Enzymatic Synthesis Of Nucleoside-5’-triphosphate And Deoxynucleoside-5’-triphosphate

Nucleoside triphosphates(NTPs)and deoxynucleoside triphosphate(dNTPs)are not only important biochemical substances,but also widely used in medicine,food and scientific research.In this study,a multienzyme synthesis system combining the nucleotide kinase(INP-N-NMKase)and acetate kinase(INP-N-ACKase)shown on the cell surface using ice nucleation protein(INP)was used to one-pot produce NTP/dNTP from nucleoside monophosphate(NMP)/deoxynucleoside monophosphate(dNMP).In this system,a ATP regeneration system was coupled which catalyzed by ACKase from ACP.With the regeneration of ATP,only a small amount of ATP(1/20 of substrate concentration and 1/5 in UMP reaction)was required at the begin of the reaction.The gene nmk(including cmk,amk,tmk and umk)and ack fragments from Lactobacillus bulgicus were amplified and inserted into the downstream of plasmid pET-28a containing the N-terminal domain of inp-n of ice nucleation protein,respectively.The surface display bacteria,E-INP-N-NMK and E-INP-N-ACK,were constructed and all the fused proteins were well expressed.The results showed that with 5 mM substrate in the reaction system,the conversion rates of CTP and dCTP were 96%and 93%,respectively,and the conversion rates of ATP and dATP were up to 96%.The dTTP conversion rate was 90%,and the UTP conversion rate was 80%.There was no obvious product degradation in the reaction process.The stability of fusion proteins displayed on cell surface as much as or even better than intracellular free enzymes at 37℃,especially in the presence of the substrate.Moreover,due to the characteristics of the whole cells,they could be reused for more than 8 times after centrifugation.