Biochemical and molecular characterization of Pseudomonas aeruginosa CTM50182 organic solvent-stable elastase

International Journal of Biological Macromolecules 60 (2013) 165–177

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Biochemical and molecular characterization of Pseudomonas aeruginosa CTM50182 organic solvent-stable elastase Bassem Jaouadi a,∗ , Nadia Zaraî Jaouadi a , Hatem Rekik a , Belgacem Naili a , Abdelhamid Beji b , Abdelhafidh Dhouib c , Samir Bejar a a Laboratory of Microorganisms and Biomolecules (LMB), Centre of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, P.O. Box 1177, Sfax 3018, Tunisia b Max-Planck-Institute of Biochemistry (MPIB), Department of Molecular Biology (DMB), Martinsried by Munich, Am Klopferspitz 18, D-82152, Germany c Environmental Bioprocesses Laboratory (EBL), AUF Regional Excellence Pole, Centre of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, P.O. Box 1177, Sfax 3018, Tunisia

a r t i c l e

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Article history: Received 11 January 2013 Received in revised form 20 May 2013 Accepted 23 May 2013 Available online 30 May 2013 Keywords: Pseudomonas aeruginosa AMPP Protease Elastase Thermostability Organic solvents

a b s t r a c t An extracellular alkaline elastase was produced from Pseudomonas aeruginosa CTM50182. It was chromatographically purified using HPLC and Mono Q Sepharose column. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme (called AMPP) was a monomer with a molecular mass of 33,015.18 Da. The N-terminal 29 amino acid sequence of AMPP showed high homology with those of Pseudomonas elastases. It showed optimal activity at pH 12 and 80 ◦ C and was stable at a pH range of 9–12 after 120 h of incubation. Its thermoactivity and thermostability were upgraded in the presence of 5 mM Co2+ . Its half-life times at 70 and 80 ◦ C were 16 and 10 h, respectively. It was completely inhibited by ethylene glycol-bis (␤-aminoethyl ether)-N,N,N ,N tetraacetic acid (EGTA), and 1,10-phenanthroline, suggesting that it belongs to the metalloprotease family. AMPP also exhibited high catalytic efficiency, organic solvent-tolerance, and hydrolysis. The lasB gene encoding AMPP was cloned, sequenced, and expressed in Escherichia coli. The biochemical properties of the extracellular purified recombinant enzyme (rAMPP) were similar to those of native AMPP. This organic solvent-stable protease could be considered a potential candidate for application as a biocatalyst in the synthesis of enzymatic peptides. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Proteases are a highly important class of enzymes that are found in all cellular forms of life. They represent one of the earliest known families of enzymes that have long been used to catalyze the hydrolysis of peptide bonds in aqueous solutions and the synthesis of peptides in the presence of organic solvents. They have been extensively characterized from a variety of sources and occupy a pivotal position in terms of industrial application. Their large scale application has, however, often been curtailed by their limited abilities to withstand alkalinity and to maintain stability in solvent media. Most enzymes, including proteases, are labile catalysts that lose their catalytic activity easily in the presence of organic solvents [1–3]. The search for solvent-stable proteases has, therefore, been an important area of research.

∗ Corresponding author. Tel.: +216 99 535253; fax: +216 74 870451. E-mail addresses: [email protected], [email protected] (B. Jaouadi). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.05.019

In this respect, Pseudomonas aeruginosa has been reported to produce at least four different types of endopeptidases. The predominant proteases secreted by this Gram-negative bacterium are (i) pseudolysin (elastase or LasB), a Zn-metalloendopeptidase with a molecular mass of 33 kDa [4]; (ii) staphylolysin (LasA), a mature Zn-metalloendopeptidase with a molecular mass of 20 kDa [5]; (iii) aeruginolysin (P. aeruginosa alkaline protease), a protease with a molecular mass of 49.5 kDa [6]; and (iv) protease Ps-1, a lysine-specific endopeptidase with a molecular mass of 30 kDa [7]. Several elastases were also described, and their enzymatic properties were extensively investigated in the literature [4,8,9]. Moreover, many genes encoding Zn-metalloendopeptidases were identified and characterized, including those of P. aeruginosa PAO1 [10], P. aeruginosa IFO 3455 [11], P. aeruginosa PST-01 [12], P. aeruginosa MN7 [13], P. aeruginosa K [14], P. aeruginosa MCM B327 [15], and P. aeruginosa A2 [16]. P. aeruginosa elastase has also been crystallized, and its 3D structure was determined [17,18]. The isolation and screening of protease-producing strains from naturally occurring habitats or alkaline wastewater is expected to provide new valuable strains for the production of organic solventstable proteases in highly alkaline conditions. This paper reports

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on the purification and biochemical characterization of an elastase (AMPP) from P. aeruginosa CTM50182 isolated from NPK chemically contaminated soil samples. The nucleotide and amino acid sequences of the corresponding gene (lasB) were also determined. 2. Materials and methods 2.1. Substrates and chemicals Unless specified otherwise, all substrates, chemicals, and reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and were of the analytical or highest grade available. 2.2. Isolation and cultivation of protease-producing microorganisms Soil samples were collected from different Tunisian sites to isolate protease-producing microorganisms. They were then dispersed in sterile distilled water. Alternatively, protease activity was screened in strains available in the bacterial collection of the Centre of Biotechnology of Sfax (CBS). The treated samples and strains were plated onto skimmed milk agar plates containing (g/l): peptone, 5; yeast extract, 3; skimmed milk 250 ml; and bacteriological agar, 20 at pH 9. The plates were then incubated for 12 h at 37 ◦ C to obtain colonial growth. The colonies with a clear zone formed by the hydrolysis of milk casein were evaluated as protease producers. Several proteolytic strains were isolated. The CTM50182 strain, available from the CBS bacterial collection and previously isolated from contaminated soil samples at the NPK (nitrogen-phosphatepotash) Chemical Company of Sfax, Tunisia showed a large clear zone of hydrolysis, and was, therefore, selected for further experimental analysis. The growth medium used for protease production by strain CTM50182 at pH 9 consisted of (g/l): casein, 15; soy peptone, 5; (NH4 )2 SO4 , 2; MgSO4 ·7H2 O, 1; CaCl2 , 1; K2 HPO4 , 0.5; KH2 PO4 , 0.5; NaCl, 1; and trace elements 2% (v/v) [composed of (g/l): ZnCl2 , 0.4; FeSO4 ·7H2 O, 2; H3 BO3 , 0.065; and MoNa2 O4 ·2H2 O, 0.135]. The Media were autoclaved for 20 min at 121 ◦ C. Growth experiments were performed in 1000-ml conical flasks, with a working volume of 100 ml. The flasks were incubated at 37 ◦ C on a rotary shaker (250 rpm) for 48 h. Growth kinetics were monitored by measuring absorbance at 600 nm. The cell-free supernatant was recovered by centrifugation (14,000 × g, 40 min) at 4 ◦ C, and employed as a protease preparation in subsequent assays. All experiments were conducted in triplicates and good reproducibility was obtained. 2.3. Identification of microorganism, DNA sequencing and phylogenetic analysis Analytical profiling index (API) strip tests and 16S rRNA gene sequence analysis (ribotyping) were carried out to identify the genus to which the CTM50182 strain belonged. API 20 NE strips (bioMérieux, SA, Marcy-l’Etoile, France) were used to investigate the physiological and biochemical characteristics of the strain in accordance with the manufacturer’s instructions. The 16S rRNA gene was amplified by polymerase chain reaction (PCR) using forward primer, 5 -AGAGTTTGATCCTGGCTCAG-3 , and reverse primer, 5 -AAGGAGGTGATCCAAGCC-3 [19]. The genomic DNA of the strain was purified, using the Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA), and then used as a template for PCR amplification (35 cycles, 94 ◦ C for 30 s denaturation, 60 ◦ C for 60 s primer annealing, and 72 ◦ C for 90 s extension). The amplified ∼1.5 kb PCR product was cloned in the pGEM-T Easy vector (Promega, Madison, WI, USA), leading to the pBJPa16S plasmid (this study). The E. coli DH5␣ [F− supE44 ˚80 ılacZ

M15 (lacZYA-argF) U169 endA1 recA1 hsdR17 (rk − , mk + ) deoR thi-1 − gyrA96 relA1] (Invitrogen, Carlsbad, CA, USA) was used as a host strain. All recombinant clones of E. coli were grown in LB broth media with the addition of Ampicillin, isopropylthio-␤-d-galactopyranoside (IPTG), and X-gal for screening. DNA electrophoresis, DNA purification, restriction, ligation, and transformation were all performed according to the method previously described by Sambrook et al. [20]. The nucleotide sequences of both strands of the cloned 16S rRNA gene sequence were determined using the BigDye® Terminator v3.1 Cycle Sequencing Kit and the automated DNA sequencer ABI Prism® 3100-Avant Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Sequence comparisons were performed using the BLAST program (NCBI, USA). Phylogenetic and molecular evolutionary analyses were conducted via the molecular evolutionary genetic analysis (MEGA) software version 4.1. Distances and clustering were calculated using the neighborjoining method. Bootstrap analysis was used to evaluate the tree topology of the neighbor-joining data by performing 100 re-samplings. 2.4. Enzymatic activity assays Caseinolytic activity was measured using the Folin-Ciocalteu method and as previously described elsewhere [21] using Hammersten casein (Merck, Darmstadt, Germany) as a substrate. One casein unit (CU) was defined as the amount of enzyme that hydrolyzed the substrate and that produced 1 ␮g of amino acid equivalent to tyrosine per min at 80 ◦ C and pH 12 in 50 mM Na2 HPO4 –NaOH buffer supplemented with 5 mM CoCl2 at pH 12 (buffer A). The same protocol was used to measure enzyme activity on natural proteins (hemoglobin, myoglobin, keratin, gelatin, albumin, fibrin, and wheat gluten). Keratinolytic activity was determined using a modified version of a protocol previously described elsewhere [22] with keratin azure as insoluble substrate [23]. One keratin unit (KU) was defined as the amount of enzyme causing an increase of 0.1 in absorbance at 440 nm in 1 min under the experimental conditions described. The same protocol was used to determine enzyme activity on azoalbumin and azo-casein. Elastolytic activity was determined according to the method of Riffel and Brandelli [24] using elastin as a substrate. Unless otherwise stated, 20 mg of elastin–orcein were suspended in 2.9 ml of 50 mM Tris–HCl buffer (pH 8.5) containing 5 mM CoCl2 . After 5 min of pre-incubation, a suitably diluted enzyme was added to the solution, and the reaction mixture was shaken at 160 strokes per min at 30 ◦ C. The reaction was terminated after 4 h of incubation by the addition of 2 ml of 500 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 6). The reaction mixture was then filtered using Whatman Grade No. 5 Filter Paper, and the absorbance of the filtrate was detected at 590 nm. The complete hydrolysis of elastin–orcein brought a 0.27 increase of absorbance. One elastin unit (EU) was defined as the amount of enzyme that produced a 0.0135 increase in absorbance per hour under the experimental conditions used. Activities against N-hippuryl-l-lysine (Hip-Lys) and Nhippuryl-l-phenylalanine (Hip-Phe) were determined according to the method recommended by Biochemica Merck (Darmstadt, Germany). Unless otherwise stated, the substrate was dissolved in 25 mM Tris–HCl buffer (pH 8) containing 5 mM CoCl2 and 500 mM NaCl. After the addition of the suitably diluted enzyme solution, absorbance was monitored at 254 nm during the first 10 min and 1 h of reaction. One hippuryl unit (HU) was defined as the amount of enzyme that split 1 ␮mol of substrate per min. Enzyme activities represent the means of at least three determinations carried out in triplicates.

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2.5. AMPP purification procedure Five hundred milliliters of a 30-h culture of P. aeruginosa CTM50182 was centrifuged for 20 min at 8000 × g to remove microbial cells. The supernatant containing extracellular protease was used as the crude enzyme preparation and was submitted to the following purification steps. The supernatant was incubated for 1 h at 60 ◦ C, and insoluble material was removed by centrifugation at 12,000 × g for 30 min. The clear supernatant was precipitated between 50 and 70% ammonium sulfate saturation. The precipitate was then recovered by centrifugation at 12,000 × g for 30 min, resuspended in a minimal volume of buffer A containing 25 mM NaCl (buffer B), and dialyzed overnight against repeated changes of buffer B. Insoluble material was removed by centrifugation at 12,000 × g for 30 min. The supernatant was loaded and applied to a high performance liquid chromatography (HPLC) system using a Bio-Sil SEC 125-5 column (7.8 mm × 300 mm) that was pre-equilibrated with 50 mM HEPES buffer supplemented with 5 mM CoCl2 and 10 mM NaCl at pH 7.5 (buffer C). Proteins were separated by isocratic elution at a flow rate of 30 ml/h with buffer C and detected using a UV-VIS Spectrophotometric detector (Knauer, Berlin, Germany) at 280 nm. The fractions containing protease activity (eluted at a void volume of 1.7, with retention time of 21 min) were pooled and then applied to a Mono Q Sepharose column (Pharmacia, Uppsala, Sweden) equilibrated with buffer C. The column was rinsed with 500 ml of the same buffer. Adsorbed material was eluted with a linear NaCl gradient (0–500 mM) in buffer C at a rate of 35 ml/h. The column (2.6 cm × 50 cm) was extensively washed with buffer C until the optical density of the effluent at 280 nm was zero. Fractions of 5 ml each were collected at a flow rate of 40 ml/h and analyzed for caseinolytic activity and protein concentration. Protease activity was eluted between 125 and 225 mM NaCl. Pooled fractions containing protease activity were concentrated in centrifugal micro-concentrators (Amicon Inc., Beverly, MA, USA) with 10-kDa cut-off membranes and were stored at −20 ◦ C in a 20% glycerol (v/v) solution for further analysis. 2.6. Protein quantification, electrophoresis, and mass spectrometry Protein concentration was determined by the method of Bradford [25] using a Dc protein assay kit purchased from Bio-Rad Laboratories (Hercules, CA, USA), with bovine serum albumin (BSA) as a standard. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 10% (w/v) acrylamide in gels as described by Laemmli [26]. Protein bands were visualized with Coomassie Brilliant Blue R-250 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) staining. Casein zymography staining was performed as previously described by Jaouadi et al. [27]. The molecular mass of the purified AMPP was analyzed in linear mode by MALDI-TOF/MS using a Voyager DE-RP instrument (Applied Biosystems/PerSeptive Biosystems, Inc., Framingham, MA, USA). Data was collected with a Tektronix TDS 520 numeric oscillograph and analyzed using the GRAMS/386 software (Galactic Industries Corporation, Salem, NH, USA). 2.7. N-terminal amino acid sequencing Bands of purified AMPP on SDS gels were transferred to a ProBlott membrane (Applied Biosystems, Foster City, CA, USA), and N-terminal sequence analysis was performed by automated Edman’s degradation using an Applied Biosystem Model 473A gasphase sequencer. The sequence was compared to those in the

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Swiss-Prot/TrEMBL database using the BLAST homology search program. 2.8. Effects of pH on the activity and stability of AMPP The activity of AMPP was measured at a pH range of 2–13 at 70 ◦ C using casein. Its pH stability was determined by preincubation in buffer solutions at 50 ◦ C and different pH values for 240 h. Aliquots were withdrawn, and residual enzymatic activity was determined at pH 12 and 80 ◦ C. The following buffer systems, supplemented with 5 mM CoCl2 , were used at 50 mM: glycine–HCl for pH 2–5, 2-(N-morpholino) ethanesulfonic acid (MES) for pH 5–6; HEPES for pH 6–8, Tris–HCl for pH 8–9, glycine–NaOH for pH 9–11, bicarbonate–NaOH for pH 11–11.5, Na2 HPO4 –NaOH for pH 11.5–12, and KCl–NaOH for pH 12–13. 2.9. Optimum temperature and thermal stability of AMPP The effect of temperature on AMPP activity was examined at 40–100 ◦ C and pH 12 for 20 min. Thermal stability was determined by incubation at 70–100 ◦ C and pH 12 for 24 h in the presence and absence of 5 mM CoCl2 . Aliquots were withdrawn at specific time intervals to test the remaining activity under standard conditions. The non-heated enzyme, which was cooled on ice, was considered as a control (100%). 2.10. Effects of inhibitors and metal ions on AMPP stability The effects of phenylmethanesulfonyl fluoride (PMSF), diiodopropyl fluorophosphates (DFP), soybean trypsin inhibitor (SBTI), benzamidine hydrochloride hydrate, N␣-p-tosyl lphenylalanine chloromethyl ketone (TPCK), N␣-p-tosyl l-lysine chloromethyl ketone (TLCK), 5,5-dithio-bis-(2-nitro benzoic acid) (DTNB), monoiodoacetic acid (MIA), ld-dithiothreitol (DTT), 2mercaptoethanol (2-ME), N-ethylmalemide (NEM), leupeptin, pepstatin A, 1,2-epoxy-3-(p-nitrophenoloxy) propane (EPNP), aprotinin bovine, 1,10-phenanthroline monohydrate, phosphoramidon disodium salt, glutathione, ethylene-diaminetetraacetic acid (EDTA), EGTA, and various monovalent and divalent metal ions (5 mM) on protease stability were investigated by pre-incubating the purified AMPP enzyme for 30 min at 25 ± 2 ◦ C with each inhibitor, and for 1 h at 50 ◦ C in the presence of metal ions. Enzyme assays were carried out under standard assay conditions. 2.11. Substrate specificity of AMPP The substrate specificity of AMPP was determined using natural (elastin–orcein hemoglobin, myoglobin, casein, gelatin, fibrin, keratin, albumin, and gluten) and modified (azo-casein, azo-albumin, and keratin azure, collagen type I: FITC conjugate, and collagen type II: FITC conjugate) protein substrates as well as ester [N-benzol-l-arginine ethyl ester (BAEE), N-benzol-l-arginine ethyl ester (BTEE), S-benzyl-l-cysteine ethyl ester hydrochloride (BCEE), and N-acetyl-l-tyrosine ethyl ester monohydrate (ATEE)] and synthetic peptide [N-succinyl-l-Tyr-l-Leu-l-Val-p-nitroanilide, N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide, Nsuccinyl-l-Ala-l-Ala-l-Pro-l-Leu-p-nitroanilide, glutaryl-l-Ala-lAla-l-Pro-l-Leu-p-nitroanilide, N-succinyl-l-Ala-l-Ala-l-Pro-lMet-p-nitroanilide, N-succinyl-l-Ala-l-Ala-l-Val-l-Ala-p-nitroanilide, N-methoxysuccinyl-l-Ala-l-Ala-l-Pro-l-Val-p-nitroanilide, N-succinyl-l-Ala-l-Ala-l-Ala-p-nitroanilide; N-succinyll-Ala-l-Ala-l-Val-p-nitroanilide, N-succinyl-l-Ala-l-Ala-l-Phe-pnitroanilide, N-succinyl-l-Ala-l-Pro-l-Ala-p-nitroanilide, l-Leu-pnitroanilide, ˛-benzoyl-l-tyrosine p-nitroanilide (BAPNA), Hip-Lys,

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and Hip-Phe] substrates. Enzymatic activities were determined on each substrate according to standard conditions. 2.12. Kinetic measurements Kinetic parameters were calculated from the initial rate activities of the purified enzymes using natural (elastin–orcein) and modified (azo-albumin) proteins and ester (BCEE) and synthetic peptide [Suc-(Ala)3 -p-NA] substrates. The purified enzymes used in this work were AMPP (this study); PPE, a porcine pancreas elastase (EC 3.4.21.36, protease type IV, Sigma Chemical Co.); KERAB, a keratinase from Streptomyces sp. strain AB1 [22,28]; thermolysin, a metalloenzyme from Bacillus thermoproteolyticus rokko (EC 3.4.24.27, protease type X, Sigma Chemical Co.); SAPBL31I/T33S/N99Y, a triple mutant enzyme from Bacillus subtilis DB430/pNZ2 [29]; nattokinase (fibrinolytic natto), a commercially available enzyme from Smart City® S.A.; and alcalase (subtilisin Carlsberg, EC 3.4.21.62, protease type VIII, Sigma Chemical Co.). The pH and temperature values used in the kinetic study were adjusted to the optimum conditions for each enzyme (AMPP, pH 12, 80 ◦ C; PPE, pH 8.5, 25 ◦ C; KERAB, pH 11.5, 75 ◦ C; thermolysin, pH 8, 70 ◦ C; SAPB-L31I/T33S/N99Y, pH 12, 70 ◦ C; nattokinase, pH 7, 37 ◦ C; and alcalase, pH 9.5, 55 ◦ C). The reaction was performed at different substrate concentrations ranging from 0.05 to 50 mM, for 15 min in assay buffer supplemented with 10% (v/v) dimethyl sulfoxide (DMSO) and 1% (v/v) Triton X-100. For the natural substrate, the complete hydrolysis of elastin–orcein brought a 0.27 increase of absorbance at 590 nm. One elastin unit was defined as the amount of enzyme that produced a 0.0135 increase in absorbance per hour under the assay conditions used. For the modified substrate, one azo-albumin unit was defined as the amount of enzyme causing an increase of 0.1 in absorbance at 440 nm in one min under the experimental conditions described. For the ester substrate, the rate of change in absorbance at 253 nm was measured for 3 min. One BCEE unit was defined as the amount of enzyme causing a rate of change in absorbance of 0.001 per min under the assay conditions. For the synthetic peptide substrate, the amount of released p-nitroanilide (p-NA) was recorded at 410 nm. One unit of enzymatic activity was defined as the amount of enzyme releasing 1 ␮mole of p-NA under standard assay conditions. Each assay was carried out in triplicate, and kinetic parameters were estimated by Lineweaver–Burk plots. Kinetic constants, Michaelis–Menten constant (Km ) and maximal reaction velocity (Vmax ) values were calculated using the Hyper32 software package offered by the Liverpool University (http://hompage.ntlword.com/john.easterby/hyper32.html). The value of the turnover number (kcat ) was calculated using the following equation: kcat =

Vmax [E]

where [E] refers to the active enzyme concentration and Vmax to maximal velocity. 2.13. Effects of organic-solvents on protease activity and stability Various organic solvents, with different Log P values (60%, v/v), were tested with shaking at 150 strokes per min and 30 ◦ C for 1 and 90 d to evaluate their effects on protease activity and stability, respectively. The proteases used were AMPP, thermolysin, and PPE. The relative and residual caseinolytic activities were assayed under the same conditions at 60 ◦ C and pH 10. The activity of the enzyme without any organic solvent was taken as 100%.

2.14. Determination of hydrolysis degree Hemoglobin hydrolysis was carried out at 50 ◦ C and pH 10. The pH was kept constant throughout hydrolysis by adding NaOH 4 N. An amount of 3 g of hemoglobin was dissolved in 100 ml of 50 mM glycine–NaOH buffer containing 2 mM CaCl2 at pH 10 and then treated with 1000 U/ml of the purified enzymes, namely AMPP, PPE, thermolysin, KERAB, nattokinase, SAPB-L31I/T33S/N99Y, and alcalase. The NaOH amount needed to maintain the pH constant was proportional to the degree of hydrolysis (DH). Enzymatic reactions were stopped when the DH became constant. The DH, defined as the percent ratio of the number of peptide bonds broken (h) to the total number of peptide bonds in the assayed substrate (htot ), was calculated for each case from the amount of base (NaOH) added to keep the pH constant during hydrolysis [30] as given below: DH(%) =

h B × Nb 1 1 × 100 = × 100 × × MP ˛ htot htot

where B refers to the amount of base consumed (ml) to keep the pH constant during the reaction, Nb to the normality of the base, MP to the mass (g) of protein (N × 6.25), and ˛ to the average dissociation degree of the ␣-NH2 groups released during hydrolysis expressed as: 10pH–pK ˛= 1 + 10pH–pK where pH and pK refer to the values at which the proteolysis was conducted. The total number of peptide bonds (htot ) in the hemoglobin protein concentrate was assumed to be 8.3 meq/g [30]. 2.15. Molecular cloning and expression of the lasB gene The preparation of plasmid DNA, digestion with restriction endonucleases, and separation of fragments by agarose gel electrophoresis were performed using general molecular biology techniques as described by Sambrook et al. [20]. Four oligonucleotides were synthesized, based on the high degree of sequence homology published for the elastase lasB gene of P. aeruginosa strains, and used for the isolation and determination of the elastase gene sequence. The complete lasB gene and its flanking regions were amplified using the forward primer F-JB100 (5 AAGCGTCGGCCGGAGTACTTCG-3 ) and the reverse primer R-JB101 (5 -GACCGGCATTCCTTCCTGGAG-3 ) to generate an approximately 1.6 kb PCR fragment using genomic DNA from P. aeruginosa CTM50182 as a template and DNA polymerase from Pyrococcus furiosus (Pfu) (Biotools, Madrid, Spain). The internal primers, namely forward primer F-JB103 (5 -ACTGTCGCGGCCGCATTTCG3 ) and reverse primer R-JB104 (5 -TATAGAACTCGGCAGCCTCG-3 ), were used to amplify and sequence the internal region of the lasB gene (0.6 kb). DNA amplification was carried out using the Gene Amp® PCR System 2700 (Applied Biosystems, Foster City, CA, USA). The amplification reaction mixtures (50 ␮l) contained 20 pg of each primer, 200 ng of DNA template, amplification buffer, and 2 U of Pfu DNA polymerase. The cycling parameters used were 94 ◦ C for 5 min followed by 35 cycles of 94 ◦ C for 30 s denaturation, 54 ◦ C for 45 s primer annealing, and 72 ◦ C for 90 s extension). The PCR products were then purified using an agarose gel extraction kit (Jena Bioscience, GmbH, Germany). The purified 1.6 kb PCR fragment was cloned in pCR-Blunt cloning vector into E. coli HB101 [F− mcrB mrr hsdS20 (rB − mB − ) recA13 leuB6 ara-14 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20 (SmR ) glnV44 − ] (Invitrogen, Carlsbad, CA, USA) host strain. Recombinant clones of E. coli were grown in LB broth media with the addition of Ampicillin (100 ␮g/ml), IPTG (160 ␮g/ml), and X-gal (360 ␮g/ml). A clone was noted to harbor a plasmid called pBJ100 and was, therefore, retained for further study. The pBJ100 plasmid

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169

Pseudomonas aeruginosa CTM50182 (JX909351)

Pseudomonas aeruginosa ATCC 27853 (AB037545) 76

87

Pseudomonas aeruginosa DSMZ 50071T (HE978271) Pseudomonas aeruginosa ATCC BAA-1006 (AY631058) Pseudomonas aeruginosa LMG 1242T (Z76651) Pseudomonas aeruginosa ATCC 27853 (AY268175)

Pseudomonas aeruginosa ATCC 10145 (AF094713)

100

Pseudomonas aeruginosa ATCC 15692 (AF094715) Pseudomonas aeruginosa DSM 50071 (X06684)

50

Pseudomonas alcaligenes IAM 1236T (D84006) Pseudomonas stutzeri CCUG 11256T (U26262)

Pseudomonas nitroreducens IAM 1439T (D84021) Pseudomonas jinjuensis Pss 26T (AF468448)

98 80

Pseudomonas citronellolis ATCC 13674T (AB021396) Pseudomonas putida IAM 1236T (D84020) Pseudomonas chlororaphis IAM 12354T (D84011)

55 99

98

Pseudomonas fluorescens IAM 12022T (D84013) Pseudomonas syringae ATCC 19310T (AF094749) Pseudomonas graminis DSM 11363T (Y11150)

79 71

94

Pseudomonas jinjuensis Ps 3-10T (AF468450) Pseudomonas jessenii CIP 105274T (AF068259)

Pseudomonas pavonaceae IAM 1155T (D84019) Escherichia coli ATCC 11775T (X80725) 0.02 Fig. 1. Phylogenetic tree based on 16S rRNA gene sequences showing the position of strain CTM50182 within the radiation of the genus Pseudomonas. The sequence of E. coli ATCC 11775T (X80725) was chosen arbitrarily as an outgroup. Bar, 0.02 nt substitutions per base. Numbers at nodes (>50%) indicate support for the internal branches within the tree obtained by bootstrap analysis (percentages of 100 bootstraps). NCBI accession numbers are presented in parentheses.

was digested with EcoRI restriction enzyme and used for expression studies. The resulting DNA fragment, which was noted to harbor the lasB encoding gene, was sub-cloned in the pTrc99A vector under the control of the inducible Ptac promoter that was previously digested with the EcoRI restriction enzyme leading to the pBJ101 plasmid.

2.18. Nucleotide sequence accession number The nucleotide sequence of 16S rRNA and lasB genes reported in this paper have been deposited in GenBank under the accession numbers of JX909351 and JX970630, respectively.

2.16. Recombinant enzyme localization and purification After reaching an optical density of 0.7 at 600 nm, the production of target protein from HB101/pJB101 was induced by the addition of IPTG. The protease crude extracts were prepared from the extracellular fraction as described in a previous work by the authors [27]. 2.17. DNA sequencing and amino acid sequence alignment The nucleotide sequence was determined on both strands by the BigDye® Terminator v3.1 Cycle Sequencing Kit and the automated DNA sequencer ABI Prism® 3100-Avant Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Multiple nucleotide sequence alignment was performed using the BioEdit version 7.0.2 software program. The amino acid sequence homology was analyzed using the BLASTP program available at the NCBI server.

3. Results and discussion 3.1. Screening of alkaline protease-producing strains About one hundred and ninety bacterial strains that were either newly isolated from Tunisian soil samples or obtained from the CBS bacterial collection were identified as protease producers based on the their patterns of clear zone formation on casein-containing media at pH 9. The ratio of the diameter of the clear zone and that of the colony served as an index for the selection of strains with high protease production ability. Sixty-seven isolates exhibited a ratio that was higher than 4, with the highest ratio being 7. Strain CTM50182 exhibited the highest extracellular protease activity (about 21,000 U/ml) after 30 h of incubation in an optimized medium and was, therefore, retained for all subsequent studies.

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Fig. 2. (a) Chromatography of the protease from P. aeruginosa CTM50182 on Mono Q-Sepharose. The column (2.6 cm × 50 cm) was equilibrated with buffer B. Adsorbed material was eluted with a linear NaCl gradient (0–500 mM in buffer B) at a flow rate of 35 ml/h, and assayed for protein content at 280 nm () and protease activity () as described in Section 2. (b) Size exclusion HPLC chromatography of the purified AMPP (20 ␮g) from P. aeruginosa CTM50182 using a Bio-Sil SEC 125-5 column (7.8 mm × 300 mm) shows a single and symmetrical peak of approximately 33 kDa (retention time = 19 min), (c) SDS-PAGE of the purified protease. Lane 1, protein markers. Lane 2, purified AMPP (30 ␮g) obtained after Q Sepharose anion-exchange chromatography (fractions 26–32), (d) zymogram activity staining of the purified protease, and (e) MALDI-TOF spectrum of 10 pmol purified AMPP protease from P. aeruginosa CTM50182. The mass spectrum shows a series of multiply protonated molecular ions. The molecular mass of the enzyme was found to be 33,015.18 Da.

3.2. Identification and molecular phylogeny of the microorganism

bacterium. API 20 NE tests revealed that it could utilize citrate, lactose, manitol, guconate, and caprate, but not l-arabinose, mannose, adipate, and malate. The 16S rRNA gene sequence (accession no: JX909351) from CTM50182 was 98% similar to that of the P.

The CTM50182 isolate was identified as a Gram-negative, catalase-positive, oxydase-positive, motile, and aerobic rod-shaped Table 1 Flow sheet for AMPP purification. Purification step

Total activity (units)a , b (×103 )

Crude extract Heat treatment (1 h at 60 ◦ C) (NH4 )2 SO4 fractionation (50–70%) HPLC (Bio-Sil SEC 125-5) Mono Q Sepharose

10,500 9135 8295 5355 1853

± ± ± ± ±

49 31 25 14 07

Total protein (mg)a , c 6750 1902 507 72 19

± ± ± ± ±

97 22 7 3 1

Specific activity (U/mg of protein)a 1555 4802 16,360 74,375 97,526

± ± ± ± ±

134 346 568 796 2154

Activity recovery rate (%)

Purification factor (fold)

100 87 79 51 17.64

1 3.08 10.52 47.82 62.71

The experiments were conducted three times and ±standard errors are reported. One CU is defined as the amount of enzyme that hydrolyzed the substrate and produced 1 ␮g of amino acid equivalent to tyrosine per minute under the experimental conditions used. c Amounts of protein were estimated by Bradford method [25]. a

b

B. Jaouadi et al. / International Journal of Biological Macromolecules 60 (2013) 165–177

aeruginosa ATCC 27853 (accession no: AB037545), the P. aeruginosa DSMZ 50071T (accession no: HE978271), and the P. aeruginosa ATCC BAA-1006 (accession no: AY631058) (Fig. 1). 3.3. Purification of AMPP The supernatant obtained by the centrifugation of the P. aeruginosa CTM50182 culture broth (500 ml) was used as the crude enzyme solution. The enzymatic preparation was heat-treated (65 ◦ C for 1 h) and precipitated at 50–70% ammonium sulfate saturation. The precipitate formed was collected by centrifugation (12,000 × g for 30 min at 4 ◦ C), dissolved in a minimum amount of buffer B, and then dialyzed overnight against repeated changes of the same buffer. Fractions corresponding to protease activity were pooled and loaded on HPLC Bio-Sil SEC 125-5 column equilibrated with buffer C. Purification to homogeneity was achieved using a Mono Q Sepharose anion exchange chromatography. Bound proteins were eluted with a linear gradient of NaCl from 0 to 500 mM in buffer C at a rate of 35 ml/h. The protein elution profile obtained at the final purification step indicated that the protease was eluted at 125–225 mM NaCl (Fig. 2a). The results of the purification procedure are summarized in Table 1. Enzyme purity was estimated to be about 62.71-fold greater than that of the crude extract. Under optimum assay conditions, the purified enzyme had a specific activity of 97,526 U/mg, with a yield of about 17.64%. This preparation was a homogeneous enzyme with high purity as it exhibited a unique symmetrical elution peak with a retention time of 19 min, corresponding to a protein of nearly 33 kDa on HPLC gel filtration chromatography (Fig. 2b). SDS-PAGE analysis showed that the pooled fractions displayed one band corresponding to an apparent molecular mass of about 33 kDa (Fig. 2c). Zymogram activity staining revealed one zone of caseinolytic activity for the purified sample co-migrating with proteins of a molecular mass of 33 kDa (Fig. 2d). MALDI-TOF/MS analysis confirmed that the purified AMPP had an exact molecular mass of 33015.18 Da (Fig. 2e). These observations strongly suggested that AMPP was a monomeric protein comparable to those previously reported for other proteases from Pseudomonas strains [13,15,16,31]. 3.4. N-terminal amino acid sequence The sequence determined for the first 29 N-terminal amino acids of the AMPP enzyme from the P. aeruginosa CTM50182, namely AEAGGPGGNQKIGKYTYGSDYGPLIVNDR, showed uniformity, indicating that it was isolated in a pure form. This sequence was submitted to comparisons with existing protein sequences in the GenBank non-redundant nucleotide database, using the BLASTP and tBlastn search programs, and the BLASTP software available at the Swiss-Prot database (http://www.expasy.ch/sprot/). The sequence showed high levels of homology with those found in other Pseudomonas proteases, reaching 100% identity with chain A of elastase PAE (PDB, code 1U4G) from P. aeruginosa, elastase LasB (accession no: AEM456371) from P. aeruginosa MCCB123, and keratinase KP2 (accession no: ADP00718) from P. aeruginosa KS1. It also showed 93% identity with elastase LasB (accession no: ABR82732) from P. aeruginosa PA7. The AMPP protease differed from LasB PA7 elastase by three residues (E2Q, T16N, and S19T for AMPP and LasB PA7, respectively). 3.5. Effects of inhibitors and metal ions on protease stability Proteases can be classified by their sensitivity to various inhibitors. The relative inhibitory effects of the various compounds assayed are listed in Table 2. The protease presented in this work was not inhibited by thiol (DTNB, NEM, iodoacetamide, leupeptin)

171

Table 2 Effects of various inhibitors, reducing agents, and metal ions on AMPP stability. Protease activity measured in the absence of any inhibitor or reducing agent was taken as control (100%). The non-treated and dialyzed enzyme was considered as 100% for metal ion assay. Residual activity was measured at pH 12 at 80 ◦ C. Inhibitor/reducing agent/metal ions

Concentration

Residual activity (%)a

None EDTA EGTA 1,10-Phenanthroline monohydrate Phosphoramidon disodium salt PMSF DFP TLCK TPCK Benzamidine Aprotinin bovine MIA SBTI DTNB EPNP 2-ME ld-DTT l-Cysteine Glutathione NEM Iodoacetamide Leupeptin Pepstatin A Co2+ (CoCl2 ) Zn2+ (ZnCl2 ) Ca2+ (CaCl2 ) Mn2+ (MnCl2 ) Mg2+ (MgCl2 ) Cu2+ (CuCl2 ) Ba2+ (BaCl2 ) Fe2+ (FeCl2 ) Hg2+ (HgCl2 ) Ni2+ (NiCl2 ) Cd2+ (CdCl2 ) Na+ (NaCl) K+ (KCl) Li+ (LiSO4 )

– 10 mM 2 mM 10 mM 10 ␮M 5 mM 5 mM 1 mM 0.5 mM 1 mM 30 ␮M 30 ␮M 1 mg/ml 5 mM 5 mM 5 mM 5 mM 5 mM 5 mM 2 mM 5 mM 50 ␮g/ml 1 ␮g/ml 5 mM 5 mM 5 mM 5 mM 5 mM 5 mM 5 mM 5 mM 5 mM 5 mM 5 mM 5 mM 5 mM 5 mM

100 25 5 10 31 100 97 99 98 102 99 100 105 94 96 43 0 97 93 97 96 93 101 271 180 143 121 114 41 97 81 0 0 0 100 100 100

a

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.5 0.9 0.1 0.2 0.3 2.5 2.4 2.5 2.5 2.5 2.5 2.5 2.6 2.4 2.4 1.1 0.0 2.5 2.4 2.4 2.4 2.4 2.5 5.1 4.2 3.7 2.9 2.7 1.1 2.4 1.7 0.0 0.0 0.0 2.5 2.5 2.5

Values represent means of four replicates, and ±standard errors are reported.

and acid (pepstatin A) reagents or by serine protease inhibitors (PMSF, DFP). While its activity was partially inhibited by sulfhydryl reagents, such as glutathione and 2-ME, (43 and 57% inhibition, respectively), it was completely inhibited by DTT (100% inhibition), strongly suggesting that the enzyme contained S S bonds as part of its monomeric structure. Other inhibitors, including TPCK and TLCK, chymotrypsin alkylating agents, benzamidine and aprotinin, serine protease competitive reagents, and SBTI, a soybean trypsin inhibitor, did not display any inhibitory effects. Enzyme activity was, however, strongly inhibited by the chelating agents EGTA (2 mM), 10-phenanthroline (the zinc specific metal chelator) (10 mM), and EDTA (10 mM) (95, 85, and 75% inhibition, respectively), which are well-known inhibitors of metalloproteases. The inhibition profile observed with those metal chelators strongly suggested that the AMPP protease was a metalloenzyme. The specific metal cofactor required for its function is, however, still unknown, which is the case for almost all P. aeruginosa endopeptidases [9,13,16]. Several metal ions were also assayed for their effects on AMPP activity (Table 2). The activity of the enzyme was essentially unaffected by monovalent cations (Na+ , Li+ , and K+ ). Its activity was enhanced by143, 180, and 275% following the addition of CaCl2 , ZnCl2 , and CoCl2 at 5 mM as compared to the control, respectively. This result indicated that the enzyme required Co2+ , Zn2+ , and Ca2+ for optimal activity. In fact, the role of Ca2+ is probably related to the stabilization of the activated form of the AMPP and to the

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(b)

(a)

(c)

125

100

75

50

25

0

250

Relative protease activity (%)

Residual protease activity (%)

Relative protease activity (%)

125

100

75

50

.

25

3

4

5

6

7

8

9

10

11

12

13

6

pH

7

8

(d)

9

pH

10

150

100

50

11

40 45 50 55 60 65 70 75 80 85 90 95 100

12

Temperature (°C)

(e)

150

40 70 °C (0 mM Co2+) 80 °C (0 mM Co2+) 90 °C (0 mM Co2+) 100 °C (0 mM Co2+)

125

70 °C (5 mM Co2+) 80 °C 5 mM Co2+) 90 °C (5 mM Co2+) 100 °C (5 mM Co2+)

PPE Thermolysin Alcalase Natokinase AMPP SAPB-L31I/T33S/N99Y KERAB

35

Degree of hydrolysis (%)

Residual protease activity (%)

5 mM Co2+

0

0 2

0 mM Co2+

200

100

75

50

25

30 25 20 15 10 5 0

0 0

2

4

6

8

10

12

14

16

18

20

22

24

Time (h)

0

15

30

45

60

75

90 105 120 135 150 165 180 195 210 225 240

Hydrolysis time (min)

Fig. 3. Effects of pH on the activity (a) and stability (b) of AMPP. The activity of the enzyme at pH 12 was taken as 100%. Buffer solutions used for pH activity and stability are presented in Section 2. Effects of the thermoactivity (c) and the thermostability (d) of AMPP. The enzyme was pre-incubated in the absence or presence of CoCl2 at various temperatures ranging from 70 to 100 ◦ C. Residual protease activity was determined from 0 to 24 h at 2 h intervals. The activity of the non-heated enzyme was taken as 100%. (e) Hydrolysis curves of hemoglobin proteins treated with various purified enzymes. Each point represents the mean (n = 3) ± standard deviation.

preservation of its structure against autolysis. Zn2+ is, on the other hand, is essential for catalysis due to the formation of zinc monohyroxide that bridges the catalytic zinc ion to the side chain of the enzyme’s active site as previously reported by Larsen and Auld [32]. Moreover, while the enzyme was slightly activated by Mn2+ and by Mg2+ , it underwent no significant inhibition in the presence of Ba2+ and Fe2+ . Protease activity was, however, completely inhibited by Hg2+ , Ni2+ , and Cd2+ and moderately inhibited by Cu2+ . In fact, the proteases purified from P. aeruginosa MN7 and P. aeruginosa A2 were partially inhibited by Ca2+ , Mg2+ , Ba2+ , and Mn2+ but strongly inhibited by Zn2+ , Cu2+ , and Hg2+ (5 mM) [13,16]. Likewise, the activity of the P. aeruginosa PseA protease was markedly decreased by Ni2+ and Cu2+ and moderately reduced by Zn2+ , Mn2+ , Mg2+ , and Ca2+ [33].

At pH 12, and using casein as substrate, the protease was optimally active at 70 ◦ C, in the absence of CoCl2 , and at 80 ◦ C, in the presence of 5 mM Co2+ (Fig. 3c). The half-life times of AMPP in the absence of additives were 14, 8, 4, and 1.5 h at 70, 80, 90, and 100 ◦ C, respectively (Fig. 3d). The addition of different concentrations of CoCl2 (1–10 mM) enhanced the thermostability of the enzyme. Maximal thermostability was achieved with 5 mM Co2+ (data not shown). As shown in Fig. 3d, the half-life times of AMPP at 70, 80, 90, and 100 ◦ C increased to 16, 10, 6, and 2.8 h in the presence of 5 mM CoCl2 . In fact, Co2+ was previously reported to improve the activity and stability of the P. aeruginosa PT121 protease [9]. The thermoactivity and the thermostability of AMPP were higher than those of several other proteases from P. aeruginosa previously reported in the literature [9,13,16,33].

3.6. Effects of pH and temperature on protease activity and stability

3.7. Substrate specificity of AMPP

Fig. 3a shows that AMPP displayed activity over a broad range of pH (2–13), with an optimum at pH 12, and that it underwent an abrupt decrease in activity above pH 12. The relative activities at pH 5 and 13 were 55 and 25%, respectively. A similar sudden drop in activity between pH 10.6 and 11 was previously observed for the SAPB enzyme in earlier works by the authors [21,27]. The pH stability profile indicated that the purified enzyme was stable in the pH range between 9 and 12 (Fig. 3b) but underwent a rapid decrease in activity at pH values of above 12. The enzyme retained 90 and 92% of its activity at pH 7 and 8, respectively, after 120 h of incubation at 50 ◦ C. This is in agreement with previous results on most proteases from P. aeruginosa, which exhibited optimum activity at pH values ranging from 7 to 9 [9,13,16,33].

The substrate specificity of proteases is often attributed to the amino acid residues preceding the peptide bond they hydrolyze. The relative hydrolysis rates of various substrates were investigated to elucidate the amino acid preference/substrate specificity of AMPP (Table 3). The highest activity was observed with elastin–orcein, casein, albumin, and azo-albumin. A relatively high activity against fibrin, gelatin, and azo-casein was also observed. The enzyme showed low collagenase activity on collagen types I and II. The purified AMPP was noted to exhibit esterase and amidase activities on BCEE, BAEE, and BAPNA, but not on BTEE and ATEE. It also displayed a preference for aromatic and hydrophobic amino acid residues, such as Phe, Met, Leu, Ala, and Val, at the carboxyl side of the splitting point in the P1 position. AMPP was, therefore, active against alalnine

B. Jaouadi et al. / International Journal of Biological Macromolecules 60 (2013) 165–177 Table 3 Substrate specificities of the purified AMPP. Substrate

Natural protein Elastin–orcein Casein Gelatin Albumin Fibrin Keratin Gluten Myoglobin Hemoglobin Modified protein Azo-albumin Azo-casein Keratin azure Collagen type I Collagen type II Ester BCEE BAEE ATEE BTEE Synthetic peptide Suc-(Ala)3 -pNA Suc-Ala-Pro-Ala-pNA Suc-(Ala)2 -Phe-pNA Suc-(Ala)2 -Val-pNA Suc-(Ala)2 -Pro-Phe-pNA Suc-(Ala)2 -Pro-Met-pNA Suc-(Ala)2 -Pro-Leu-pNA Glu-(Ala)2 -Pro-Leu-pNA Met Suc-(Ala)2 -Pro-Val-pNA Suc-(Ala)2 -Val-Ala-pNA Suc-(Ala)2 -Pro-Phe-pNA Suc-Tyr-Leu-Val-pNA l-Leu-pNA BAPNA Hip-Lys Hip-Phe

Concentration

Monitoring wavelength (nm)a

Relative activity (%)b

20 g/l 30 g/l 30 g/l 30 g/l 30 g/l 30 g/l 30 g/l 30 g/l 30 g/l

660 660 660 660 660 660 660 660 660

100 95 75 90 86 81 25 50 71

± ± ± ± ± ± ± ± ±

2.5 2.4 2.0 2.3 2.2 2.1 0.9 1.4 2.0

30 g/l 30 g/l 30 g/l 3 mg/ml 3 mg/ml

440 440 440 490 490

100 90 72 35 23

± ± ± ± ±

2.5 2.3 2.0 1.2 1.0

5 mM 5 mM 5 mM 5 mM

253 253 253 253

100 80 0 0

± ± ± ±

2.5 2.1 0.0 0.0

4 mM 4 mM 4 mM 4 mM 4 mM 4 mM 4 mM 4 mM 4 mM 4 mM 4 mM 4 mM 4 mM 5 mM 5 mM 5 mM

410 410 410 410 410 410 410 410 410 410 410 410 410 410 254 254

100 94 90 86 71 83 65 62 51 58 45 38 47 77 60 71

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.5 2.4 2.3 2.2 2.0 2.1 1.6 1.6 1.4 1.5 1.3 1.2 1.3 2.1 1.5 2.0

Values represent means of three replicates, and ± standard errors are reported. The unit activity of each substrate was determined by measuring absorbance at specified wavelengths as described in Section 2. a

173

Suc-(Ala)3 -pNA was used as a synthetic substrate, AMPP was also noted to exhibit kcat /Km values that were 11.73, 5.20, 3.16, 2.91, 1.92, and 1.41 times higher than those of SAPB-L31I/T33S/N99Y, alcalase, thermolysin, KERAB, nattokinase, and PPE, respectively (Table 4). 3.9. Effects of organic solvents on proteolytic activity and stability Various organic solvents were examined for their effects on the activity and stability of the purified AMPP, thermolysin, and PPE proteases. The enzyme solutions containing 60% (v/v) an organic solvent were incubated at 30 ◦ C with shaking (Table 5). When compared to the control, AMPP, PPE, and thermolysin were noted to retain at least 91, 75, and 52% of their activities after 90 d of incubation in the presence of hexane as a hydrophobic solvent and 96, 62, and 71% of their activities in the presence of ethanol as a hydrophilic solvent, respectively. By contrast, the proteases were noted to be completely deactivated by acetonitrile and ethyl acetate, which have previously been reported to be quite harmful polar aprotic solvents to other solvent-stable proteases from Bacillus licheniformis RSP-09-37 [35] and Bacillus cereus WQ9-2 [36,37]. In media containing organic solvents, enzyme deactivation is most probably caused by the disruption of the protein molecule hydrophobic core due to the change of medium hrdrophobicity [38]. In particular, polar solvents (such as acetonitrile and ethyl acetate), which can penetrate into the protein, are more capable of inducing structural changes for the interaction between the active site and substrate than non-polar solvents, as previously reported by Serdakowski and Dordick [39]. This suggested that the stability of enzymes in the presence of organic solvents depends on both the enzyme and the nature of the organic solvent being used. The activity and stability shown by AMPP in the presence of DMSO, N,N-dimethylformamide (DMF), chloroform, and N-heptane, were higher than those of thermolysin and PPE, which further supported the potential candidacy of the AMPP metalloprotein for future application as a biocatalyst for the synthesis of peptide reactions in low water activity systems.

b

peptide bonds, a quality that was previously demonstrated for SAPB [27,28,34] and KERAB [22]. When Suc-(Ala)n -pNA was used as the synthetic oligopeptide substrate, a minimum length of two residues was required for hydrolysis. Enzymatic activity was noted to largely depend on secondary enzyme substrate contacts with amino acid residues (P2, P3, etc.) more distant from the scissile bond, as illustrated by the differences observed between the kinetic parameters of Suc-(Ala)2 -Val-pNA and Suc-Tyr-Leu-Val-pNA. AMPP was noted to attain the highest hydrolysis rates of 100 and 90% with Suc(Ala)3 -pNA and Suc-Ala-Pro-Ala-pNA, respectively. Its preference for lager hydrophobic amino acids could presumably be due to the active site cleft or the crevice lined with hydrophobic amino acids residues.

3.10. Determination of the hydrolysis degree The hydrolysis curves of hemoglobin protein after 4 h of incubation are shown in Fig. 3e. The purified enzymes were used at the same levels of activity (1000 U/ml) for the production of protein hydrolysates from hemoglobin and for the subsequent comparisons of hydrolytic efficiencies. Hemoglobin was noted to attain high rates of hydrolysis during the first 1 h. The enzymatic reaction was then noted to decrease and to reach a steady-state phase where no apparent hydrolysis took place. As shown in Fig. 3e, the purified AMPP was the most efficient (32%) protease used during hydrolysis and nattokinase was the least efficient (10%) one. After 4 h of hydrolysis, the DH reached about 28% with PPE, 25% with KERAB, 22% with thermolysin, 18% with SAPB-L31I/T33S/N99Y, and 15% with alcalase. 3.11. Cloning and sequencing of the lasB gene

3.8. Determination of kinetic parameters AMPP, KERAB, PPE, thermolysin, SAPB-L31I/T33S/N99Y, nattokinase, and alcalase exhibited the classical kinetics of Michaelis–Menten for the four substrates used. The order of the catalytic efficiency (kcat /Km ) values of each enzyme was almost the same, i.e., elastin–orcein < azo-albumin < BCEE < Suc-(Ala)3 -pNA (Table 4). When elastin–orcein was used as a protein substrate, the kcat /Km exhibited by AMPP were 7.47, 5.84, 3.04, 2.62, 1.86, and 1.63 times higher than those of alcalase, SAPB-L31I/T33S/N99Y, nattokinase, KERAB, PPE, and, thermolysin, respectively. When

Using the elastase gene sequences of P. aeruginosa strains, two primers, called F-JB100 and R-JB101 , were designed and used to amplify a fragment of about 1.6 kb that could contain the lasB gene. This PCR fragment was purified and cloned in a pCR-Blunt cloning vector using an E. coli HB101 host strain, thus leading to pJB100. The complete nucleotide sequence and amino acid sequence deduced for the lasB gene are shown in Fig. 4. The analysis of the nucleotide sequence of the lasB gene and its flanking DNA regions revealed the presence of an open reading frame (ORF) of 1497bp that encoded a pre-pro-enzyme consisting of 498 aa with a

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Table 4 Kinetic parameters of purified proteases: AMPP, PPE, thermolysin, KERAB, SAPB-L31I/T33S/N99Y, nattokinase, and alcalase for the hydrolysis of protein, ester, and synthetic peptide substrates. kcat (×104 min−1 )

kcat /Km (×104 min−1 mM−1 )

Catalytic efficiency relative to AMPP

0.72 0.30 0.35 0.29 0.10 0.65 0.12

27.05 14.54 16.58 10.30 4.63 8.87 3.62

1.00 0.53 0.61 0.38 0.17 0.32 0.13

± ± ± ± ± ± ±

0.71 0.20 0.17 0.18 0.07 0.29 0.14

32.12 15.62 10.49 8.26 5.33 9.75 4.87

1.00 0.48 0.32 0.25 0.16 0.30 0.15

± ± ± ± ± ± ±

3.10 1.90 2.61 1.60 1.51 2.42 0.62

57.21 35.44 30.63 14.44 16.69 27.31 7.31

1.00 0.61 0.53 0.25 0.29 0.47 0.12

42.17 ± 6.10 31.16 ± 3.08 19.34 ± 1.52 23.10 ± 1.57 10.14 ± 0.69 32.29 ± 3.15 19.01 ± 1.23

61.83 43.58 19.51 21.19 5.27 32.17 11.87

1.00 0.70 0.31 0.34 0.08 0.52 0.19

Substrate

Enzyme

Km (mM)

Elastin–orcein

AMPP PPE Thermolysin KERAB SAPB-L33I/T33S/N99Y Nattokinase Alcalase

0.425 0.526 0.513 0.691 1.110 1.032 1.420

± ± ± ± ± ± ±

0.010 0.018 0.014 0.026 0.037 0.031 0.040

11.50 7.65 8.51 7.12 5.14 9.16 5.15

± ± ± ± ± ± ±

Azo-albumin

AMPP PPE Thermolysin KERAB SAPB-L33I/T33S/N99Y Nattokinase Alcalase

0.301 0.416 0.571 0.754 0.924 0.733 1.201

± ± ± ± ± ± ±

0.005 0.011 0.019 0.028 0.030 0.027 0.038

9.67 6.50 5.99 6.23 4.93 7.15 5.85

BCEE

AMPP PPE Thermolysin KERAB SAPB-L33I/T33S/N99Y Nattokinase Alcalase

0.492 0.630 0.850 1.490 1.156 0.921 1.545

± ± ± ± ± ± ±

0.013 0.024 0.029 0.050 0.038 0.030 0.053

28.15 22.33 26.04 21.53 19.30 25.16 11.30

Suc-(Ala)3 -pNA

AMPP PPE Thermolysin KERAB SAPB-L33I/T33S/N99Y Nattokinase Alcalase

0.682 ± 0.025 0.715 ± 0.027 0.991 ± 0.033 1.322 ± 0.044 1.921 ± 0.085 1.004 ± 0.031 1.601 ± 0.060

Values represent means of three replicates, and ±standard errors are reported.

predicted molecular weight of 53,675.1 Da. This ORF started with an ATG codon at nucleotide position 1 and terminated with a TAA stop codon. A Shine–Dalgarno (SD)-like sequence was observed 10–16 bps upstream from the ATG codon. The G + C content of the mature elastase gene (64%) was typical of the P. aeruginosa genome. This ORF was confirmed as the gene encoding AMPP since, as

determined by the Edman degradation method, the deduced amino acid sequence was noted to include the 29 N-terminal amino acid sequence of the purified AMPP. This sequence was identical to those of elastases from other P. aeruginosa strains [11–13]. This ORF was also noted to count the stacking region (SR) (1557–1592 bp), which supported the existence of a termination codon at this site.

Table 5 Effects of organic solvents on the activity and stability of the purified AMPP, thermolysin, and PPE proteases. The non-treated enzyme was considered as 100%. The activity is expressed as a percentage of activity level in the absence of organic solvents. Organic solvent (60%, v/v)

Log P

Relative activity (%)

None N-hexadecane N-decane Isooctane N-octane N-heptane N-hexane Cyclohexane Toluene Benzene Chloroform 1-Hexanol 1-Buthanol Ethyl acetate Isopropanol Isopropyl alcohol Acetonitrile Ethanol Methanol DMF DMSO

– 8.8 5.6 4.5 4.5 4.0 3.5 3.2 2.5 2.0 2.0 1.8 0.88 0.73 0.28 0.05 −0.15 −0.24 −0.76 −1.03 −1.35

100 115 110 123 141 150 120 125 111 122 161 101 70 0 103 104 0 108 114 180 225

AMPP ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Residual activity (%) Thermolysin

2.5 2.6 2.6 3.5 3.7 4.0 3.5 3.5 2.6 3.5 4.2 2.5 2.0 0.0 2.5 2.5 0.0 2.6 2.6 4.6 6.0

100 105 95 120 133 140 150 138 85 70 103 66 50 0 101 102 0 80 92 125 155

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Values represent means of three replicates, and ±standard errors are reported.

2.5 2.5 2.4 3.5 3.6 3.7 4.0 3.9 2.1 2.0 2.5 1.8 1.4 0.0 2.5 2.5 0.0 2.1 2.4 3.5 4.1

PPE 100 101 91 111 120 130 110 108 101 104 120 85 76 0 121 125 0 77 83 135 180

AMPP ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.5 2.5 2.3 2.6 3.5 3.6 2.6 2.6 2.5 2.5 3.5 2.1 2.0 0.0 3.5 3.5 0.0 2.0 2.1 3.6 4.6

100 105 101 112 122 135 110 113 102 112 142 91 60 0 94 90 0 96 105 155 185

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Thermolysin 2.5 2.5 2.5 2.6 3.5 3.6 2.6 2.6 2.5 2.6 3.7 2.3 1.8 0.0 2.4 2.3 0.0 2.4 2.5 4.1 4.7

100 95 77 108 112 127 135 125 72 60 83 52 38 0 91 88 0 71 75 115 127

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.5 2.4 2.0 2.6 2.6 3.5 3.6 3.5 2.0 1.8 2.1 1.4 1.2 0.0 2.3 2.1 0.0 2.0 2.0 2.6 3.5

PPE 100 81 85 102 110 115 101 99 93 91 112 75 50 0 111 110 0 62 71 121 150

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.5 2.1 2.1 2.5 2.6 2.6 2.5 2.5 2.4 2.3 2.6 2.0 1.4 0.0 2.6 2.6 0.0 1.8 2.0 3.5 4.0

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175

Pre 1 AAGCGTCGGCCGGAGTACTTCGGCCTGAAAAAACCAGGAGAACTGAACAAGATGAAGAAGGTTTCTACGCTTGACCTGTTGTTCGTTGCG 90 -204 SD M K K V S T L D L L F V A -185 F-JB100

Pro

Signal peptide

91 ATCATGGGTGTTTCGCCGGCCGCTTTTGCCGCCGACCTGATCGACGTGTCCAAACTCCCCAGCAAGGCTGCCCAGGGCGCGCCCGGCCCG 180 -184 I M G V S P A A F A A D L I D V S K L P S K A A Q G A P G P -155

SPR

Pro-sequence

181 GTCACCTTGCAAGCCGCGGTCGGCGCCGGCGGTGCCGACGAACTGAAAGCGATCCGCAGCACGACCCTGCCCAACGGCAAGCAGGTCACC 270 -154 V T L Q A A V G A G G A D E L K A I R S T T L P N G K Q V T -125 271 CGCTACGAGCAATTCCACAACGGCGTACGGGTGGTCGGCGAAGCCATCACCGAAGTCAAGGGTCCCGGCAAGAGCGTGGCGGCGCGGCGC -124 R Y E Q F H N G V R V V G E A I T E V K G P G K S V A A R R

360 -95

361 AGCGGCCATTTCGTCGCCAACATCGCCGCCGACCTGCCGGGCAGCACCACCGCGGCGGTATCCGCCGAGCAGGTGCTGGCCCAGGCCAAG -94 S G H F V A N I A A D L P G S T T A A V S A E Q V L A Q A K

450 -65

451 AGCCTGAAGGCCCAGGGCCGCAAGACCGAGAATGACAAAGTGGAACTGGTGATCCGCCTGGGCGAGAACAACATCGCCCAACTGGTCTAC -64 S L K A Q G R K T E N D K V E L V I R L G E N N I A Q L V Y

540 -35

541 AACGTCTCCTACCTGATTCCCGGCGAGGGACTGTCGCGGCCGCATTTCGTCATCGACGCCAAGACCGGTGAAGTGCTCGATCAGTGGGAA -34 N V S Y L I P G E G L S R P H F V I D A K T G E V L D Q W E

630 -5

631 GGCCTGGCCCACGCCGAGGCGGGCGGCCCCGGTGGCAACCAGAAGATCGGCAAGTACACCTACGGTAGCGACTACGGTCCGCTGATCGTC -4 G L A H A E A G G P G G N Q K I G K Y T Y G S D Y G P L I V

720 26

Mature protein

F-JB103

-1 +1 721 AACGACCGCTGCGAGATGGACGACGGCAACGTCATCACCGTCGACATGAACGGCAGCACCAACGACAGCAAGACCACGCCGTTCCGCTTC 27 N D R C E M D D G N V I T V D M N G S T N D S K T T P F R F

810 56

811 GCCTGCCCGACCAACACCTACAAGCAGGTCAACGGCGCTTATTCGCCACTGAACGACGCGCATTTCTTCGGCGGCGTGGTGTTCAAACTG 57 A C P T N T Y K Q V N G A Y S P L N D A H F F G G V V F K L

900 86

901 TACCGGGACTGGTTCGGCACCAGCCCGCTGACCCACAAGCTGTACATGAAGGTGCACTACGGGCGCAGCGTGGAGAACGCCTTCTGGGAC 87 Y R D W F G T S P L T H K L Y M K V H Y G R S V E N A F W D

990 116

991 GGCACGGCGATGCTCTTCGGCGACGGCGCCACCATGTTCTATCCGCTGGTGTCGCTGGACGTGGCGGCCCACGAGGTCAGCCACGGCTTC 1080 117 G T A M L F G D G A T M F Y P L V S L D V A A H E V S H G F 146

Ca

Zn 141 ZnB

Zn

1081 ACCGAGCAGAACTCCGGGCTGATCTACCGCGGGCAATCCGGCGGAATGAACGAGGCGTTCTCCGACATGGCCGGCGAGGCCGCCGAGTTC 1170 147 T E Q N S G L I Y R G Q S G G M N E A F S D M A G E A A E F 176

155

Zn

Ca R-JB104 Ca

1171 TACATGCGCGGCAAGAACGACTTCCTGATCGGCTACGACGTCAAGAAGGGCAGCGGTGCGTTGCGCTACATGGACCAGCCCAGCCGTGAC 1260 177 Y M R G K N D F L I G Y D V K K G S G A L R Y M D Q P S R D 206

Ca

Ca

1261 GGGCGATCCATCGACAACGCCTCGCAGTACTACAACGGTATCGACGTGCACCACTCCAGCGGCGTGTACAACCGTGCGTTCTACCTGCTG 1350 236 207 G R S I D N A S Q Y Y N G I D V H H S S G V Y N R A F Y L L

223 1351 GCCAACTCGCCGGGCTGGGATACCCGCAAGGCCTTCGAGGTGTTCGTCCGCGCCAACCGCTACTACTGGACCGCCACCAGCAACTACAAC 1440 237 A N S P G W D T R K A F E V F V R A N R Y Y W T A T S N Y N 266 1441 AGCGGTGCCTGTGGAGTGATTAGCTCGGCGCAGAACCGCTACTACTCGGCGGCTGACGTCACCCGGGCGTTCAGCACCGTCGGCGTGACC 1530 267 S G A C G V I S S A Q N R Y Y S A A D V T R A F S T V G V T 296 1531 TGCCCGAGCGCGTTGTAAGCTCGGTGGCCCCGGCCAGCACTCCAGGAAGGAATGCCGGTCGG 1592 297 C P S A L End SR R-JB101 SR Fig. 4. Nucleotide sequence of the lasB gene and the deduced amino acid sequence of the elastase of P. aeruginosa CTM50182. Translation starts at a nucleotide position 1. The first amino acid of the mature elastase, Ala, is counted as +1. Numbers written on both sides of the lines indicate the positions of nucleotides and amino acids. The putative starting residues of the prepeptide (pre), propeptide (pro), and mature elastase and the active site residues E141, Y155, and H223 are indicated. The nucleotide sequences ATG and TAA (both highlighted) indicate the initiation and terminal codon of translation, respectively. Amino acids shaded in gray with ‘Ca’ and ‘Zn’ show residues of calcium and zinc ligands. The black box indicates the N-terminal amino acid sequence of the purified AMPP. SD: Shine–Dalgarno-like sequence; SPR: signal peptide recognition site; ZnB: zinc-binding motif; SR: stacking region.

3.12. Amino acid sequence inspection SignalP, version 3.0 (http://www.cbs.dtu.dk/services/SignalP/) predicted a signal peptide (pre-sequence) of 23 aa bordered with the signal peptidase recognition (SPR) site A-F-A-A, indicating that a group of strongly hydrophobic amino acids was conserved. Belonging to the signal sequence, the pro-sequence consisting of 174 aa had to be cleaved by autoproteolytic processing in the periplasm. The active mature elastase consisted of 301 aa (A1-L301), with a predicted molecular weight of 33,102.6 Da and a predicted isoelectric point of 6.17. The apparent molecular weight of the purified enzyme (33 kDa) determined by SDS-PAGE, MALD-TOF/MS, and HPLC gel filtration chromatography was in good agreement with the predicted value. The PROSITE (Swiss Institute of Bioinformatics) analysis of the AMPP protease (498 aa) revealed a conserved

Zn-binding motif (VAAHEVSHGF), a feature typical to all zinc metallopeptidases, starting at amino acid position 136. The ligands of the zinc atom were H140, H144, and E164, and those of the calcium ion were carboxyl groups of D136, E172, E175, and D183, carbonyl group of L185, and one water molecule. The typical metalloendopeptidases catalytic residues (E141, Y155, and H223) in the active site were also conserved in the lasB gene. The amino acid sequence deduced from the nucleotide sequence of the lasB gene was compared to those of other known elastases from P. aeruginosa strains (Fig. 5). The findings revealed 99% identity with the sequence of elastase from P. aeruginosa MN7 [13] and 98% identity with those from P. aeruginosa PST-01 [12] and P. aeruginosa IFO 3455 [11]. The regions from amino acids 135–146 of the mature AMPP enzyme show 100% similarity to the zinc-binding or active site regions of PST-01 elastase (Pseudolysin) and other

176

B. Jaouadi et al. / International Journal of Biological Macromolecules 60 (2013) 165–177 Pre

Pro

AMPP MN7 PST-01 IFO 3455

MKKVSTLDLLFVAIMGVSPAAFAADLIDVSKLPSKAAQGAPGPVTLQAAVGAGGADELKAIRSTTLPNGKQVTRYEQFHNGVRVVGEAITEVKGPGKSVA -197 MKKVSTLDLLFVAIMGVSPAAFAADLIDVSKLPSKAAQGAPGPVTLQAAVGAGGADELKAIRSTTLPNGKQVTRYEQFHNGVRVVGEAITEVKGPGKSVA MKKVSTLDLLFVAIMGVSPAAFAADLIDVSKLPSKAAQGAPGPVTLQAAVGAGGADELKAIRSTTLPNGKQVTRYEQFHNGVRVVGEAITEVKGPGKSVA MKKVSTLDLLFVAIMGVSPAAFAADLIDVSKLPSKAAQGAPGPVTLQAAVGAGGADELKAIRSTTLPNGKQVTRYEQFHNGVRVVGEAITEVKGPGKSVA

AMPP MN7 PST-01 IFO 3455

ARRSGHFVANIAADLPGSTTAAVSAEQVLAQAKSLKAQGRKTENDKVELVIRLGENNIAQLVYNVSYLIPGEGLSRPHFVIDAKTGEVLDQWEGLAHAEA 3 ARRSGHFVANIAADLPGSTTAAVSAEQVLAQAKSLKAQGRKTENDKVELVIRLGENNIAQLVYNVSYLIPGEGLSRPHFVIDAKTGEVLDQWEGLAHAEA AQRSGHFVANIAADLPGSTTAAVSAEQVLAQAKSLKAQGRKTENDKVELVIRLGENNIAQLVYNVSYLIPGEGLSRPHFVIDAKTGEVLDQWEGLAHAEA AQRSGHFVANIAADLPGSTTAAVSAEQVLAQAKSLKAQGRKTENDKVELVIRLGENNIAQLVYNVSYLIPGEGLSRPHFVIDAKTGEVLDQWEGLAHAEA

AMPP MN7 PST-01 IFO 3455

GGPGGNQKIGKYTYGSDYGPLIVNDRCEMDDGNVITVDMNGSTNDSKTTPFRFACPTNTYKQVNGAYSPLNDAHFFGGVVFKLYRDWFGTSPLTHKLYMK 103 GGPGGNQKIGKYTYGSDYGPLIVNDRCEMDDGNVITVDMNGSTNDSKTTPFRFACPTNTYKQVNGAYSPLNDAHFFGGVVFNLYRDWFGTSPLTHKLYMK GGPGGNQKIGKYTYGSDYGPLIVNDRCEMDDGNVITVDMNSSTDDSKTTPFRFACPTNTYKQVNGAYSPLNDAHFFGGVVFKLYRDWFGTSPLTHKLYMK GGPGGNQKIGKYTYGSDYGPLIVNDRCEMDDGNVITVDMNSSTDDSKTTPFRFACPTNTYKQVNGAYSPLNDAHFFGGVVFKLYRDWFGTSPLTHKLYMK

AMPP MN7 PST-01 IFO 3455

VHYGRSVENAFWDGTAMLFGDGATMFYPLVSLDVAAHEVSHGFTEQNSGLIYRGQSGGMNEAFSDMAGEAAEFYMRGKNDFLIGYDVKKGSGALRYMDQP 203 VHYGRSVENAYWDGTAMLFGDGATMFYPLVSLDVAAHEVSHGFTEQNSGLIYRGQSGGMNEAFSDMAGEAAEFYMRGKNDFLIGYDIKKGSGALRYMDQP VHYGRSVENAYWDGTAMLFGDGATMFYPLVSLDVAAHEVSHGFTEQNSGLIYRGQSGGMNEAFSDMAGEAAEFYMRGKNDFLIGYDIKKGSGALRYMDQP VHYGRSVENAYWDGTAMLFGDGATVFYPLVSLDVAAHEVSHGFTEQNSGLIYRGQSGGMNEAFSDMAGEAAEFYMRGKNDFLIGYDIKKGSGALRYMDQP

AMPP MN7 PST-01 IFO 3455

SRDGRSIDNASQYYNGIDVHHSSGVYNRAFYLLANSPGWDTRKAFEVFVRANRYYWTATSNYNSGACGVISSAQNRYYSAADVTRAFSTVGVTCPSAL 301 SRDGRSIDNASQYYNGIDVHHSSGVYNRAFYLLANSPGWDTRKAFEVFVDANRYYWTATSNYNSGACGVISSAQNRNYSAADVTRAFSTVGVTCPSAL SRDGRSIDNASQYYNGIDVHHSSGVYNRAFYLLANSPGWDTRKAFEVFVDANRYYWTATSNYNSGACGVIRSAQNRNYSAADVTRAFSTVGVTCPSAL SRDGRSIDNASQYYNGIDVHHSSGVYNRAFYLLANSPGWDTRKAFEVFVDANRYYWTATSNYNSGACGVIRSAQNRNYSAADVTRAFSTVGVTCPSAL

Mature

Fig. 5. Amino acid sequence alignment of AMPP elastase with those from P. aeruginosa MN7, P. aeruginosa PST-01, and P. aeruginosa IFO 3455. The first amino acid of the mature elastase, Ala, is counted as +1. X (highlighted character) shows amino acid changes in AMPP elastase with other metalloenzymes.

P. auriginosa elstases (Fig. 5). On the basis of those similarities, H140, H144, and E164 could perform the function of zinc ligands and carboxyl groups of D136, E172, E175, and D183. Furthermore, the carbonyl groups of L185 could engage calcium ions in the mature AMPP. Similarity, E141, Y155, and H’s-223, which form the active center in pseudolysin, could perform the same catalytic function in the case of AMPP. The classification analysis of the deduced amino acid sequence demonstrated that the mature protease was a zinc metalloproteinase of the M4 family. The alignment of the deduced amino acid sequence of lasB with those of known proteases revealed high homology with the extracellular zinc metalloproteases previously isolated and characterized from P. aeruginosa strains. Nevertheless, one amino acid in the propeptide and 5 aa (K85N, F114Y, V190I, R253D, and Y280N), 7 aa (G44S, N47D, F114Y, V190I, R253D, S274R, and Y280N), and 8 aa (G44S, N47D, F114Y, M128V, V190I, R253D, S274R, and Y280N) in the mature AMPP elastase were noted to differ from the MN7, PST-01, and IFO 3455 elastase residues, respectively. More pronounced differences were noted between the biochemical properties of the three proteases as compared to AMPP 12/80 ◦ C. In fact, MN7 [13], PST-01 [12], and IFO 3455 [11] elastases showed pH and temperature optima of 8/60 ◦ C, 8.5/55 ◦ C, and 8/50 ◦ C, respectively. The MN7 elastase [13] also showed a halflife time of 0.42 h at 70 ◦ C, which was much shorter than the 14 h half-life time displayed by AMPP. The optimum temperature of AMPP was 80 ◦ C, which is higher than the 55 ◦ C of PST-01 [12]. This variation may be attributed to differences between those enzymes in terms of seven amino acid residues, thus indicating the influential role played by G44, N47, F114, V190, R253, S274, and Y280 in determining AMPP thermoactivity. 3.13. Expression of the lasB gene in E. coli and characterization of the recombinant enzyme To express AMPP, the corresponding gene was cloned downstream of PT7 or Ptac promoters in pBJ100 and pBJ101, respectively, and then introduced in HB101 strain. No alkaline protease activity was detected in the periplasmic fraction neither in the intracellular fraction for all recombinant strains. Relatively high levels of specific activity of 3800 and 25,000 U/mg were, however, detected in the extracellular fractions of HB101/pBJ100

and HB101/pBJ101, respectively. Based on this study, the AMPP protease was most efficiently expressed with the construction of Ptac-lasB (pBJ101). The latter was, therefore, retained for the purification of the recombinant protease (rAMPP). Extracellular rAMPP was purified using the same strategy for the native enzyme from P. aeruginosa CTM50182. All the biochemical characteristics identified from rAMPP were almost similar to those of the original one. The large scale preparation of rAMPP as a biocatalyst for biotechnological application can, therefore, be easily performed. 4. Conclusions The extracellular solvent-stable alkaline metalloprotease (AMPP) from P. aeruginosa CTM50182 was purified and characterized. The nucleotide sequence of lasB gene and its flanking regions were determined. The lasB gene consisted of 1497 bp, encoding a pre-pro-protein of 498 aa. The AMPP mature elastase (301 aa) differed from P. aeruginosa MN7 elastase by 6 aa. Overall, the findings indicate that AMPP is endowed with a number of promising properties that might open new opportunities for the synthesis of non-aqueous peptides for use in several biotechnological applications. This would require further studies on the structure-functions relationship of the enzyme using site-directed mutagenesis and 3D structure modeling. Acknowledgments This work was funded by the Tunisian Ministry of Higher Education and Scientific Research (contract program LMB-CBS, grant no. LR10CBS04) and the Agence Universitaire de la Francophonie (AUF)/Regional Excellence Pole for Interuniversity Scientific Cooperation: “Environmental and Industrial Bioprocesses” contract no. 6313PS652 coordinated by Pr. Sami Sayadi (EBL-CBS). The authors wish to express their gratitude to Mr. Anis Akal (ISBS), Miss Houda Ouni (ISBS), Mr. Kamel Walha (SA-CBS), Mrs. Najla Masmoudi (SACBS), and Mr. Rafik Hmidi (SCCM-CBS) for their technical assistance. They are also grateful to Pr. Abdelmalek Badis (University of Saad Dahlab of Blida, Algeria) and Pr. Hafedh Belghith (LVBPPE-CBS) for their constructive discussions and valuable help during the preparation of this study. Special thanks are also due to Pr. Anouar Smaoui

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