From the Antarctic soil metagenomic library [24], one clone producing a blue halo after one week incubation at 18 °C on SBA-tributyrin media was selected as a potential esterase/lipase producer.

Complete DNA sequencing of the insert (3 kb) revealed a unique putative gene, named mhlip (Genbank access. number. GU550075). The gene encodes a 262 amino acids hypothetical protein with a theoretical molecular mass of 28,075 Da and a pI of 5.2. The encoded protein, MHlip, displayed 83% amino acid identity to a putative cytosolic α/β hydrolase derived from Acidovorax delafieldii 2AN (Acc. Numb. ZP-04765427). The sequence also showed significant identity with many other mesophilic proteins including the arylesterase from Pseudomonas fluorescens (PFE-ESTE 26% id., acc. no. P22862.4), the non-heme bromoperoxidase (BPOA2, 30% id., acc. no. P29715.3) and the non-heme chloroperoxidase (PRXC-STRAU, 29% id., acc. no. O31168.1) from Streptomyces aureofaciens, and with the crystallized esterase YTXM from Bacillus subtilis (id. 26%, acc. no. P23974).

We constructed a phylogenetic tree using the MHlip sequence and some sequences derived from various esterase-related families [25]. Next, Pfam numbers were assigned to each sequence using Pfam_scan (http://pfam.sanger.ac.uk/) [26] (Figure 1). MHlip was associated with the α/β hydrolase family 6 (Pf12697). This fold is observed in many enzymes having multiple functions (i.e., transacetylase, lipase, hydroxynitrile lyase, methylesterase, peptidase, haloperoxidase, carboxylesterase) [27].

Sequences alignment highlighted all the residues considered as involved in substrate binding and catalysis (Figure 2) [29]. In addition, residues W₂₉ and M₉₅ assumed to limit the active site of PFE-ESTE are displayed. These two residues limit the diffusion of large substrates in the catalytic cleft, independently of any lid structure. Thus resulting in an enzyme having high affinity for short substrates (i.e., pNPA) [29].

The MHlip-encoding gene was PCR-amplified and introduced in the pET22b vector in order to express a C-terminal His-tagged MHlip. The recombinant protein was purified from the cytoplasmic fraction of transformed E. coli BL21(DE3) using Ni-NTA columns.

Since MHlip was first isolated on tributyrin containing media, its activity was investigated on various p-nitrophenyl (pNP)-esters at pH 8.0 (Figure 3). MHlip is only active on small substrates such as pNPA; activity on pNPB is less than 5% of the pNPA hydrolysis and no activity was detected on substrates with longer acyl-chain lengths.

The pH-dependence of the MHlip activity on pNPA was investigated by monitoring the absorbance at 348 nm between pH 3.5 and 11.5, in a 20 mM pH-adjusted poly-buffer. MHlip was significantly active over a wide range of pH, from 4.5 to 9 (Figure 3).

The thermal sensitivity of the MHlip activity towards pNPA was recorded at pH 8.0 in a thermal gradient ranging from 4 to 55 °C. The rate of pNPA hydrolysis rapidly increased from 4 to 35 °C and then quickly decreased to few percents of residual activity at 45 °C. Interestingly, MHlip remained significantly active when temperature decreased (20% at 4 °C) (Figure 4).

Kinetic parameters for pNPA hydrolysis were determined under initial rate conditions using a nonlinear regression analysis of the Michaelis–Menten equation. Hydrolysis was measured at 30 °C using pNPA as substrate at final concentrations ranging from 0 to 10 mM in 20 mM Tris-HCl (pH 8). For MHlip, the kinetic constants were: K_m = 1.2~0.1 mM, k_cat = 3.13.10⁻²~0.2.10⁻² sec⁻¹ and k_cat/K_m = 0.025 mM⁻¹ sec⁻¹.

MHlip fluorescence spectra recorded at 20 °C and 90 °C were compared. When MHlip was incubated at 80 °C its fluorescence emission spectrum, compared to the spectrum obtained at 20°C, revealed its unfolding. A bathochromic effect was observed when the temperature increased as the maximum fluorescence was shifted to higher wavelengths (from 338 to 350 nm). In order to determine the MHlip melting temperature, the fluorescence emission at 338 nm was recorded in a thermal gradient, from 20 to 90 °C (data not shown).

This approach appeared to be inappropriate since the increase of temperature caused a decrease of the fluorescence emission at 338 nm, with no clear transition. The determination of the apparent melting temperature (Tm) was eventually achieved by recording the wavelength of the maximum fluorescence emission (λ_max) as a function of the temperature. At an excitation wavelength of 280 nm, a clear transition from a native to an unfolded state was observed at ~50 °C (Figure 4).

A previously described short-insert metagenomic library (average insert size of 5.1 kb) was used for the present work [19]. Screening for lipolytic activity was carried out by plating 3,000 recombinant E. coli clones on Spirit-Blue Agar (SBA) Media (BD-Difco, MD) containing chloramphenicol (12.5 µg/mL) and 1% emulsified Tributyrin (Sigma-Aldrich). The plates were incubated at 37 °C for 24 hours and kept at 18 °C for one week.

Forward primer (5’-GAGCACATATGCCTTTCGCGCA-3’) and the reverse primer (5’-GCTTCAGAGGCGCTCTCGAGCTTGTCGAGA-3’), containing NdeI and XhoI restriction sites, respectively (underlined), were used for PCR amplification of the full length MHlip-encoding gene. The obtained amplicon was ligated in an NdeI/XhoI double digested pET22b plasmid (Novagen, NJ) that allowed the production of a C_term-6×HisTag fused protein. After confirmation of the sequence, the pET22b:MHlip was introduced in competent E. coli BL21(DE3) cells (Novagen). Transformed cells were cultivated at 18 °C in LB media containing ampicillin (100 µg/mL). Heterologous protein expression was carried out for four hours by adding 0.4 mM isopropyl-D-1-thiogalactopyranoside (IPTG) when the OD_600nm reached ~0.5. After centrifugation, the cell pellet was resuspended in 20 mM Tris-HCl (pH 8.0) and the cells were disrupted by sonication (3 cycles of 30 sec at amplitude of 10–12 µm). Proteins from the cytoplasmic fractions were recovered by centrifugation at 20,000 × g for 40 min, and MHlip was purified by affinity chromatography on a 5 mL Ni-NTA columns (GE Healthcare) in a linear gradient of imidazole (from 0 to 250 mM). Protein concentration was determined by the Bicinchoninic Acid (BCA)-protein quantitation assay (Pierce) using serum albumin as standard. Finally, 30 mg of pure protein was obtained per litre of culture. The purified protein was subjected to automatic Edman degradation for the determination of the N-terminal amino acid sequence on an Applied Biosystems 492 Protein Sequencer (Perkin Elmer, Waltham, MA, USA). The resulting sequence (MPFAH) was found to match the predicted one. The purified protein was used for further characterization.

Lipase/esterase activity assays were carried out on p-nitrophenyl-esters (pNP-Acetate, pNPA; pNP-Butyrate, pNPB; pNP-Caprilate, pNPC; and pNP-Laurate, pNPL; all purchased from Sigma) by spectrophotometric methods. The assay mixture was prepared as follow: 940 µL of 20 mM Tris-HCl (pH 8.0), 10 µL of 10 mM p-nitrophenyl-ester in acetonitrile, and 50 µL of enzyme solution. The release of p-nitrophenol was followed taking into account a molar extinction coefficient at 405 nm (ε_405nm) of 16,500 M⁻¹.cm⁻¹.

The pH-dependence of the enzyme’s activity on pNPA, between pH 3.5 and 11.5, was investigated by monitoring the absorbance at 348 nm and replacing the Tris-HCl buffer by a 20 mM pH-adjusted polybuffer consisting of Tris, KCl, Na₂HPO₄, CH₃COO.Na and dihydrogen citrate [30].

The thermal sensitivity of the MHlip activity toward pNPA was recorded as described above, over a temperature range between 4 and 55 °C.

In order to investigate the thermal stability of MHlip, the purified protein was submitted to increasing temperatures and the process of denaturation was followed by intrinsic fluorescence spectra. The spectrum of the native MHlip at 20 °C was recorded on a Perkin Elmer LS50B spectrofluorometer using a 1 cm cell path length. Maximum fluorescence emission was observed at a wavelength of 338 nm using an excitation wavelength of 280 nm. The Tm of the enzyme was determined by plotting the wavelength of the maximum emission (λ_max), measured at temperatures ranging from 20 to 80 °C.