High-level expression and molecular characterization of a recombinant prolidase fromEscherichia coliNovaBlue

Abstract

Long-term use of organophosphorus (OP) compounds has become an increasing global problem and a major threat to sustainability and human health. Prolidase is a proline-specific metallopeptidase that can offer an efficient option for the degradation of OP compounds. In this study, a full-length gene fromEscherichia coliNovaBlue encoding a prolidase (EcPepQ) was amplified and cloned into the commercially-available vector pQE-30 to yield pQE-EcPepQ. The overexpressed enzyme was purified from the cell-free extract of isopropyl thio-β-D-galactoside IPTG-inducedE. coliM15 (pQE-EcPepQ) cells by nickel-chelate chromatography. The molecular mass ofEcPepQ was determined to be about 57 kDa by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and the result of size-exclusion chromatography demonstrated that the enzyme was mainly present in 25 mM Tris–HCl buffer (pH 8.0) as a dimeric form. The optimal conditions forEcPepQ activity were 60 °C, pH 8.0, and 0.1 mM Mn2+ion. Kinetic analysis with Ala-Pro as the substrate showed that theKmandkcatvalues ofEcPepQ were 8.8 mM and 926.5 ± 2.0 s−1, respectively. The thermal unfolding ofEcPepQ followed a two-state process with one well-defined unfolding transition of 64.2 °C. Analysis of guanidine hydrochloride (GdnHCl)-induced denaturation by tryptophan emission fluorescence spectroscopy revealed that the enzyme had a [GdnHCl]0.5,N-Uvalue of 1.98 M. The purified enzyme also exhibited some degree of tolerance to various water/organic co-solvents. Isopropanol and tetrahydrofuran were very detrimental to the enzymatic activity ofEcPepQ; however, other more hydrophilic co-solvents, such as formamide, methanol, and ethylene glycol, were better tolerated. Eventually, the non-negative influence of some co-solvents on both catalytic activity and structural stability ofEcPepQ allows to adjust the reaction conditions more suitable forEcPepQ-catalyzed bioprocess.