Environmental factors modulating the stability and enzymatic activity of the Petrotoga mobilis Esterase (PmEst)

Enzymes isolated from thermophilic organisms found in oil reservoirs can find applications in many fields, including the oleochemical, pharmaceutical, bioenergy, and food/dairy industries. In this study, in silico identification and recombinant production of an esterase from the extremophile bacteria Petrotoga mobilis (designated PmEst) were performed. Then biochemical, bioinformatics and structural characterizations were undertaken using a combination of synchrotron radiation circular dichroism (SRCD) and fluorescence spectroscopies to correlate PmEst stability and hydrolytic activity on different substrates. The enzyme presented a high Michaelis-Menten constant (KM 0.16 mM) and optimum activity at ~55°C for p-nitrophenyl butyrate. The secondary structure of PmEst was preserved at acid pH, but not under alkaline conditions. PmEst was unfolded at high concentrations of urea or guanidine through apparently different mechanisms. The esterase activity of PmEst was preserved in the presence of ethanol or propanol and its melting temperature increased ~8°C in the presence of these organic solvents. PmEst is a mesophilic esterase with substrate preference towards short-to medium-length acyl chains. The SRCD data of PmEst is in agreement with the prediction of an α/β protein, which leads us to assume that it displays a typical fold of esterases from this family. The increased enzyme stability in organic solvents may enable novel applications for its use in synthetic biology. Taken together, our results demonstrate features of the PmEst enzyme that indicate it may be suitable for applications in industrial processes, particularly, when the use of polar organic solvents is required

On the structural stability of guanosine-based supramolecular hydrogels

Supramolecular hydrogels formed from the self-assembly of low molecular weight derivatives are very attractive systems, because of their potential applications in nano- and bio-technology. In this paper, the stable and transparent hydrogels observed in binary mixtures of guanosine derivatives (G), namely guanosine 5′-monophosphate (GMP) and guanosine (Gua), dissolved in water (at volume fractions larger than 0.95), were investigated by microscopy techniques and Small Angle X-ray Scattering (SAXS). The results confirm the presence of G-quadruplexes, chiral cylindrical rods obtained by the regular stacking of self-assembled planar cyclic guanosine quartets. However, the addition of Gua determines the formation of very stable hydrogels able to trap large amounts of water (up to a volume fraction of 0.99) and characterised by an unusual anisotropic order. A modified lateral helix-to-helix interaction pattern, tuned by Gua, is suggested to be responsible for the supramolecular gelation and the stability of the hydrogels during swelling

Esterase activity assays.

<p>a) The relative hydrolytic activities of PmEst on substrates with different acyl chain sizes were calculated with respect to C4. b) Determination of the optimum temperature of PmEst on <i>p</i>NB, and c) Residual activity normalized as a function of pre-incubation time at 25, 35, 45 and 55°C. PmEst was inactive when pre-heated at temperatures higher than 55°C. Error bars represent the standard deviations from the triplicate measurements.</p

Thermal stability of PmEst evaluated by spectroscopic methods.

<p>a) Synchrotron radiation circular dichroism spectra as a function of temperature overlaid in the Z direction; b) emission spectra of PmEst as a function of temperature in sodium phosphate pH 7.4. c) fluorescence emission maxima data as a function of temperature. d) thermal denaturation curves of PmEst, taken from SRCD data at 196, 208, and 222 nm, and e) Phase delay (bottom curves) and modulation ratio (top curves) of the excited state lifetime of PmEst in PBS as a function of temperature, from 15°C (solid) to 45°C (dot) in10°C steps; and f) their respective phasor diagram, showing the point at 55°C is not on the same trajectory of the other point at below temperatures.</p

Characterization of the PmEst enzyme.

<p>a) Sequence alignment between PmEst and esterases from other organisms. Conserved residues among esterases are highlighted in gray, and residues from esterase domains (GxSxGG) are in bold font. Secondary structure prediction of PmEst using Psipred and based on the 3-D homology model. Secondary structure elements are labeled as h, helix; e, extended β-strand; c, disordered/other. CbEst, acetyl esterase from <i>Clostridiaceae bacterium</i> GM1; BwEst acetyl esterase from <i>Blautia wexlerae</i>; RsEst, esterase from <i>Ruminococcus sp</i>. From JC304; BaEst, acetyl esterase from <i>Bacillus akibai</i>; CspEst, esterase from <i>Coprobacillus sp</i>.D7; and estA, esterase (PDB ID: 2UZ0, chain B) from <i>Streptococcus pneumoniae</i>. b) SDS-PAGE showing: 1) SUMO-PmEst fusion protein after affinity chromatography on Ni-NTA; 2) fusion protein after digestion with SUMO protease; 3) purified PmEst after rechromatography on Ni-NTA column; on the left, molecular weight markers in kDa; black arrow is indicating PmEst protein; c) Size exclusion chromatography on Superdex 200 10/300 GL column (13 μm average particle size) with MW calibration curve inset: carbonic anhydrase (29 kDa), ovalbumin (44 kDa), bovine serum albumin (66 kDa), conalbumin (75 kDa), and aldolase (158 kDa).</p

Structural properties of PmEst.

<p>SRCD spectra of the enzyme in aqueous solution (black) and in dehydrated films (gray).</p

Esterase activity of PmEst in organic solvents.

<p>a) Relative activity of PmEst in the presence of 10%, 20%, and 30% of ethanol (black) and propanol (gray). Error bars represent the standard deviation from the triplicate measurements at each temperature.</p

Effect of urea and guanidine on the structure of PmEst.

<p>Curves of the chemical denaturation of PmEst with addition of urea (black) and GndHCl (gray), taken from the normalized ellipticity value at 220 nm of the CD spectrum registered at each condition. Sigmoidal fit was performed on each curve, considering two different states for PmEst: native and denatured.</p