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.