Plant materials and growth conditions
A lowland-type switchgrass cultivar, Alamo, was employed for detoxification of DNT and DNTS. According to the criteria described by Hardin (Hardin et al. 2013), the development of our switchgrass plants at vegetative phase was divided into three vegetative stages (V1, V2, and V3) and five elongation stages (E1, E2, E3, E4, and E5) before they entered the reproductive phase. Plants were grown in a greenhouse with a duration period of 16-hours of light (390 µE·m-2·S-1).
Codon optimization ofNfsI
The coding sequence of NfsI characterized in Enterobater cloacae was downloaded from GenBank (accession No. M6308.1) (Bryant and DeLuca, 1991). The codon optimization of NfsI was based on the code preference of switchgrass and was subjected to chemical synthesis.
Enzyme kinetics analysis of recombinantNfsI
The codon optimizedNfsI was cloned into pET32a vector after digested by Eco RI. The recombinant plasmid pET32a-NfsI was transformed into Rosetta E. coli for production of recombinant NfsI protein (Xiong et al., 2019). The purified recombinant protein was subjected to enzyme kinetics analysis of NfsI against 2,4-DNT and DNTS, as described by Bryant (Bryant and DeLuca, 1991). Moreover, 2,4-DNT, DNTS, and their transformed products were identified and quantified by reversed phase high-performance liquid chromatography along with a photo diode array and electrospray ionization tandem mass spectrometry (LC-PDA/ESI-MS/MS). The authentic 2,4-DNT and DNTS were used as external criteria to identify the 2,4-DNT and DNTS, and their content was quantitated based on the absorbance at 254 nm and 205 nm, respectively. The 2,4-DNT and DNTS were ordered from Aladdin Industrial Corporation, Shanghai, China (CAS No. 121-14-2) and Synchem OHG, Germany (CAS No. 63348-71-0), respectively.