High-level expression and molecular characterization of a recombinant prolidase from Escherichia coli NovaBlue

Tzu-Fan Wang; Meng-Chun Chi; Kuan-Ling Lai; Min-Guan Lin; Yi-Yu Chen; Huei-Fen Lo; Long-Liu Lin

doi:10.7717/peerj.5863

High-level expression and molecular characterization of a recombinant prolidase from Escherichia coli NovaBlue

PeerJ. 2018 Oct 31:6:e5863. doi: 10.7717/peerj.5863. eCollection 2018.

Authors

Tzu-Fan Wang^#¹, Meng-Chun Chi^#¹, Kuan-Ling Lai^{1

2}, Min-Guan Lin³, Yi-Yu Chen¹, Huei-Fen Lo², Long-Liu Lin¹

Affiliations

¹ Department of Applied Chemistry, National Chiayi University, Chiayi, Taiwan.
² Department of Food Science and Technology, Hungkuang University, Taichung, Taiwan.
³ Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.

^# Contributed equally.

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 from Escherichia coli NovaBlue 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-induced E. coli M15 (pQE-EcPepQ) cells by nickel-chelate chromatography. The molecular mass of EcPepQ 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 for EcPepQ activity were 60 °C, pH 8.0, and 0.1 mM Mn²⁺ ion. Kinetic analysis with Ala-Pro as the substrate showed that the K _m and k _cat values of EcPepQ were 8.8 mM and 926.5 ± 2.0 s^-1, respectively. The thermal unfolding of EcPepQ 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-U value 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 of EcPepQ; 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 of EcPepQ allows to adjust the reaction conditions more suitable for EcPepQ-catalyzed bioprocess.

Keywords: Chaotropic agent-induced denaturation; Escherichia coli; Gene expression; Molecular characterization; Organic co-solvents; Prolidase.

Grants and funding

This work was supported by a college student research scholarship (MOST 106-2815-C-241-037-B) and research grant (MOST 107-2313-B-415-013) from the Ministry of Science and Technology of Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.