-4344/13/2/300 |
| work_keys_str_mv | AT tobiasheinks comparisonoffourimmobilizationmethodsfordifferenttransaminases AT nicolaimontua comparisonoffourimmobilizationmethodsfordifferenttransaminases AT michelleteune comparisonoffourimmobilizationmethodsfordifferenttransaminases AT janliedtke comparisonoffourimmobilizationmethodsfordifferenttransaminases AT matthiashohne comparisonoffourimmobilizationmethodsfordifferenttransaminases AT uwetbornscheuer comparisonoffourimmobilizationmethodsfordifferenttransaminases AT gabrielefischervonmollard comparisonoffourimmobilizationmethodsfordifferenttransaminases |