One-step Purification and Immobilization of Nucleoside Deoxyribosyltransferase for Continuous-flow Biosynthesis of 2'-deoxyadenosine.
Nucleoside analogs are commonly used drugs for the treatment of cancer and viral infections. Nucleoside deoxyribosyltransferase (NDT) is one of the key enzymes required for the biosynthesis of deoxynucleotides. The feasibility of biochemical transformation processes is usually greatly dependent on the cost of biocatalysts. Therefore, immobilizing and reusing biocatalysts is an approach to be considered to bring biotransformations closer to industrial feasibility, since it not only allows enzymes to be reused but can also improve their stability under several reaction conditions. In this work, histidine tags have been successfully fused to a Nucleoside deoxyribosyltransferase from Lactobacillus reuteri (LrDNT), which has been produced in a bench-scale reactor using a facile araBAD promoter-based protein expression system in an industrial microorganism, the E. coli BL21(DE3) strain. The overexpressed LrNDT was bound to a nickel-chelating agarose affinity chromatography medium via a one-step purification and immobilization process with a 99% yield, retaining about 97% of its initial activity. The optimal conditions for the immobilized LrNDT maintain high enzyme activity within the range of 20-45°C and pH 6.0-7.5. Finally, the immobilized enzyme reactor (IMER) of LrNDT was applied for the continuous-flow biosynthesis of 2'-deoxyadenosine and retained 90% of its activity after 16 hours, providing a theoretical basis for the industrial production of nucleoside analogs..
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Preprint| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
Preprints.org - (2024) vom: 14. März Zur Gesamtaufnahme - year:2024 |
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| Sprache: |
Englisch |
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| Beteiligte Personen: |
Li, Menglei [VerfasserIn] |
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| Links: |
Volltext [kostenfrei] |
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| doi: |
10.20944/preprints202403.0795.v1 |
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| PPN (Katalog-ID): |
preprintsorg042918626 |
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| 520 | |a Nucleoside analogs are commonly used drugs for the treatment of cancer and viral infections. Nucleoside deoxyribosyltransferase (NDT) is one of the key enzymes required for the biosynthesis of deoxynucleotides. The feasibility of biochemical transformation processes is usually greatly dependent on the cost of biocatalysts. Therefore, immobilizing and reusing biocatalysts is an approach to be considered to bring biotransformations closer to industrial feasibility, since it not only allows enzymes to be reused but can also improve their stability under several reaction conditions. In this work, histidine tags have been successfully fused to a Nucleoside deoxyribosyltransferase from Lactobacillus reuteri (LrDNT), which has been produced in a bench-scale reactor using a facile araBAD promoter-based protein expression system in an industrial microorganism, the E. coli BL21(DE3) strain. The overexpressed LrNDT was bound to a nickel-chelating agarose affinity chromatography medium via a one-step purification and immobilization process with a 99% yield, retaining about 97% of its initial activity. The optimal conditions for the immobilized LrNDT maintain high enzyme activity within the range of 20-45°C and pH 6.0-7.5. Finally, the immobilized enzyme reactor (IMER) of LrNDT was applied for the continuous-flow biosynthesis of 2'-deoxyadenosine and retained 90% of its activity after 16 hours, providing a theoretical basis for the industrial production of nucleoside analogs. | ||
| 700 | 1 | |a Fan, Tiantian |4 aut | |
| 700 | 1 | |a Hou, Xuewen |4 aut | |
| 700 | 1 | |a Ding, Chenghui |4 aut | |
| 700 | 1 | |a Qiu, Min |4 aut | |
| 700 | 1 | |a Lv, Yaoyao |4 aut | |
| 700 | 1 | |a Liang, Rui |4 aut | |
| 700 | 1 | |a Xia, Yu |4 aut | |
| 700 | 1 | |a Zhang, Kunxiao |0 (orcid)0000-0001-5548-4127 |4 aut | |
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