Fig 1.
Lipolytic activity and gene annotation of the Pl17.E10 clone.
(A) Clones obtained from the metagenomic approach were cultivated in a Petri dish containing LB media supplemented with 1% (v/v) tributyrin, 1% (w/v) gumarabic, 0.00125% (v/v) chloramphenicol and 0.001% (v/v) arabinose. The plate was maintained at 37°C for three days and for four more days at 4°C. A clear halo was observed around the P117.E10 fosmid clone, evidencing its ability to degrade tributyrin. A detailed physical map of Pl17.E10 is shown, which consists of 25 identified ORFs depicted as arrows according to their location and direction in the fosmid vector. The black arrow indicates ORF16, which encodes the Est16 protein. (B) The characteristics of each ORF were obtained after using ORF Finder and the BLASTP search. The putative function of each ORF and their GenBank accession numbers are also shown.
Fig 2.
Phylogenetic tree and comparison of Est16 with esterases/lipases showing their biotechnological applications.
(A) A phylogenetic tree of 18 sequences from patented esterases/lipases, the sequence of aryl-esterase (PDB code 1VA4) and Est16 was generated using the Maximum Likehood method (MEGA6.0) with a bootstrap of 1,000 replicates. The estimated value of the shape parameter for the discrete Gamma Distribution is 16.1381. Substitution pattern and rates were estimated under the WAG model (+G) with 5 categories. (B) The amino acid sequence alignment of Est16 with Anti-Kazlauskas lipase (patent number US20050153404) and aryl-esterase (PDB code 1VA4). The residues involved in the substrate-pocket and family classification are shown in bold and the catalytic triad residues are denoted with asterisks (*). The consensus motif of family V is shown.
Fig 3.
Structural features of the Est16 model in comparison with an esterase from P. fluorescens and the Anti-Kazlauskas lipase.
(A) Ribbon representation of the Est16 model (green) superposed on the structures of P. fluorescens esterase (blue) and the patented lipase (patent number US20050153404) (purple) revealing the conservation of the alpha/beta fold. N is the N-terminal. (B) A detailed picture of the residues from the catalytic triad of the three enzymes showing the structural superposition. Residues are shown in the following order: Est16, Pfl (PDB code 1VA4) and Anti-Kazlauskas lipase. A comparative analysis of the domains and pockets of Est16 (C), the Anti-Kazlauskas lipase model (D) and the structure of the P. fluorescens aryl-esterase (E) are shown in the cartoon. Helices are blue, β-sheets are cyan and the residues in the active site are shown as yellow sticks. The large and small domains are delimited in the three proteins.
Fig 4.
Production steps of Est16 as a soluble and stable protein.
(A) 12% SDS-PAGE of the expression fractions and IMAC samples of Est16 from E. coli BL21 (DE3) cells carrying the pET28a-est16 vector, which contained the est16 gene. E. coli cells were grown in LB up to O.D.600nm = 0.5 at 37°C and then induced with 0.1 mM IPTG at 28°C for 20 hours. Lane 1: non-induced cells; Lane 2: induced cells; Lane 3: molecular weight marker (the kDa values are indicated in the picture); Lane 4: soluble extracts; Lane 5: flow-through fraction from the IMAC purification; Lane 6: 10 mM imidazole wash fraction; Lanes 7, 8 and 9: 50 mM, 100 mM and 1 M imidazole elution fractions, respectively. (B) The chromatogram obtained after the size-exclusion chromatography using a HiLoad 16/60 Superdex 200 column (GE Healthcare Bio-Sciences). The insets show the samples related to the peaks loaded into the 12% SDS-PAGE polyacrylamide gels. Lane 1, molecular weight standards; Lane 2: sample before SEC; Lanes 3–4: fractions from the first peak; Lanes 5–12: fractions from the second peak, which were concentrated for further spectroscopic and biochemical analyses.
Fig 5.
Tributyrin assay in Petri dishes and spectroscopic analyses of Est16.
Tributyrin assays of Est16 (50 μg/mL) in Petri dishes containing 0.23% Tris-HCl (w/v), 1.2% agar (w/v) and 1% tributyrin at pH 7.0 (A), pH 8.0 (B) and pH 9.0 (C). Circular dichroism assays of Est16 at pH 7.0 (D), pH 8.0 (E) and pH 9.0 (F). The samples contained 5.64 μmol of enzyme and the spectra were recorded using a Jasco J-810 spectropolarimeter with a 10 mm path length. Similarly, the thermal denaturation of the enzyme was measured at 222 nm as a function of the temperature at pH 7.0 (G), pH 8.0 (H) and pH 9.0 (I). The melting temperature (Tm) is shown for each spectrum.
Fig 6.
(A) Substrate specificity against p-NP esters of various lengths (C4 to C16); (B) The effect of pH on Est16 activity against p-NP butyrate; (C) The effect of temperature on Est16 activity against p-NP valerate (■) and octanoate (●); (D) Est16 activity against p-NP butyrate was evaluated in the presence of Ca2+, Co2+, K+, Mg2+, Mn2+, Ni2+, Zn2+ and EDTA; (E) The influence of detergents and (F) organic solvents on the enzyme catalytic activity torward p-NP butyrate. In E, the striped bar is triton X-100 and the white bar is tween 20. In F, the black bar is the control, the white bar is DMF and the striped bar is DMSO. In the graphics (A-F), the small letters on the top indicates the significant difference between each condition performed in the experiment, according to ANOVA and Tukey’s test at 5% probablility.
Table 1.
Kinetic parameters and maximum enzyme rate (Vmax) of Est16 activity against p-nitrophenyl substrates.