Cloning and co-expression of recombinant N-demethylase B and N-demethylase D genes in Escherichia coli
Li, Dengchao | Han, Qiumin
NdmB and ndmD genes encoding N-demethylase (Ndm) B and NdmD from Pseudomonas putida CBB5 were successfully cloned in pET32a, designated as pET32a-ndmB-His-His-ndmD (pET32a-BHHD) and electroporated into Escherichia coli strain BL21 (DE3). Polymerase chain reaction (PCR), restriction enzyme digestion and DNA sequencing were subsequently employed to confirm the success of the procedure. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the estimated molecular weight of NdmB and NdmD co-expressed in recombinant E. coli was 35 and 60 kDa, respectively. A one-step purification of Ndms using a Ni-affinity column resulted in a 10.3-fold purification with 33.6% yield. The enzyme activity of NdmB and NdmD showed that 1000 μmol/L theobromine was completely converted into 7-methylxanthine within 90 min by resting cells containing pBHHD.
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