High-Throughput Screening of Signal Peptide Library with Novel Fluorescent Probe

Nitrile hydratase (NHase) is an excellent biocatalyst for the synthesis of amide compounds and is composed of two heterologous subunits. However, the secretory expression of NHase has been difficult to achieve because of its complex expression mechanism. In this work, a novel fluorescent probe Rho-IDA-CoII was synthesized by a one-pot method. Rho-IDA-CoII could specifically label His-tagged proteins in vitro, such as for staining in-gel, Western blot, and ELISA analysis. Furthermore, Rho-IDA-CoII combined with dot blots could quantitatively detect His-tagged proteins at between 1-10 pmol and perform high-throughput screening for the NHase signal peptide library. Recombinant Bacillus subtilis WB800/phoB-HBA with the extracellular expression of NHase was screened (ca. 6500 clones). After optimization of fermentation conditions, the NHase activity in the culture supernatant reached 17.34±0.16 U/mL. This is the first time that secretory NHase has been expressed in B. subtilis successfully.