Supplementary Data
FT-IR spectrum of DAB and the degraded DAB
The FT-IR spectrum of DAB (Fig.1a) showed the characteristic peaks. The spectrum
shows major stretching bands owing to the peptide group occurred in the spectral region
1200–1700 cm-1. The bands at 1501.85 cm-1 and 1592.20 cm-1 are due to the aromatic ring
stretching vibrations of C=C-C. The band at 748.37 cm-1 is attributed to the aromatic C-H
bending out of plane 1, 2 disubstitution (ortho). The bands at 3364.91 cm-1 and 3386.58 cm-1
are due to the N-H stretching in aromatic primary amine. The bands at 1336.16 cm-1, 1321.06
cm-1, 1274.38 cm-1 are due to the C-N stretching in aromatic primary amine. The stretching of
aromatic C-H was observed at 3056.07 cm-1, 3038.19 cm-1 and 3027.68 cm-1. The bending of
primary amine N-H was found at 1633.55 cm-1.
The FT-IR spectrum of the degraded DAB by MICE of Citrobacter freundii clearly
shows that the DAB was degraded (Fig.2b). The bands at 2922.26 cm-1 and 2853.03 cm-1 are
attributed to the asymmetric and symmetric stretching of CH3 group respectively. The peak at
1751 cm-1 may be due to aliphatic ketone group and the asymmetric stretching of carboxylate
ion was found at 1650.06 cm-1. The bands at 1450.3 cm-1 and 1380 cm-1 are attributed to
asymmetric and symmetric bending of CH3 group respectively. The band at 2953.6 cm-1 is
due to the stretching of O-H group. The peak at 1082.15 cm-1 may be attributed to stretching
of C-O group. The FT-IR spectrum confirms the presence of functional groups in pyruvic
acid.
Fig.1. FT-IR spectrum of (a) DAB and (b) DAB after treatment with MICE
H1 & 13C NMR spectroscopy
The 1H NMR spectrum of DAB was scanned between 0 and 12 ppm. The 1H-NMR
spectrum showed signals in the aromatic region (6–7 ppm) which indicates a 1,2-disymmetrically substituted benzene skeleton. The chemical shift around δ 4.3 ppm can be
assigned to the amine bonded protons (Fig. 2a). The enzyme degraded DAB shows the
presence of aliphatic proton at δ 3.4 and δ 2.5 ppm. The compound after degradation has been
identified as pyruvic acid containing aliphatic hydrogen atoms.
Fig.2. H1NMR spectra of (a) DAB and (b) DAB after treatment with MICE.
The
13
C NMR spectrum of DAB was scanned between δ 0.0 and 200 ppm. The
chemical shift around δ 130–140 ppm indicates the evidence of carbon in the aromatic ring
(Fig. 3a). The occurrence of chemical shift around δ 117.8 ppm confirms the presence of
carbon atoms attached to amine groups. The
13
C-NMR spectrum of DAB degraded sample
shows that the peaks at δ 110-140 ppm were disappeared. The chemical shift around δ 31.15
ppm indicates the evidence for methyl carbon. The ketone carbon showed a chemical shift at
δ 207ppm (Fig. 3b). The absence of carboxylic peak at δ 180 ppm may be due to the masking
effect of the solvent. This confirms the presence of pyruvic acid after the degradation of DAB
using MICE.
Fig.3. C13 NMR spectra of (a) DAB and (b) DAB after treatment with MICE
GC-MS analysis of metabolic products of DAB
GC-MS analysis was used to the identify end products of DAB degradation. The
molecular ion [M+] at m/z 108 with retention time 6.08 min was found to be identical to the
mass spectral properties of the authentic DAB (Fig. 4a). The mass spectrum data of DAB
shows that the fragment peak at C6H4+ (80) .The degraded sample showed the presence of
pyruvic acid, giving the molecular ion peak at m/z 88 with the retention time of 1.75 min.
Pyruvic acid on fragmentation of COOH group yielded CH3CO+ corresponding to peak at m/z
43 and on elimination of CH3 group gave a peak corresponding to m/z 33(Fig. 4b). The
fragments with molecular ions confirm the presence of pyruvic acid as the product of
degradation of DAB by MICE.
Fig.4. GC-MS spectra of the (a) DAB and (b) DAB after treatment with MICE.