Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coli
© Torres et al.; licensee BioMed Central Ltd. 2012
Received: 17 May 2012
Accepted: 3 August 2012
Published: 9 August 2012
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.
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.
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.
KeywordsPenicillin acylase Thermus thermophilus Pre-pro-protein Autoprocessing Thermophile Thermozyme
Penicillin acylase (PAC, EC 18.104.22.168) is one of the most relevant enzymes in the pharmaceutical industry. It is used in the production of 6-amino penicillanic acid (6-APA), which is subsequently used in the chemical synthesis of new lactams with greater effectiveness. PACs belong to the N-terminal nucleophile hydrolase family, whose members undergo a complex maturation process. In this process, the pre-pro-protein is synthesized and translocated to the periplasm with the concomitant removal of the signal peptide. The resulting pro-protein is then autoproteolyzed in the periplasm rendering the β-subunit. A second autoproteolysis detaches the α-subunit and uncovers the active site by elimination of a spacer peptide . The precision of the maturation process is extremely relevant for the functionality of the final heterodimeric protein (α- plus β-subunits).
Industrially, the penicillin G acylase (PGA) from Escherichia coli is the enzyme of choice, whether recombinant or native. Although the optimum temperature for the hydrolysis of penicillin G is 50°C, the enzyme loses stability above 30°C and must be used in immobilized form. Other described PGAs with higher stability are those from Alcaligenes faecalis (AfaePGA, t1/2 of 15 min at 55°C), Bacillus badius (t1/2 of 20 min at 55°C) and Achromobacter xylosoxidans (t1/2 of 55 min at 55°C). Their thermostability arises from different reasons such as additional disulfide bonds, more salt bridges or additional buried ionic pairs, respectively [2, 3]. However, operational stability of the catalytic process could be strongly improved by the use of enzymes with a longer half-life under the reaction conditions regularly used.
Bearing in mind the above-mentioned limitations of penicillin acylases and the naturally superior operational stability of thermozymes, a putative pac gene (NCBI_accession number TTC1972) was identified in the genome of the thermophilic bacterium Thermus thermophilus (Tth) HB27, a microorganism with an optimum growth temperature of 75°C . Enzymes isolated from extreme thermophiles (thermozymes) show an optimum temperature similar to that of maximum growth rate of its source. In addition, thermozymes exhibit an above-average resistance to chemical denaturation, for instance, caused by organic solvents, detergents or pH. Thus, the Tth PAC protein would be an extremely stable catalyst suitable for the industrial production of semi-synthetic penicillins. Generally speaking, fermentation of the natural thermophilic host is not economically viable due to the nutritional and energy requirements of the process. Thus, expression of the protein of interest in a recombinant system is usually preferred. However, recombinant expression of proteins from thermophiles in mesophiles is frequently a challenge. In fact, it is estimated that less than 20% of the ORFs of any extreme thermophile or hyperthermophile genome may be expressed and folded correctly in E. coli. Strategies to improve folding of thermozymes expressed recombinantly in E. coli include growth of the host at higher temperature , refolding of the denatured protein in the presence of cofactors, or co-expression with chaperones from E. coli.
In this work, we report the first characterization of a penicillin acylase from a thermophile. With the objective of a potential industrial application in mind:
we performed differential fractionation of the TthPAC combined with detergent solubility and trypsin accessibility assays and characterized the enzyme as a periplasmic, membrane-bound heterodimer in its native host, which makes TthPAC overproduction in Thermus impractical.
TthPAC was expressed recombinantly in the cytoplasm of E. coli devoid of its signal peptide but nevertheless, the existence of autoproteolytic maturation leading to a functional enzyme was achieved.
we improved productivity and activity of TthPAC in E. coli by coexpression with chaperones and Ca2+ supplementation, respectively.
finally, we characterized the enzyme with different amides and determined the enzyme to be most proficient in the hydrolysis of penicillin K, similarly to the acylases of Streptomyces lividans and Actinoplanes utahensis.
Results and discussion
Tth PAC is produced and processed in T. thermophilus
Subcellular localization of Tth PAC
In mesophilic bacteria, PACs have been reported either as cytosolic or periplasmic proteins [10, 11]. However, the data presented here show that Tth PAC is attached to the external face of the cytoplasmic membrane, thus broadening the possible subcellular locations of the PACs to be studied hereafter. Based on the sGFP fusion experiments, we suggest that Tth PAC is being directed to punctual secretion points of the cytosolic membrane, through the twin-arginine system (TAT), that has been shown to form foci near the cell poles in other bacteria . This is because sGFP folds in the cytoplasm  and the only secretion system that enables the transport of folded proteins is the TAT system. Also, the presence of two arginines in the N-terminal signal peptide of Tth PAC protein supports this idea. In summary, we suggest Tth PAC carries a non-cleavable TAT signal peptide that operates as a signal anchor domain. A similar case has been described for a Rieske Fe-S protein, whose TAT signal peptide is not processed but ultimately forms a transmembrane helix through the lipid bilayer . Whether the membrane location of PACs is a feature that could be extended to other thermophilic bacteria is an issue to be elucidated.
Heterologous expression of Tthpac in E. coli cells
The use of cytoplasmic or periplasmic chaperones in the folding assistance of PACs overproduced in heterologous hosts has been widely reported [22–26]. As for Tth PAC, TF and GroEL/ES proved to be the best choices at improving Eco PGA production . However, in disagreement with the results reported by Xu et al. , we found that Tth PAC activity in the presence of these chaperones was reduced when compared to the absence of folding modulator co-expression. We hypothesize that Tth PAC remains attached to the chaperone after its folding assistance, so a proper Tth PAC catalysis is prevented. Chaperone detaching experiments based on the use of ATP/Mg+2 incubations were carried out, and a Tth PAC release from GroEL/ES complex was observed [Additional file 2.
Effect of Ca+2 on the production and maturation of Tth PAC
Purification of recombinant Tth PAC and evaluation of its maturation
Enzymatic characterization of Tth PAC
Kinetic parameters of Tth PAC for the hydrolysis of different substrates
R (acyl chain)
0.32 ± 0.05
5.16 ± 0.13
Penicillin dihydro F
0.98 ± 0.22
1.46 ± 0.06
0.94 ± 0.18
0.90 ± 0.03
0.74 ± 0.11
0.57 ± 0.01
While the actinomycetal PKAs share 40% identity, Tth PAC has only between 16-18% of identical residues with the former proteins. However when comparing the amino acid sequence of these three proteins with the ones of PACs with aromatic active centers (such as Eco PGA, Afae PGA, P. rettgeri PGA), some replacements that could explain the preference for an aliphatic or aromatic penicillin become evident. Two of the Phe identified as stabilizing residues of the phenylacetyl moiety of penicillin G in Eco PGA (α172 and β57) , are replaced by Ser/Gly and Ile, respectively, in PKAs [Additional file 3.
A PAC enzyme from the extreme thermophile Tth HB27 strain was identified in the outer side of the cytoplasmic membrane. This is the first reported PAC from a extreme thermophile and owing to its potential use as a thermostable biocatalyst, an N-terminal 32-amino acid deletion version of Tth PAC was successfully overexpressed in E. coli cells using chaperone co-expression and calcium supplementation of the culture medium in order to enhance productivity and activity. The recombinant protein was purified and the maturation of the precursor in the cytoplasm of E. coli leading to an active enzyme was confirmed by MALDI-TOF analysis of the individual subunits. In the light of these data, a similar maturation process and mechanistic features as EcoPAC (N-terminal nucleophile Ser, structural calcium, activity-modulating linker peptide) can be hypothesized for this enzyme. Enzymatic characterization of Tth PAC was performed and its preference for aliphatic-chain penicillins was established, in good correlation with the aromatic for aliphatic substitutions found in multiple alignments for well-characterized. Industrial preparations of PenG obtained as bulk materials by fermentation contain up to 3% aliphatic penicillins, which cannot be hydrolyzed by PenG acylases and represent a significant amount in large-scale operations . These “impurities” can now be hydrolyzed by PKAs, such as the one described in this work. Furthermore, penicillin acylases are not limited to penicillin hydrolysis and have a wide substrate specificity finding uses in other biocatalytic reactions, such as amide and ester formation under kinetic control or kinetic resolutions of amides . Tth PAC showed an optimum reaction temperature of 75°C and thermal stability assays confirm that this enzyme clearly surpasses the stability of all other reported (native) acylases up to date, making it an interesting addition to the existing biocatalytic toolbox.
Materials and bacterial strains
All reagents used were of analytical grade and were purchased from Sigma (st. Louis, MO, USA) or Merck (Darmstadt, Germany). Penicillin G, V, K, F and dihydroF were kindly provided by Antibioticos S.A. (León, Spain). Tth Δpac is a knockout in the pac gene (NCBI accession number TTC1972) derived from Tth HB27 strain. Tth ΔslpA is a knockout in the slpA gene derived from Tth NAR1 strain . E. coli DH5α [F– Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK–, mK+) phoA supE44 λ– thi-1 gyrA96 relA1] was used for subcloning steps and E. coli BL21(DE3) [F– ompT hsdSB(rB–, mB–) gal dcm (DE3)] was used as the host for recombinant protein expression.
All the primers used in this work are listed in [Additional file 7: Table S2]. In order to produce antibodies against the α- and β-subunits of PAC, each subunit was amplified from Tth HB27 genome using the primers αNdeIFw and αEcoRv for the α-subunit and βNdeIFw and βEcoRv for the β-subunit. The DNA fragments were cloned into the pET28b + plasmid between the Nde I and EcoR I restriction sites, yielding plasmids pET28αPAC and pET28βPAC. For the expression of the fusions with sGFP , Tthpac leader sequence, the entire Tthpac gene, Tthpac devoid of its leader sequence and Tth β-glycosidase codifying gene (NCBI accession number TTP0042) were amplified from Tth HB27 genome using the pairs of primers lsFw/lsRv, pacFw/pacRv, ΔSppacFw /pacRv, and βglyFw/βglyRv. The gene fusions were cloned into the plasmid pMKPnqosGFP . For the expression of the chimeric protein Sp Eco -PAC Tth , the codifying sequence for the signal peptide of E. coli PGA was amplified with the primers EcoFw and EcoRv. The Tth pac gene devoid of its leader sequence was amplified from Tth HB27 genome with the primers ΔSppacFw and ΔSppacRv. The gene fusion was assembled by overlap extension PCR and ligated to the pMKPnqo expression plasmid. For the homologous expression of Tth PAC, the pac gene was amplified from Tth HB27 genome with the primers pac2Fw and pac2Rv. The gene was ligated to the pWUR112/77-1  expression plasmid and the construction was named pWURPAC. For the heterologous expression of Tthpac, the ΔSppac2Fw primer and the ΔSppac2Rv primer were used in a PCR reaction for the amplification of a 2223 bp fragment corresponding to the Tthpac gene without the 5´ 96-nucleotide codifying sequence. The amplified gene was cloned into the pET28a plasmid between the Nde I and EcoR I restriction sites, yielding plasmid pET28a-ΔSpPAC. The construction was then transformed into E. coli DH5α competent cells. Plasmids were extracted and their inserts were sequenced to confirm the absence of mutations in Tthpac gene.
Fluorescence microscopy was performed using a Zeiss Inverted LSM510 confocal microscope. Z-stacks were obtained using a Zeiss 100x/1.3 oil Plan-Neofluar objective lens and parameters appropriate to comply with the Nyquist criteria for image sampling. Images were subjected to linear deconvolution using the Huygens System 2.2 software (Scientific Volume Imaging B.V., Hilversum. the Netherlands). Adobe Photoshop and Image J (Wayne Rasband, NIH, USA) were used for final assembly of the images. For sGFP fusion experiments, Tth HB27 strain was transformed with each one of the sGFP fusions (β-glycosidase-sGFP, ΔSpTth PAC-sGFP, Tth PAC-sGFP and SpTthPAC-sGFP) and was grown aerobically in TB liquid media (Ramirez-Arcos et al., 1998) at 70°C with mild shaking (150 rpm) up to an OD550 of 0.2-0.4 before cell harvesting by low-speed centrifugation (3,000 x g, 5 min). Mowiol was added after washing the cells with Milli-Q water.
Isolation of the membrane fraction of Tth cells
Bacteria in mid-log growth phase were harvested by centrifugation at 5000 x g for 5 min at room temperature. The pellet was washed in TE 1X (Tris 10 mM pH 8, EDTA 1 mM) buffer and resuspended in TE. Cells were disrupted in a LABSONIC U sonicator (B. Braun) (2 times 30 seconds – 1 minute). Intact cells were removed by centrifugation at 5000 x g for 5 min. The supernatant was ultracentrifuged 30 min at 201,240 x g at 4°C in a Beckman TL-100. The pellet was washed in 10 mM Tris–HCl pH 7.5 and was centrifuged again in the same conditions. The final pellet corresponded to the membrane fraction.
Selective solubilization of membrane components
Selective solubilization of the cytoplasmic membrane by non-ionic or weakly-ionic detergents is a widely used method to separate the inner and outer membrane components in Gram negative bacteria [14–17]. The membrane fraction was resuspended in 10 mM Tris–HCl pH 7.5 and 1% Triton X-100 or 1% Sarkosyl and incubated for 30 min at 37°C. Samples were then ultracentrifuged 30 min at 201,240 x g and 4°C. Supernatant was stored and the pellet was washed once again in the same buffer and centrifuged in the same conditions. Both fractions were subjected to SDS-PAGE and immunodetection of Tth PAC subunits.
Tth NAR1 ΔslpA strain was grown at 70°C under aerobic conditions up to saturation. The bacteria were harvested by centrifugation at 5000 x g for 5 min at room temperature, washed once with 10 mM Tris–HCl pH 8.0, and the resulting pellet was resuspended in the same buffer up to a final cell concentration of 1010 cells/ml. Trypsin treatment was carried out using two different concentrations of enzyme at 37°C in the presence or absence of 5 or 10 mM EDTA. Tth PAC integrity was analyzed by western blot.
The Tth HB27 Δpac mutant strain was transformed with the plasmid pWUR112/77-1  carring the pac gene (pWURPAC) and was plated onto TB (Ramírez-Arcos et al., 1998) agar with bleomycin . The constitutive expression of PAC was carried out growing the clones aerobically in TB liquid media (Ramírez-Arcos et al., 1998) with bleomycin at 70°C and 150 rpm until saturation. Cells were harvested by centrifugation at 4,000 × g and 4°C for 15 min. Cell pellets were resuspended in 50 mM Tris buffer pH 7.5 and 50 mM NaCl and disrupted by sonication on ice bath (2 rounds of 45 secs), using a sonicator LABSONIC U (B. Braun).
Heterologous expression and purification of PAC
E. coli BL21(DE3) strain was transformed with the plasmid pET28a carring the ΔSp-Tthpac gene (pET28a-ΔspPAC) and was plated on LB agar with kanamycin (30 μg mL-1). The strain E. coli BL21(DE3) pET28a-ΔspPAC was then transformed with the vectors pGTf2, pTf16, pGro7 or pKJE7 (Takara Bio Inc), carrying the genes for GroEL-GroES, Trigger factor, GrpE and DnaK-DnaJ chaperone systems. Transformants were plated onto LB agar containing kanamycin (30 μg mL-1) and chloramphenicol (30 μg mL-1). Both transformations were based on the calcium temperature shock method.
The inoculums for batch protein production were prepared by overnight cultivation of the selected clone in 100 mL shake flaks with 20 ml of LB medium at 37°C. For protein production 1.5 mL of the corresponding inoculum culture was transferred to 150 mL of fresh LB medium supplemented with 10 mM CaCl2, and containing 0.5 g.L-1 of L-arabinose for chaperone induction. Cells were cultivated at 37°C and 200 rpm until they reached the Tth PAC over-expression induction point, corresponding to an OD600 ≈ 0.6 ± 0.5. Induction was performed by addition of IPTG (isopropyl-β-D thiogalactopyranoside) to a final concentration of 1 mM, and cultivation was continued for 17 hours at 22°C and 200 rpm. Cells were harvested by centrifugation (4,000 × g for 15 min) at 4°C. Cell pellets were resuspended in 10 mL of lysis buffer (50 mM sodium phosphate pH 7.0, 300 mM NaCl) and disrupted by sonication on ice bath (3 rounds of 10 min, 0.6 pulse and 40% power), using a sonicator LABSONIC U (B. Braun). Soluble and insoluble protein fractions were separated by centrifugation at 12,000 × g for 15 min at 4°C. The supernatant containing the His-tagged protein was partially purified from thermolabile E. coli proteins by a heat-shock treatment of 20 min at 65°C. Thermostable proteins were recovered from the supernatant after centrifugation (12,000 × g for 30 min) at 4°C, and subjected to IMAC chromatography using a Talon Cell-through resin (BD Biosciences) previously equilibrated with lysis buffer. The column was washed once with 1 volume of the lysis buffer. The His-tagged protein was eluted with 150 mM imidazole, diafiltrated and concentrated with Amicon Ultra-15 10 kDa centrifugal devices ((Millipore) using 50 mM sodium phosphate pH 7.0, 5 mM NaCl, 0.5 mM CaCl2, and stored at −20°C until use. The use of stabilizing additives like polyols was avoided because we observed interference with components of the reaction mixture. Protein content was determined using the Bio-Rad protein assay dye reagent concentrate (Bio-Rad, USA) with bovine serum albumin as standard. Samples for SDS-PAGE separation were prepared in 5× SDS-PAGE loading buffer and heated for 5 min at 95°C. Electrophoresis of protein samples was done with 12% (w/v) SDS-PAGE and the gel was stained with Coomassie Brilliant Blue R-250. Identical samples were electroblotted on PVDF membranes and incubated with polyclonal rabbit antibodies against the α- and β-subunit of Tth PAC, and later with an alkaline phosphatase-conjugated goat anti-rabbit antibody.
Metal affinity purified His6::ΔSpTth PAC was subjected to SDS-PAGE on 12% polyacrylamide gels and the proteins were stained with Coomassie Brillant Blue R-250 solution. The polypeptide bands corresponding to α- and β-subunits of Tth PAC protein were excised from the gel and subjected to MALDI-TOF mass spectrometer analysis. The peptide identification by LC/MS/MS analysis was carried out in the ‘CBMSO Protein Chemistry Facility’, a member of ProteoRed network.
Determination of Tth PAC enzymatic activity and thermal stability
Fluorescamine was used to follow the kinetics of Tth PAC against penicillins, cephalosporins or homoserine-lactones, through the reaction with the primary amine group of the corresponding reaction products. Aliquots of 40 microliter of reaction mixture were taken at regular intervals and were immediately frozen in dry ice after which 140 μl of 200 mM acetate buffer pH 4.5 and 20 μl of 1 mg.mL-1 fluorescamine in acetone were added. After a 60-min incubation at room temperature fluorescence was determined (Exc. 380 nm - Em. 530 nm) in a FLUOStar OPTIMA plate reader (BMG LabTech). All experiments were performed in duplicate, and the effect of non-enzymatic hydrolysis of substrates was subtracted. Substrate selectivity was analyzed using penicillin K, F, DHF, V or G, glutaryl-7 amino cephalosporanic acid (glutaryl-7ACA), cephalosporine C, and butyryl- or octanoyl-homoserine lactones (C4- or C8-HSL, respectively). Kinetic parameters Vmax and Km were determined by measuring the initial rate of hydrolysis on a range of substrate concentrations from 0.1 to 50 mM, using 0.8-2.0 μg of purified enzyme in 20 mM MES pH 5.5 for up to 1 h at 65°C. Optimum pH for the hydrolysis of 2.5 mM penicillin K was assayed at 65°C in 20 mM Britton-Robinson buffer at pH 3, 4, 5, 6, 7, 8 or 9 . The temperature giving maximum penicillin K hydrolysis was determined by incubating 0.2 μg of enzyme with 0.5 mM of penicillin K in 20 mM MES pH 5.5, in a reaction volume of 50 μl at various temperatures within the range of 57.5°C to 82.5°C. The reaction was carried out in a 96-well PCR plate on a Thermal Cycler (Bio-Rad) running in gradient mode. After the reaction, the tubes were immediately chilled on ice. Thermal stability was determined incubating 0.08 mg/ml solutions of purified Tth PAC in 50 mM phosphate buffer pH 7.5 at 65 and 75°C and measuring the residual hydrolytic activity at regular intervals as detailed above using 5 mM penicillin K as substrate.
- Tth :
penicillin G acylase
penicillin K acylase
- t 1/2 :
open reading frame
superfolder green fluorescent protein
nitrate respiration system regulatory protein
twin arginine transporter
L.T., A.H. and J.B. acknowledge funding from the Spanish Ministry of Science (grants CIT 010000-2009-29, RyC2006-02441) and an institutional grant from Fundación Ramón Areces to CBMSO. A.H. and J.B. are thankful to M.J. de Soto and E. Sánchez for their assistance. We also acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).
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