Successful expression of the Bordetella petrii nitrile hydratase activator P14K and the unnecessary role of Ser115.

BACKGROUND The activator P14K is necessary for the activation of nitrile hydratase (NHase). However, it is hard to be expressed heterogeneously. Although an N-terminal strep tagged P14K could be successfully expressed from Pseudomonas putida, various strategies for the over-expression of P14K are needed to facilitate further application of NHase. RESULTS P14K was successfully expressed through fusing a his tag (his-P14K), and was over-expressed through fusing a gst tag (gst-P14K) at its N-terminus in the NHase of Bordetella petrii DSM 12804. The stability of gst-P14K was demonstrated to be higher than that of the his-P14K. In addition, the Ser115 in the characteristic motif CXLC-Ser115-C of the active center of NHase was found to be unnecessary for NHase maturation. CONCLUSIONS Our results are not only useful for the NHase activator expression and the understanding of the role of Ser115 during NHase activation, but also helpful for other proteins with difficulty in heterologous expression.