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.