Dataset Information

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

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-?-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.

SUBMITTER: Wang TF

PROVIDER: S-EPMC6215446 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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

Wang Tzu-Fan TF Chi Meng-Chun MC Lai Kuan-Ling KL Lin Min-Guan MG Chen Yi-Yu YY Lo Huei-Fen HF Lin Long-Liu LL

PeerJ 20181031

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 wa ...[more]

PMID: 30402354

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Project description:Pullulanase plays an important role in specific hydrolysis of branch points in amylopectin and is generally employed as an important enzyme in starch-processing industry. So far, however, the production level of pullulanase is still somewhat low from wide-type strains and even heterologous expression systems. Here the gene encoding Bacillus naganoensis pullulanase was amplified and cloned. For expression of the protein, two recombinant systems, Escherichia coli BL21(DE3)/pET-20b(+)-pul and E. coli BL21(DE3)/pET-22b(+)-pul, were constructed, both bearing T7 promoter and signal peptide sequence, but different in the existance of lac operator and lacI gene encoding lac repressor. Recombinant pullulanase was initially expressed with the activity of up to 14 U/mL by E. coli BL21(DE3)/pET-20b(+)-pul with IPTG induction in LB medium, but its expression level reduced continually with the extension of cryopreservation time and basal expression was observed. However, E. coli BL21(DE3)/pET-22b(+)-pul , involving lac operator downstream of T7 promoter to regulate foreign gene transcription, exhibited pullulanase activity consistently without detected basal expression. By investigating the effect of lac operator, basal expression of foreign protein was found to cause expression instability and negative effect on production of target protein. Thus double-repression strategy was proposed that lac operators in both chromosome and plasmid were bound with lac repressor to repress T7 RNA polymerase synthesis and target protein expression before induction. Consequently, the total activity of pullulanase was remarkably increased to 580 U/mL with auto-induction by lac operator-involved E. coli BL21(DE3)/pET-22b(+)-pul. When adding 0.6% glycine in culture, the extracellular production of pullulanase was significantly improved with the extracellular activity of 502 U/mL, which is a relatively higher level achieved to date for extracellular production of pullulanase. The successful expression of pullulanase with lac operator regulation provides an efficient way for enhancement of expression stability and hence high-level production of target protein in recombinant E. coli.

Dataset Information

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

Publications

High-level expression and molecular characterization of a recombinant prolidase from <i>Escherichia coli</i> NovaBlue.

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