Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coli

Abstract

Background: Penicillin acylases (PACs) are enzymes of industrial relevance in the manufacture of β-lactam antibiotics. Development of a PAC with a longer half-life under the reaction conditions used is essential for the improvement of the operational stability of the process. A gene encoding a homologue to Escherichia coli PAC was found in the genome of the thermophilic bacterium Thermus thermophilus (Tth) HB27. Because of the nature of this PAC and its complex maturation that is crucial to reach its functional heterodimeric final conformation, the overexpression of this enzyme in a heterologous mesophilic host was a challenge. Here we describe the purification and characterization of the PAC protein from Tth HB27 overexpressed in Escherichia coli.

Results: Fusions to a superfolder green fluorescent protein and differential membrane solubilization assays indicated that the native enzyme remains attached through its amino-terminal end to the outer side of the cytoplasmic membrane of Tth cells. In order to overexpress this PAC in E. coli cells, a variant of the protein devoid of its membrane anchoring segment was constructed. The effect of the co-expression of chaperones and calcium supplementation of the culture medium was investigated. The total production of PAC was enhanced by the presence of DnaK/J and GrpE and even more by trigger factor and GroEL/ES. In addition, 10 mM calcium markedly improved both PAC specific and volumetric activities. Recombinant PAC was affinity-purified and proper maturation of the protein was confirmed by SDS-PAGE and MALDI-TOF analysis of the subunits. The recombinant protein was tested for activity towards several penicillins, cephalosporins and homoserine lactones. Hydrophobic acyl-chain penicillins were preferred over the rest of the substrates. Penicillin K (octanoyl penicillin) was the best substrate, with the highest specificity constant value (16.12 mM-1.seg-1). The optimum pH was aprox. 4 and the optimum temperature was 75 °C. The half-life of the enzyme at this temperature was 9.2 h.

Conclusions: This is the first report concerning the heterologous expression of a pac gene from a thermophilic microorganism in the mesophilic host E. coli. The recombinant protein was identified as a penicillin K-deacylating thermozyme.

Figure 1

Presence of PAC protein in Tth cells. Total proteins from Tth cells of HB27 and NAR1 strains were separated by SDS-PAGE and the presence of α- and β-PAC subunits was determined individually by western blot assays.

Figure 2

Tth PAC subcellular location. Fluorescence confocal microscopy images were taken from exponential cultures of Tth HB27 Δpac mutant strain harboring β-glycosidase-sGFP (A), ΔSpTthPAC-sGFP (B), TthPAC-sGFP (C) or Sp_TthPAC-sGFP (D) protein fusions.

Figure 3

Immunodetection of Tth PAC in the membrane fraction. The presence of α- and β-PAC subunits and of the periplasmic protein DrpA in the membrane fraction (MB), the soluble fraction (SB) and the periplasmic fraction (PE) of Tth NAR1 ΔslpA multicellular bodies was analyzed by western blot assays.

Figure 4

Tth inner membrane solubilization and immunodetection of Tth PAC. PAC was immunodetected in Tth HB27 isolated membranes after a 30-min treatment with Sarkosyl at 37°C. T, total membranes; P, insoluble membrane fraction after detergent treatment; SB, fraction of soluble proteins after detergent treatment. SlpA, outer membrane protein marker; Nqo6, inner membrane protein marker.

Figure 5

Trypsin accessibility assays. Tth PAC α- and β-subunits were immunodetected in Tth NAR1 Δ slpA strain before trypsin treatment (C), and after a digestion with 1.2 mg/ml of trypsin (1) or with 5.5 mg/ml of trypsin (2). EDTA at 5 mM was used to get a partial solubilization of the outer membrane. Nqo1 was used as a cytoplasmic marker.

Figure 6

Subcellular location of the quimeric protein Sp _Eco -PAC _Tth in Tth cells. The overexpression was performed in the Δ pac Tth mutant strain. Immunodetection of α- and β-PAC subunits was performed on the soluble protein fraction (SB) or in the membrane fraction (MB). Tth PAC matured in Tth cells was used as control (C).

Figure 7

Tth PAC overexpression in Tth Δ pac cells.TthPAC β-subunit was immunodetected in the Tth HB27 wild type strain (C+), in the Tth Δpac mutant strain transformed with pWURPAC (PAC) and in the Tth Δpac mutant (C-).

Figure 8

Effect of chaperone co-expression on Tth PAC overproduction in E. coli cells. (A) Western blot against α- and β-TthPAC subunits. Lane 1 (C), TthPAC matured in Tth; lane 2, TthPAC overexpression without chaperone co-expression; lane 3 to 6, co-expression of TthPAC with GroEL/ES-trigger factor (TF), TF alone, GroEL/ES, and DnaK/J-GrpE, respectively. (B) Optical density at 600_nm (dots), specific (white bars) and volumetric (grey bars) activity of TthPAC in the absence (none) or presence of chaperones (GroEL/ES-TF, TF alone, GroEL/ES or DnaK/J-GrpE).

Figure 9

Effect of calcium on Tth PAC overproduction in E. coli cells. CaCl₂ concentrations up to 50 mM were added to the LB media at the beginning of E. coli cells cultivation. TthPAC maturation and activity were evaluated. (A) Western blot against α- and β-TthPAC subunits. Lane 1–5, increasing calcium concentrations; lane 6 (C), TthPAC matured in Tth cells. (B) Optical density at 600_nm (circles), specific (squares) and volumetric (triangles) activity of TthPAC.

Figure 10

HIS₆::TthPAC purification. Fractions of total soluble protein (lane 1), protein flow through (lane 2), 5 mM imidazol protein elution (lane 3), and 150 mM imidazole protein elution were subjected to SDS-PAGE and stained with Coomasie Brillant Blue.

Figure 11

Optimum reaction temperature of HIS₆::TthPAC. The TthPAC enzymatic activity was assayed in 20 mM MES pH 5.5, in the presence of 0.5 mM PenK and in a temperature range from 57.5 to 82.5°C.