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Conceptualization, D.P.; Data curation, D.P., S.R., M.A. and P.S.; Formal analysis, D.P., S.R., M.A. and P.S.; Project administration, J.J. and P.R.; Resources, J.J.; Supervision, J.J.; Visualization, S.R.; Writing—original draft, D.P., M.A. and P.S.; Writing—review & editing, D.P., S.R., J.J. and P.R. All authors have read and agreed to the published version of the manuscript.
Figure 1. Profile of protein expression in E. coli BL21 (DE3) pLysS cells with and without IPTG. Lane 1: BS—Without IPTG, Before Sonication; Lane 2: AS—Without IPTG, After Sonication; Lane 3: 1 mM IPTG, Before Sonication; Lane 4: 1 mM IPTG, After Sonication; Lane 5: M—Marker; Lane 6: E1—Elute from HisTag affinity chromatography; Lane 7: P—Purified protein from size exclusion chromatography. Final lane: WB—western blot of the purified protein.
Figure 1. Profile of protein expression in E. coli BL21 (DE3) pLysS cells with and without IPTG. Lane 1: BS—Without IPTG, Before Sonication; Lane 2: AS—Without IPTG, After Sonication; Lane 3: 1 mM IPTG, Before Sonication; Lane 4: 1 mM IPTG, After Sonication; Lane 5: M—Marker; Lane 6: E1—Elute from HisTag affinity chromatography; Lane 7: P—Purified protein from size exclusion chromatography. Final lane: WB—western blot of the purified protein.
Figure 2. (a) Schematic stepwise working principle of malachite green adenylation assay. (b) Malachite green adenylation assay: detection of free phosphate liberated from the adenylation process is captured by malachite green solution at 620 nm. Standard phosphate of 0.5 M was used as positive control, and two different concentrations of enzymes (45 µg/mL and 25 µg/mL) were used to determine the enzyme.
Figure 2. (a) Schematic stepwise working principle of malachite green adenylation assay. (b) Malachite green adenylation assay: detection of free phosphate liberated from the adenylation process is captured by malachite green solution at 620 nm. Standard phosphate of 0.5 M was used as positive control, and two different concentrations of enzymes (45 µg/mL and 25 µg/mL) were used to determine the enzyme.
Figure 3. Chromatographic adenylation assay: adenylated streptomycin retains the positive charge even after the attachment of adenyl group from donor ATP are absorbed at 260 nm in GE ÄKTA™ (FPLC).
Figure 3. Chromatographic adenylation assay: adenylated streptomycin retains the positive charge even after the attachment of adenyl group from donor ATP are absorbed at 260 nm in GE ÄKTA™ (FPLC).
Figure 4. Retention analysis in HPLC: (a) elution chromatogram of ATP; time interval: 2.3 to 3.3. (b) Elution chromatogram of streptomycin; time interval: 2.0 to 2.50. (c). Elution chromatogram of adenylated streptomycin; time interval: 2.40 to 3.30.
Figure 4. Retention analysis in HPLC: (a) elution chromatogram of ATP; time interval: 2.3 to 3.3. (b) Elution chromatogram of streptomycin; time interval: 2.0 to 2.50. (c). Elution chromatogram of adenylated streptomycin; time interval: 2.40 to 3.30.
Figure 5. Mass spectrometry analysis: (a) MS/MS spectrum of AMP; adenine peak m/z at 136.2 is highlighted in blue color. (b) MS/MS spectrum of streptomycin; two peaks m/z at 263.2 and 176.2 are highlighted in green color (c) MS/MS spectrum of adenylated streptomycin. The stronger spectrum of m/z at 136.2 from AMP and m/z at 263.2 and 176.2 from streptomycin, commonly observed in adenylated streptomycin, is highlighted in blue and green color, respectively.
Figure 5. Mass spectrometry analysis: (a) MS/MS spectrum of AMP; adenine peak m/z at 136.2 is highlighted in blue color. (b) MS/MS spectrum of streptomycin; two peaks m/z at 263.2 and 176.2 are highlighted in green color (c) MS/MS spectrum of adenylated streptomycin. The stronger spectrum of m/z at 136.2 from AMP and m/z at 263.2 and 176.2 from streptomycin, commonly observed in adenylated streptomycin, is highlighted in blue and green color, respectively.
Table 1. Protein identification: MALDI-TOF/TOF mass spectrometry analysis data of trypsin-digested peptides of the targeted protein were matched against data in the Mascot database. Mass information of two peptides of the targeted protein showed a Mascot score of 70.
Table 1. Protein identification: MALDI-TOF/TOF mass spectrometry analysis data of trypsin-digested peptides of the targeted protein were matched against data in the Mascot database. Mass information of two peptides of the targeted protein showed a Mascot score of 70.
General InformationDatabaseNCBIproScore70Monoisotopic mass (Mr)29,095Calculated pI4.93Theoretical mass of protein29,936Matches3Matching proteinAminoglycoside nucleotidyltransferase Matching source organismSerratia sp. FGI94Peptide InformationPeptide fragment56APLDNTQR63175ETADLQGDER184Score5967Mr (Expt)913.45641132.4988Mr (Calc)913.49811132.4996Observed914.4636567.2567Table 2. Antibiotic susceptibility and minimal inhibitory concentration (MIC) tests: seven antibiotics in discs and Ezy MIC™ strips were tested against the cell control, vector control, and recombinant cell. Cell control (CC), empty E. coli pLysS cell; vector control (VC), E. coli pLysS cell with null pET28a vector; recombinant cell (SM), E. coli pLysS cell bearing recombinant vector expressing SMATase protein. Tested against the empty E. coli pLysS cell (cell control), E. coli pLysS cells bearing empty pET28a(+) vector (vector control) and recombinant E. coli pLysS cell bearing target gene expressing SMATase (recombinant cell).
Table 2. Antibiotic susceptibility and minimal inhibitory concentration (MIC) tests: seven antibiotics in discs and Ezy MIC™ strips were tested against the cell control, vector control, and recombinant cell. Cell control (CC), empty E. coli pLysS cell; vector control (VC), E. coli pLysS cell with null pET28a vector; recombinant cell (SM), E. coli pLysS cell bearing recombinant vector expressing SMATase protein. Tested against the empty E. coli pLysS cell (cell control), E. coli pLysS cells bearing empty pET28a(+) vector (vector control) and recombinant E. coli pLysS cell bearing target gene expressing SMATase (recombinant cell).
MIC (µg/mL)AntibioticE. coli pLysS Cells
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