Fig 1.
Protein sequences alignment between CarEW and homologs from the carboxylesterase family.
ESPript outputs obtained with the sequences from the SWISSPROPT databank and alignment with CLUSTAL W. Sequences are grouped according to similarity. PNBA_BACSU, PNB (Para-nitrobenzyl esterase) carboxylesterase from B. subtilis subsp. Subtilis str. 168; CarEW, carboxylesterase from B. subtilis K91; EST1_THEFU, carboxylesterase from Thermobifida fusca; EST2A_MOUSE, pyrethroid hydrolase Ces2e from Mus musculus; EST1_MESAU, liver carboxylesterase from Mesocricetus auratus; and EST5A_FELCA, carboxylesterase 5A from Felis catus. Conserved motifs are highlighted. Residues strictly conserved among groups are shown in white font with red background. The possible catalytic triad (serine (S), glutamic acid (E), histidine (H)) is shown at the bottom of the alignment in green font. A conserved pentapeptide (GXSXG), containing the serine residue of the catalytic triad, was framed by a dotted box. Symbols above blocks of sequences represent the secondary structure, springs represent helices, and arrows represent β-strands.
Fig 2.
Neighbor-joining tree of esterases.
Protein sequences were aligned using the built-in CLUSTAL W (default parameters), the tree was built using the neighbor-joining method with default parameters and 1000 bootstrap replications.
Fig 3.
Analysis of the protein expressed in E. coli BL21 cells following purification on a 12% SDS-PAGE.
Lane M, protein molecular marker; Lane 1, before induction with 0.7 mM IPTG; Lane 2, after induction with IPTG and grown at 20°C for 20 h; and Lane 3, purified recombinant CarEW (∼53.76 kDa).
Table 1.
Kinetic parameters of the recombinant CarEW on different substrates at pH 7.5 and 45°C.
Fig 4.
Effect of temperature and pH on CarEW activity and stability.
Relative activity of purified CarEW was determined at different temperatures (A) or pH (C) using ρ-NP butyrate (ρ-NPC4) as the substrate at 405 nm. Remaining enzyme activity was measured at 45°C and pH 7.5 after incubating purified CarEW at different temperatures (B) or pH for 60 min. Error bars represent the mean±standard deviation (n = 3).
Table 2.
Effect of various metal ions and chemical agents on enzyme activity.
Table 3.
Effect of organic solvents on enzyme stability after incubation at 45°C for 1 h.
Fig 5.
ESI-MS analysis of products identified in the CarEW catalyzed reaction.
(A) and (B) The full ion scan of DiBP degradation after incubation with CarEW and the control without CarEW. m/z 301, DiBP; DiBP incubation with CarEW. m/z 189, PTH; m/z 245, MiBP. (A) and (B) were both tested under the positive mode ([M+Na]+). (C) and (D) Daughter ion scan for metabolites derived from the biodegradation of DiBP. (C) m/z 121, phthalate acid (PTH); (D) m/z 221, monoisobutyl phthalate (MiBP). Both were tested under negative mode ([M-H]−).
Fig 6.
The results of HPLC analyses of catabolic intermediates.
(A) DiBP, MiBP and PTH mixture for the standard sample. (B) The results of DiBP degradation after incubation with CarEW. (C) Reaction of MiBP with 10 mM citric acid-Na2HPO4 solution (pH 7.5) buffer instead of CarEW. (D) The results of MiBP degradation after incubation with CarEW.
Fig 7.
Proposed pathway for incomplete diisobutyl phthalate degradation by CarEW.