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Enzymatic Synthesis of Nucleoside Triphosphates and Deoxynucleoside Triphosphates by Surface-Displayed Kinases

  • Yi Ding
  • Ling OuEmail author
  • Qingbao DingEmail author
Article
  • 57 Downloads

Abstract

Nucleoside triphosphates and deoxynucleoside triphosphates are important biochemical molecules. In this study, recombinant Escherichia coli that could display nucleotide kinases (INP-N-NMKases) and acetate kinase (INP-N-ACKase) on the cell surface were constructed by fusing an enzyme (NMKase/ACKase) to the N-terminus of ice nucleation protein (INP-N). By using intact recombinant bacteria cells as a catalyst coupled with an ACKase-catalyzed adenosine-5′-triphosphate (ATP) regeneration system, nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) could be synthesized efficiently. In a reaction system with 5 mmol/l substrate, the conversion rates of cytidine-5′-triphosphate (CTP) and deoxycytidine-5′-triphosphate (dCTP) were 96% and 93%, respectively, the conversion rate of ATP and deoxyadenosine-5′-triphosphate (dATP) was 96%, the conversion rate of deoxythymidine-5′-triphosphate (dTTP) was 91%, and the conversion rate of uridine-5′-triphosphate (UTP) was 80%. There was no obvious degradation. At 37 °C, the stability of the surface-displayed fusion protein, especially in the presence of the substrate, was significantly improved. Each whole cell could be reused more than 8 times.

Keywords

Nucleoside triphosphates Deoxynucleoside triphosphates Nucleotide kinase Acetate kinase Ice nucleation protein Surface display ATP regeneration 

Notes

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed Consent

Informed consent was obtained from all individual participants included in the study.

Supplementary material

12010_2019_3138_MOESM1_ESM.docx (444 kb)
ESM 1 (DOCX 444 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Bioreactor EngineeringEast China University of Science and TechnologyShanghaiChina
  2. 2.Department of Microbiology, Biochemistry and Molecular Genetics, New Jersey Medical SchoolRutgers UniversityNewarkUSA

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