Environmental Factors Modulating the Stability and Enzymatic Activity of the Petrotoga mobilis Esterase (PmEst)

Abstract

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.

Going deep into protein secondary structure with synchrotron radiation circular dichroism spectroscopy

“…An example of a protein containing this mixed α-β folding was studied by Lopes et al (2016) with SRCD spectroscopy. The protein (named PmEst) was originally produced by the extremophile organism Petrotoga mobilis and exhibits hydrolytic activity on short acyl chains esters.…”

“…The protein (named PmEst) was originally produced by the extremophile organism Petrotoga mobilis and exhibits hydrolytic activity on short acyl chains esters. While no crystal structure for PmEst is available, the homology model predicted for its 3D structure was used in comparison with the structural content estimated with the SRCD method and bioinformatics analyses (Lopes et al 2016). Estimations of structural content for PmEst based on the SRCD method were more closely correlated with the homology model than those performed with laboratory-bench CD (see values in Table 1).…”

“…Many research groups have used SRCD spectroscopy to examine and estimate the secondary structure content of proteins (Lees et al 2006;Matsuo and Gekko 2013;Garcia et al 2013;Hussain et al 2016;Lopes et al 2016;Miler et al 2016). The technique has been successfully employed to characterize the insertion and orientation of helical peptides in lipid bilayers (Bürck et al 2016) or compare the behavior of globular and natively unfolded proteins (Ruskamo et al 2012;Yoneda et al 2017), investigate protein/peptide structure in model membranes (Miles et al 2008;Dreschler et al 2009), assess the dynamic and flexible conformation of unordered proteins (Lopes et al 2014a, b;Bremer et al 2017), evaluate protein:protein complex formation (Cowieson et al 2008;Lopes et al 2013), detect conformational changes induced by the binding to ligands (Lima et al 2013), perform fold recognition and obtain an accurate distinction between parallel and antiparallel β-sheets (Micsonai et al 2015) and identify the conformational changes associated with a mutant protein (Wallace et al 2004).…”

How to Lengthen the Long-Term Stability of Enzyme Membranes: Trends and Strategies

“…For inducing mutations, protein engineering is a key technology [12,13]; examples are presented for improvements, such as pH stability [14] and heat tolerance [15]. Stability of enzymes produced via protein engineering can be examined using in silico prediction and actual measurement [16]. Enhancement of pH stability is not so easy because amino acids with electrically charged side chains exist at the enzyme surface and pH change makes slight changes of enzyme structure by charge distribution change caused by dissociation and association of the side chains.…”

Structural and dynamic characterization of the Golgi Reassembly and Stacking Protein (GRASP) in solution

“…Since we did not observe structural changes of CnGRASP in the presence of phospholipid vesicles presumably due to weak binding (see above), we decided to utilize alcohol/water mixtures to model the membrane field as ε changes in the medium. This is a widely used strategy in protein structural studies [212,222,223], especially in the case of IDPs [91,99]. In this work, we used methanol, ethanol and isopropanol as agents to change the dielectric constant of the medium and, thus, to separate the alterations promoted by ε in the protein structure from artefacts induced by nonspecific protein/alcohol interaction.…”