A new lipase as a pharmaceutical target for battling with infections caused by Staphylococcus aureus- Gene cloning and biochemical characterization Aişe Ünlü1, Aziz Tanriseven1, İ. Yavuz Sezen2 and Ayhan Çelik1 1 Gebze Institute of Technology, Department of Chemistry, 41400, Gebze-Kocaeli, TURKEY 2 Gebze Institute of Technology, Department of Molecular Biology and Genetics, 41400, Gebze-Kocaeli, TURKEY

Correspondence Ayhan Çelik, Department of Chemistry, Gebze Institute of Technology, 41400, Gebze, Kocaeli, TURKEY Tel: +90 262 6053087 Fax: +90 262 6053005 Email: [email protected] or Aziz Tanrıseven Department of Chemistry, Gebze Institute of Technology, 41400, Gebze, Kocaeli, TURKEY Tel: +90 262 6053081 Fax: +90 262 6053005 Email: tanrı[email protected]

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/bab.1316. This article is protected by copyright. All rights reserved.

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Abstract Staphylococcus aureus lipases along with other cell wall associated proteins and enzymes (i.e. catalase, coagulase, protease, hyaluronidase and ß-lactamase) play important roles in the pathogenesis of S. aureus and important subject of drug targeting. The appearance of antibiotic-resistant types of pathogenic S. aureus (e. g. MRSA) is a worldwide medical problem. In the present work, a novel lipase from a newly isolated Methicillin-resistant S. aureus strain from a cow with subclinical mastitis was cloned and biochemically characterized. The mature part of the lipase was expressed in Escherichia coli, purified by nickel affinity chromatography. It displays a high lipase activity at pH 8.0 and 25 °C using pnitrophenyl palmitate and has a preference for medium-long chain substrates of p-nitrophenyl esters (pNPC10-C16). Furthermore, in search of inhibitors, the effect of farnesol on growth of S. aureus and lipase activity was also studied. Farnesol inhibites the growth of S. aureus and is a mixed-type inhibitor with Ki and Ki' values of 0.2 mmol l−1 and 1.2 mmol l−1 respectively. A lipase with known properties could not only serve as a template for developing inhibitors for S. aureus but also a valuable addition to enzyme toolbox of biocatalysis. The discovery of this lipase can be potentially important and could provide a new target for pharmaceutical intervention against S. aureus infection.

Keywords: lipases, subclinical mastitis, S. aureus, expression, farnesol, MRSA

Abbreviations: MRSA, Methicillin-resistant S. aureus; pNPP, p-nitrophenyl palmitate; pNP, p-nitrophenol

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Introduction Lipases (EC. 3.1.1.3. glycerol ester hydrolases) represent one of the most important classes of enzymes used in biotechnology, particularly useful for a wide variety of different biocatalytic applications involving hydrolysis, esterification, interesterification, acidolysis, alcolysis, and aminolysis reactions [1]. They have been utilised in food, dairy, detergent, agro-chemicals, and pharmaceutical industries [2]. Lipases are widely produced by all kingdoms of life including plant, animal and microorganisms.

Lipases from microbes, especially bacterial ones play a pivotal role not

only in commercial venture but in the lipid metabolism and in disease related cases.

The

latter is particularly important since it is linked with pathological activities of certain bacteria. Staphylococcal species, for instance, have been reported to produce extracellular lipases, which may involve in pathology related lipolytic activities [3-5]. What is more, fatty acid modifying enzyme (FAME) produced by staphylococcal species may work together with lipases for inactivation of bactericidal lipids produced by host organism, resulting in colonisation and invasion of pathogenic bacteria [6]. S. aureus, a facultative anaerobic Gram-positive coccus, is one such staphylococcal pathogen species, which causes both nosocomial (a patient under medical care) and communityacquired infections in population and is also accountable for diseases in animals [7]. Since its discovery by Sir Alexander Ogston dated back in 1880 [8], numerous studies have focused on the complex battery of virulence factors and regulators that allow for its swift transition between commensalism and pathogenic states, and escape from host immune defences [9]. While, around 20–30% of the human population has been reported to carry this ubiquitous organism asymptomatically [10], it causes a wide range of infections including skin and soft tissue infections (SSTI), mastitis, bone, joint and implants infections, pneumonia, and septicaemia [11]. In animals, S. aureus can be the cause of a number of diseases including the bovine mastitis, an inflammation of the mammary gland [12]. substantial economic consequence [13].

Mastitis in dairy cows is an infection of

Occurrence rates of mastitis over the course of

lactation can reach 100% in dairy herds with typical rates of 30–50% filed in several countries [14]. Ever since the introduction of β-lactamase–resistant antibiotics into clinical applications, Methicillin-Resistant S. aureus (MRSA) strains have surfaced globally as one of the main nosocomial pathogens; their occurrence in the community is rising, considerably. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/bab.1316. This article is protected by copyright. All rights reserved.

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The immune system of the host organism is defeated by S. aureus using several mechanisms including the action of S. aureus lipases, probably.

Fully understanding of these defeat

mechanisms of S. aureus, is crucial for developing strategies that could be useful in battling with the pathogen. As a part of the strategies, role of lipase in pathogenesis may present an opportunity for tackling S. aureus related diseases, in which a direct involvement of lipase in pathogenesis was reported [15]. The aim of the current study was to identify and biochemically characterise a new surface enzyme such as a lipase from S. aureus. By doing so, we will gather information for possible understanding the role of lipases in pathogenesis. Furthermore, lipases are one of highly valued enzyme classes for biocatalystic applications for the generation of fine chemicals.

We have characterised the mature form of a lipase from a local isolate of S. aureus found in dairy cows with subclinical mastitis. Its cloning, recombinant protein production together with biochemical characterization with regard to substrate specificity, pH, and temperature optimum as well as farnesol inhibition studies have been performed. Even though the information gathered about this new lipase will be partial to connect the pathogenicity of S. aureus and role of its lipases, which we are aware of, very limited studies have been carried out only for lipolitic activities. There is no work reported for characterization of a recombinant lipase from a cow with subclinical mastitis. This work will, therefore, serve as a foundation for further studies (which are underway) in this subject.

Materials and methods Organism and growth conditions S. aureus from subclinical mastitis was grown in nutrient broth.

E. coli TOP10 and

BL21(DE3) (both from Invitrogen) have been used as host for cloning and expression of the Salip35 gene, respectively. The plasmid employed for both cloning and expression was pET14b (Novagen).

All organisms were cultivated in LB broth or on LB-agar and

supplemented with 100 g/ml of ampicillin if necessary (for plasmid maintenance). Isolation of mastitic Methicillin-resistant S. aureus and selection of lipolytic strain In order to isolation of S. aureus, milk samples from cows diagnosed as subclinical mastitis were collected aseptically. Before sampling, teat ends were disinfected with cotton swabs soaked in 70% alcohol and the first streams of milk were discarded. Sterile tubes were filled with samples about 8-10 ml by the veterinarian and transported in an icebox to the

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Laboratory of Microbiology, Molecular Biology Department at GIT. After serial dilution of milk samples with PBS, it was plated onto 7% sheep blood agar and nutrient agar plates. The plates were incubated at 37 ºC for 48 h under aerobic conditions.

The interested

colonies were identified using the classical characteristics (colony morphology, haemolysis, gram stain, catalase, coagulase) and 16S rRNA gene sequencing [8, 16-18]. Methicillin resistance test was carried out using the disc diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) [19]. Methicillin resistance was determined by cefoxitin (30 µg), oxacillin (1 µg) and methicillin (5 µg) disks. S. aureus overnight culture suspension was adjusted to The McFarland 0.5 standard and plated onto Muller-Hinton agar. Antibiotic disks were applied using sterile forceps. The plates were incubated at 35 ˚C for 24 h and zone diameters were measured. Inhibition zone diameters ≤ 21 mm indicated cefoxitin resistance, ≤ 10 mm indicated oxacillin resistance and ≤ 9 mm indicated methicillin resistance. Susceptibility of the strain to methicillin resistance was reconfirmed using the cefoxitin E-test (AB Biodisk, Solna, Sweden) according to the recommendations of the CLSI and the manufacturer. The CLSI values set for MRSA are as follows: sensitive ≤ 8 μg/ml, intermediate = 16 μg/ml, and resistant ≥ 32 μg/ml. Lipase producing S. aureus strain was screened by the following method [20]. Nutrient broth medium was supplemented with 1.5% agar and 2.5% olive oil and autoclaved. After sterilization, rhodamine B (0.1 mg ml-1) was added and thoroughly blended prior to pouring into petri dishes. Then, 10 μl of crude extracts from overnight culture were applied on plate and incubated at 37 °C for 24 h. Putative lipase producing strains were identified as orange halos around colonies under UV light at 350 nm. Cloning of Salip35 gene S. aureus 35 genomic DNA was obtained using phenol/chloroform extraction method with minor modifications according to a well established procedure [21]. Briefly, bacterial pellets were initially re-suspended in a lysis solution (250 μl, containing lysostaphin (200 μg ml-1), Tris-HCl (20 mmol l−1, pH 8.0), 2 mmol l−1 EDTA and 1.2% (w/v) Triton X-100). Purified genomic DNA (0. 4 mg ml-1) was diluted at a concentration of 3.97 ng μl-1 in TE buffer (10 mmol l−1 Tris-HCl, pH 8.0, 1 mmol l−1 EDTA). A 1188 bp portion of Salip35 gene was amplified from S. aureus genomic DNA using the following degenerated forward and reverse primers: FP (5'-GCGCGGCATATGGCGAATCAAGTACAACCAC-3') and RP (5'GCCGGCCTCGAGTTAACTTGCTTTCAATTG-3'), respectively.

The PCR reaction

mixture consisted of 2× Tag Master Mix™ (25 μl), forward and reverse primers (4 μl, 100 pmol μl-1), genomic DNA (2 μl, 3.9 ng μl-1) and double distilled water (19 μl) in a reaction This article is protected by copyright. All rights reserved.

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volume of 50 μl. The PCR reaction, carried out using Veriti®96 well Thermal CyclerTM (Applied Biosystems), involved in an initial denaturation step of 5 min at 95 °C, and then subjected to 29 cycles of amplification in two steps (95 °C, 1 min; 50 °C, 1 min; 68 °C, 2 min for 5 cycle and 95 °C, 1 min; 55 °C, 1 min; 68 °C, 2 min for 24 cycles) followed by a final extension at 68 °C for 10 min. The resulting PCR product was gel purified using QIAprep Kit (Qiagen, ATQ Biotechnology, Ankara, TR). The PCR product and pET14b vector were digested with NdeI/XhoI restriction enzymes and gel purified using QIAprep Kit (Qiagen). Ligation was achieved using T4 DNA ligases according to the manufacturer instructions (NEB). Ligation mixture (5 μl) was transferred into E. coli TOP10 (50 μl) competent cell lines according to the manufacturer instructions. Overnight cultures were prepared each of resulting colonies on LB-agar medium containing 100 μg ml-1 ampicillin. The plasmid containing Salip35 gene (pSalip35) was purified from transformants using a commercial plasmid isolation kit (Roche High Pure™) and was sequenced on both strands (RefGen, Ankara, TR). The Salip35 plasmid was then used to transform E. coli BL21(DE3) (Invitrogen) competent cells for expression purposes. Sequence analysis Multiple sequence alignment was performed using CLUSTRAL W program with the default parameters [22]. The nucleotide sequence determined in this study has been deposited in the GenBank database under access number KJ789388. Expression and purification of Salip35 For protein expression, E. coli BL21(DE3) containing the expression construct of pSalip35 was grown in LB medium containing 100 μg ml-1 of ampicillin at 37 °C in a orbital shaker at 180 rpm. After induction with 0.1 mmol l−1 IPTG at an optical density (OD600) of 0.5, growth was continued for up to 3 h at 30 °C before harvesting. The cell pellet was obtained via centrifugation at 8000 xg for 10 min and stored at −20 °C until use. Aliquots were withdrawn at regular time points. Expression was assessed by comparing the banding pattern obtained by SDS–PAGE analysis of whole cell extracts with that of a negative control (i. e., Escherichia coli BL21(DE3) containing pET14b plasmid only) For the auto induction, the procedure was adapted from Studier [23]. Briefly, a single colony was used to inoculate a 5 ml of starter culture for 500 ml of auto induction medium containing 100 μg ml-1 of ampicillin, which was incubated at 30 °C and 180 rpm for at least 16 h, until stationary phase was reached. The cell pellet was obtained via centrifugation at 8000 xg for 10 min and stored at −20 °C until use. Approximately, every gram of wet cell paste from either induction procedures was suspended in 4 ml of ice-cold Buffer A (50 This article is protected by copyright. All rights reserved.

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mmol l−1 Tris-HCl, pH 7.0, 0.5 mol l−1 NaCl, 10 mmol l−1 imidazole) containing 0.1 mmol l−1 PMSF. The cells were incubated with 80 μl of lysozyme (10 mg ml-1) for 30 min in ice at an angle of 45° for mixing readily, before disruption by sonication using a sonicator (Sonopuls HD2200, Bandelin Electronics, Berlin, Germany) fitted with a 3-mm diameter probe. The cell suspension was kept on ice and sonicated with a 15 s burst followed by a 45 s cooling for six times. The cell debris was separated from supernatant by centrifugation (8000 xg, 1 h, 4 °C) in order to obtain the crude extract. The cell-free extract was carefully removed and the clarified extract was then loaded onto a nickel HiTrap™ column (1 ml, Amersham Biosciences), equilibrated in Buffer A (minor modification with changing 200 mmol l−1 NaCl), at a flow rate of 1 ml min-1. Column chromatography was performed manually at 4 °C. The column was washed with several times with Buffer A (10 column volumes) to remove unbound material. The majority of contaminating proteins were then removed by washing with 20 mmol l−1 imidazole in Buffer A (10 column volumes) before elution of the recombinant his-tagged Salip35 by 200 mmol l−1 and 400 mmol l−1 imidazole in Buffer A.

The sample volume was reduced using a Ultracel TM ultrafiltration discs

regenerated cellulose membranes (NMWL 30 kDa) (Millipore, Germany). The buffer was exchanged to Tris-HCl (20 mmol l−1, pH 7.0) using a PD-10 desalting column (Amersham Biosciences) at 4 °C. Glycerol was added to a final concentration of 50% (v/v) and the sample stored at −20 °C until its use. SDS–PAGE analysis and protein determination SDS-PAGE electrophoresis was performed according to the method of Laemmli [24] in a Mini-PROTEAN II electrophoresis cell (Bio-Rad) using 4% (w/v) stacking and 12% (w/v) separating gels to determine the purity and approximate molecular mass of the enzyme. The molecular mass of the enzyme was approximated using β-galactosidase (116 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), lactate dehydrogenase (35.5 kDa), restriction endonuclease Bsp 981 (25 kDa), β -lactoglobulin (18.4 kDa), and lysozyme (14.4 kDa) as molecular mass standards.

Proteins on the polyacrylamide gel were stained with

0.2% (w/v) Coomassie brilliant blue R-250. Protein concentration was measured according to the method of Bradford assay [25], using bovine serum albumin as standards. Spectroscopic determination of the protein was also carried out for confirmation using the calculated (based on protein sequence) extinction coefficient value of 69790 mol−1 l cm−1 at 280 nm [26]. Protein mass spectrometry of Salip35

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Mass spectrometry of the recombinant Salip35 was performed by MALDI-TOF MS using MicroFlex instruments (Bruker Daltonics, Billerica, MA). A protein sample (50 μl, 4 mg ml-1) was concentrated by precipitation using freshly prepared trichloroacetic acid (20% (v/v), 500 μl). The resulting pellet was washed twice with cold acetone (250 μl), and centrifuged at 10000 xg for 10 min. The pellet was air dried, and re-dissolved in 40 μl of formic acid/ddH2O/isopropanol (1:3:2) mixture. The resulting solution was mixed with 2× equivalent volume of matrix (saturated solution of sinapinic acid in 50% (v/v) isopropanol), and applied on a sample target surface. The sample was air dried at room temperature to crystallize. The MS was operated in positive ion mode (20 kV, 50 shots) and calibrated with the Bruker’s protein standards II kit including cytochrome c (12 kDa), myoglobin (16 kDa), trypsinogen (23 kDa), protein A (44 kDa) and bovine serum albumin (66 kDa). Lipase activity measurements Enzyme activity was assayed using p-nitrophenylpalmitate (pNPP) as the substrate. Isopropanol (10 ml) containing p-nitrophenylpalmitate (38 mg) was mixed with 90 ml of sodium phosphate buffer (50 mmol l−1, pH 8. 0) containing 0.4% (w/v) Triton X-100 and 0.1% (w/v) gum arabic. A 990 µl amount of this freshly prepared substrate solution was mixed with lipase (10 µl) at 25 °C and the absorbance changes at 410 nm were followed for 5 minutes using UV-Vis spectrophotometer (Shimadzu UV-3600) equipped with a circulating water bath (VWR model 1162A). Absorption coefficient of 14.93 mmol−1 l cm-1, determined experimentally, was used for all calculation. One unit of activity was defined as the amount of enzyme that releases 1 μmol of p-nitrophenol per minute at 25 °C. Characterization of Salip35 lipase Effects of pH and temperature on enzyme activity and thermal stability Optimal pH and temperature were determined by individually changing the conditions of activity assays [pH from 6.5 to 10.0 using following buffer solutions: sodium phosphate (50 mmol l−1, pH 6.5–8.0), Tris-HCl (50 mmol l−1, pH 7.4–9.0) and sodium bicarbonate (50 mmol l−1, pH 9.2–10.0); temperature from 5 to 35 °C]. The molar extinction coefficient of p-NP was determined at each pH. Also, an effect of buffer ionic strength was determined using NaCl ranging from 0.05 to 2 mol l−1. In thermal stability determinations, the enzyme was kept at temperatures (25- 55 °C) for 0– 60 min and then the remaining activity was measured using standard assay method. Substrate specificity and kinetic parameters Substrate specificity was determined using p-nitrophenyl esters (pNPC2-16) as substrates under the standard assay conditions. The kinetic parameters of Salip35 were determined This article is protected by copyright. All rights reserved.

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using different concentrations (0.05-1 mmol l−1) of

p-nitrophenyl esters.

Initial rate

determinations were carried out at least three times and kinetic parameters (kcat and KM) were determined by a fit of averaged data at each concentration to the Michaelis-Menten equation using a nonlinear regression analysis program (Origin 7, OriginLab, Northampton, MA).

Effect of farnesol on growth of S. aureus and recombinant lipase activity Effect of farnesol on growth of S. aureus “Broth dilution with shaking” (BDS) method was used in the experiment [27].

Farnesol

solution in methanol (1 ml, 5-300 µmol l−1) was added to Brain Heart Infusion broth (100 ml) in Duran bottles to which an aliquot of an overnight culture of S. aureus (approximately 1 x 105 colony-forming units per ml) were supplemented. Each culture was incubated, with shaking at 40 rpm, at 37 °C for 24 h.

The farnesol inhibitions were determined by

measuring the optical density at 600 nm (OD600). The delay in proliferation (DP) was calculated from a comparison with the growth curve generated from a control culture, in which farnesol is not present. Nutrient agar plates containing Tween 80 (the oleic acid monoester of polyoxyethylene sorbitan) was used to confirm lipase activity during growing of S. aureus 35. (Fig S3). S. aureus 35 strain was cultivated on nutrient agar plates which were supplemented with Tween 80 (1% ) and farnesol (sub-MIC 4.54 mmol l−1) at 37 °C for 48 h. The method is based on the appearance of a zone of opacity, due to the precipitation as the calcium salt of the fatty acids released by hydrolysis of tweens. After incubation on nutrient agar supplemented with Tween 80, the presence or absence of opalescent zones around the colonies were recorded.

Effect of farnesol on recombinant lipase activity The inhibition kinetic parameters of Salip35 were determined using different concentrations pNPP (0.1-0.5 mmol l−1) and farnesol (0.57-1.14 mmol l−1). pNPP and farnesol solutions were prepared in dimethyl sulfoxide. Briefly, 10 ml of DMSO containing pnitrophenylpalmitate (0.5-10 mmol l−1) was mixed with 90 ml of sodium phosphate buffer (50 mmol l−1, pH 8.0) containing Triton X-100 (0.4%), gum arabic (0.1%), and farnesol (0.57-1.14 mmol l−1). The mixture (990 µl) was pre-incubated at 25 °C for 1 min, followed by the addition of Salip35 lipase (10 μl) and inverted thoroughly. The absorbance changes at 410 nm were followed for 2.0 minutes. The kinetic parameters (Km, Vmax, Ki and Ki') were determined by employing the Lineweaver–Burk plot.

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Results Cloning of Salip35 gene and recombinant enzyme production Initial screening of lipase activity produced by S. aureus strain was carried out using a plate assay in a medium containing olive oil and the fluorescent dye, Rhodamine B [20]. Lipase producing strains produced orange halos around colonies under UV light at 350 nm. The strain produced the largest halo, numbered as S. aureus 35, was used for cloning a gene product for mature part of S. aureus lipase (Fig. S1). The genomic DNA from S. aureus strain was used with the degenerative primers, based on the sequence of S. xylous lipase [28] for amplification of the mature region of Salip35 gene, which was subsequently inserted into pET14b by aid of NdeI/XhoI restriction enzymes followed by ligation using T4 DNA ligase. The positive colonies with a gene insert in the plasmids were identified by digestion of the plasmids with XhoI and using colony PCR, followed by agarose gel analysis and sequencing. This construct contains the complete lipase coding region and N-terminal His-tag together with a thrombin cleavage site. The resulting recombinant plasmid was named as pSalip35. E. coli BL21(DE3) strain was transformed with the expression plasmid (pSalip35) to express the recombinant lipase with a 6xHis tag. For the recombinant enzyme production, the best condition was found to be 3 h of growth at 30 °C followed by induction using 0.1 mmol l−1 IPTG in the LB-medium. Auto induction procedure [23] with a small alteration has been utilised for the regular protein production in E. coli BL21(DE3) in a cost effective manner. The cell-free extract was examined by SDSPAGE. A protein of the expected size (~ approximately 45 kDa) was readily noticed on a Coomassie-stained gel (Fig. 1). The recombinant lipase was then purified to homogeneity from cell lysates by nickel-affinity chromatography in a HiTrap™ column with a gradient of imidazole solution in a stepwise approach ranging from 20 to 400 mmol l−1. A single step purification protocol yields 60-70 mg protein from a liter culture (Studier media) with over 90% purity based on SDS-PAGE analysis (Fig. 1, lane 8 and 9). The Salip35 exhibits a single band corresponding to a molecular mass of about 45 kDa. The deduced product of S. aureus Lip without N-terminus modification is a protein of 391 amino acid residues, 43734.1 Da, and pI of 8.43. The addition of 6xHis tag raised the amino acid residues to 412 (46028.6 Da) and pI of 8.68. [Insert Fig. 1 here.] Protein mass spectrometry of Salip35

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The molecular mass of the mature Salip35 (single charged) was determined by MALDI-TOF spectrometer and found to be 46246 Da (Fig. 2). The calculated mass of 6xHis tagged Salip35 from its amino acid sequences is 46028 Da. The measured and calculated masses are in agreement with each other and the difference in masses is well within the instrument’s resolution power and measurements errors. [Insert Fig. 2 here.] Characterization of Salip35 lipase Effects of pH and temperature on enzyme activity and thermal stability The activity-pH profile showed an optimum at pH 8.0 in sodium phosphate (50 mmol l−1) and pH 8.5 in Tris-HCl (50 mmol l−1) buffer solutions for the hydrolysis of pNPP at 25 °C (Fig. 3). The activity-temperature profile showed maximum specific activity of 35.29 U mg1

at 25 °C (Fig. 4A). Upon increasing temperature, the enzyme activity is decreased sharply

and only 53% of maximum activity was observed at 30 °C. The optimal temperature is 25 °C, indicating that the enzyme is not thermostable. The thermostability of the enzyme was determined by incubating the enzyme solution at different temperatures (25-55 ºC) for 0–60 min and determining the remaining activity using standard assay method (Fig. 4B). The half life of the purified lipase was 2 and 1 min at 45 °C and 50 °C, respectively. The enzyme was completely inactivated at 55 °C within 5 min. [Insert Fig. 3 here.] [Insert Fig. 4A and 4B here.] Substrate specificity and kinetic parameters Substrate specificities were assessed by testing the enzymatic activity against p-nitrophenyl alkanoate esters of varying alkyl chain lengths (C2-16).

Kinetic parameters for the

recombinant S. aureus lipase (Salip35) were determined by measuring rates of hydrolysis of substrates. Maximal velocity (Vmax), affinity constant (KM), turnover number (kcat), and catalytic efficiency (kcat/KM) were determined (Table 1). The kinetic analysis of Salip35 performed on standard assay substrates has produced a hyperbolic plot corroborating the Michaelis–Menten behavior of the enzyme. The high affinity of the recombinant enzyme for pNPM (36.85 U mg-1) is reflected in the relatively low KM value. Activity was decreased with decreasing chain length to only 1.58 U mg-1 with p-nitrophenylacetate (C2) as a substrate. The highest hydrolysis rates were obtained with pNP-myristate (C14) and pNPpalmitate (C16), indicating the enzyme’s preference for medium-long-size acyl chain lengths.

The specificity of Salip35 towards p-nitrophenyl esters of longer fatty acids

indicates that it is a true lipase. This article is protected by copyright. All rights reserved.

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[Insert Table 1 here.] Effects of farnesol on S. aureus cells and lipase We have investigated the antimicrobial activity of farnesol on S. aureus (isolate 35) growth and the inhibitory effect of farnesol on lipase activity. The effect of farnesol on S. aureus (isolate 35) growth was evaluated, in which growth assays of S. aureus cells incubated in the presence of farnesol were carried out by measuring the optical density of the culture (Figure 5A). It was observed that farnesol (50 μmol l−1 to 300 μmol l−1) strongly inhibited S. aureus growth. To compare antibacterial activities of farnesol, the delay in proliferation (DP) was determined [29]. DP was defined as the difference between the experimental and control cultures in terms of the time required to reach an OD600 of 0.9. When the concentration of farnesol was (50 μmol l−1), DP was calculated as 13.1 h. The proliferation of the cells was totally inhibited by farnesol at concentration of 300 μmol l−1. The inhibitory effect of farnesol on lipase activity was also evaluated. The mode of farnesol inhibitory action against enzyme was determined using Lineweaver-Burk plots (Figure 5B).

It was found

that farnesol is a mixed-type inhibitor for the lipase with Ki and Ki' values of 0.2 mmol l−1 and 1.2 mmol l−1 respectively. [Insert Figure 5A and 5B here.] Discussion Surface proteins of invading bacteria play a pivotal role in the pathogenesis of diseases. Such proteins expressed in various size and shapes with many distinct functionalities engage with organisms invaded in sequential manner that involves bacterial adherence, colonisation, and internalisation.

Like many other pathogens, staphylococcal species are ones of

pathogens and remained such major health concerns as pustules, furuncles, fatal infections including endocarditis, osteomyelitis, and septic shock syndrome [30]. S. aureus causes disease by two major mechanisms, which are invasion and inflammation. During the bacterium-host cell interaction, bacterial surface proteins play a key role in the pathogenesis of disease. They have functions in the bacterial adherence, colonization and internalization.

S. aureus produces a wide range of

virulence factors that promote in evading the host’s immune response including cell wall associated proteins and enzymes [31]. Furthermore, exotoxins produced by S. aureus are responsible for damaging host tissue and promoting dissemination. A number of membrane damaging toxins and superantigen toxins can result in tissue damage and symptoms of septic shocks secreted toxins, respectively. The main

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function of these proteins may be the conversion of host tissues into nutrients needed for bacterial growth, to invade and destroy host tissues and metastasize to other locations during infections [3, 15, 32]. What is more, a third virulence mechanism that is important in certain infections is the ability to form biofilm [33]. The surface enzymes of staphylococci are generally divided into two categories, enzymes having potential virulence property (catalase, coagulase, protease, lipase, hyaluronidase and β-lactamase) and enzymes possessing with no known virulence property (lysostaphin, nuclease, and phosphatase). One of the enzymes with potential virulence property is the surface lipase.

Although the

role of lipase is not fully understood in staphylococcal pathogenicity, several reports have indicated that they are involved in the release of free fatty acids (octadecanoic acid) in blood plasma, skin colonisation and related diseases [34, 35].

The pathogenic role of lipases

during an S. aureus infection remains imprecise; however, it has been shown that S. aureus strains isolated from deep infections generally produce elevated amounts of lipase than those derived from superficial locations and it is suggested that they improve the adherence and colonization of pathogenic bacteria by degrading lipids on the external surface of the host, after that the liberated free fatty acids function as nutrients for the persistence of colonized bacteria and free fatty acids, the end products of lipolytic activity, are known to harm several immune system functions thus might have an indirect influence on their pathogenic potential [4, 15, 36, 37]. The most important data of that secreted lipases are took part in pathogenesis is the discovery of anti-lipase IgG antibodies in patients experiencing of infections by S. aureus, enriching the virulent potential of extracellular lipases [38, 39]. A recent report, on the other hand, has findings which contradict with role of lipases in S. aureus (USA300) [40]. In this work, the investigators has mutated strains of CA-MRSA so that it could not make SAL2 lipase, and evaluated how the mutant and the wild-type responded to triglycerides. The mutants cultivated well in the presence of triglycerides. But, due to the activity of SAL2 lipase in wild-type, it produced high concentrations of fatty acids from triglycerides, which inhibited bacterial growth, in turn. They have concluded that efforts are focussed toward finding out why these bacteria have developed a growthinhibiting system of liberating large amounts of fatty acids from skin secretions. In short, it is suggested that the evolutionary preservation of huge production of SAL2 lipase's activity must be aiding the bacterium to colonize and continue to live on human This article is protected by copyright. All rights reserved.

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skin. The difficulty, however, is the determining how it works, needing to study persistence and virulence not only on the skin, but in abscesses underneath the skin, and in bacteremia. Due to their biological significance in battle with the bacterial pathogens as well as biotechnological significances, biochemical characterization of a new lipase from a novel Methicillin-resistant S. aureus, named as Salip35, was reported. enzyme as a recombinant lipase was produced.

The mature form of the

The enzyme was characterised

biochemically with respect to pH, optimal temperature, substrate specificity. The inhibition studies with Salip35 using farnesol were carried out in order to find out the potential connection between lipase inhibition and the growth of S. aureus. Since recent studies have shown that farnesol acts as a quorum-sensing molecule and has showed to inhibit the growth of some microorganisms, [41], including the coagulase positive Staphylococcus aureus [11, 42] and the coagulase negative Staphyloccous epidermidis [43]. The hydrophobic character of farnesol favours its accumulation in microbial cell membranes, resulting in interference with membrane integrity. (Farnesol works as reduction of cell membrane integrity.) Also, Kaneko and co-workers [44] showed that farnesol affects the mevalonate pathway, which is related with cell membrane preservation and protein anchoring, among other cell functions. Farnesol causes at high concentration, extensive disruption of the cell membrane heading to instant cell death. Thus, farnesol seems not only serve as an inhibitor for Salip35, but also play role in many other cell functions, as mentioned, leading to cell death. The determined properties may provide new insight information about the lipase with potential virulence property in the battle against pathogenicity caused by S. aureus. In the production of novel lipase, the degenerative primers based on the sequence of S. xylous [28] was used, resulting in a gene product for mature region of Salip35. This gene, inserted into pET14b, was used for creating expression plasmid, from which the recombinant, a hexa-histidine tagged lipase was produced. A search using Protein BLAST database showed high scoring similarities (98.98% identity and 99.49% similarity) between amino acid sequences of newly produced lipase from S. aureus 35 and lipases from S. aureus NCTC8530 [45] and S. xylosus [28]. The phylogenetic analysis suggested SaLip35 to be relatively close to lipases from S. aureus NCTC8530 [45] and S. xylosus [28] ( Figure 6). [Insert Figure 6 here.]

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14

The purified enzyme showed maximal activity at pH range of 8.0-8.5 for the hydrolysis of pNPP. The closely related lipase from S. xylosus lipase also presented highest activity at alkaline pH of 8.2 [28]. Lipases generally exert their highest hydrolysis activities at alkaline pH; Salip35 is no different from them.

The optimal pH value for lipases from S.

haemolyticus L62 [46], S. simulans [47] and S. aureus B56 [48] is 8.5. However, it is pH 8.2 for lipase from S. xylosus [28]. Although S. aureus NCTC8530 [45] and Salip35 have high homology in their amino acid sequences, the optimal pH (6.0) for lipase from S. aureus NCTC8530 was quite different from that of Salip35 (8.0-8.5). The activity of Salip35 depended not only on the pH, but also on the type of buffer used. The hydrolysis rate at pH 8.0 was 6.0% higher in phosphate buffer than in Tris-HCl buffer. The activation of lipases by phosphate buffer has already been observed previously [49]. The hydrolysis activity of Salip35 decreases at acidic pH values and undergoes a complete inactivation at pH 5.5 (data not shown). Similarly, decreases in activities at acidic pH values were reported for lipases from S. haemolyticus L62 [46] and S. warneri [50].

Also, the effect of buffer ionic

strength on hydrolytic activity was studied. Salip35 showed that the optimum activity was attained at 0.5 mol l−1 NaCl in Tris-HCl and sodium phosphate buffers (Figure S2). Salip35 has a low optimal temperature (25 °C) which is similar to that of lipase from S. warneri [50].

However, most of the lipases from staphylococci have higher optimal

temperatures (30-55 °C) [51]. Substrate specificity of Salip35 was evaluated by testing hydrolytic activity using pnitrophenyl alkanoate esters of varying alkyl chain lengths (pNPC2-16). Staphylococcal lipases have different substrate chain length selectivity; both S. aureus NCTC8530 [45] and S. epidermidis [52] hydrolyse triacylglycerols but they have a strong preference for shortchain substrates. However, S. hyicus [53], S. simulans [47] and S. xylosus [28] lipases hydrolyse triacylglycerols or p-nitrophenyl esters equally well irrespective of their chain lengths. Salip35 hydrolyse p-nitrophenyl esters almost irrespective of their chain lengths. However, it has the highest activity for p-nitrophenyl myristate (36.85 U mg-1), followed by p-nitrophenyl palmitate (35.29 U mg-1), indicating the enzyme’s preference for mediumlong-size acyl chain lengths. The lowest activity was observed when p-nitrophenyl acetate used as substrate (1.58 U mg-1). In the present work, effect of farnesol on a newly isolated Methicillin-resistant S. aureus and the novel lipase was also studied. It was found that farnesol was a mixed type inhibitor (Ki and Ki' values of 0.2 mmol l−1 and 1.2 mmol l−1, respectively) for the enzyme.

In the

literature, there is only one work on farnesol inhibition of a lipase from S. aureus [54]. This article is protected by copyright. All rights reserved.

15

Farnesol was found to be a competitive inhibitor with a Ki of 0.4 mmol l−1 based on unconvincing data. Conclusion In this work, a novel lipase from a newly isolated Methicillin-resistant S. aureus strain from a cow with subclinical mastitis was cloned and biochemically characterized. In the fight with S. aureus pathogenicity, it appears that the role of lipase is important. Developing effective inhibitors for this enzyme could help to tackle S. aureus infections. The discovery of this lipase was potentially important and could provide a new target for pharmaceutical intervention against Staphylococcus aureus infection. Cloning and characterization of this novel lipase could provide a subject for fighting with S. aureus pathogenesis in future. Acknowledgments We would like to thank Dr. Ali Turkan for his help with protein mass spectrometry. We also would like to thank Dr. Mine Gül Şeker (GIT) and Dr. Nezahar Gürler (IU) the identification of the strain. . Funding This work was funded by Gebze Institute of Technology [Grant Number: 2008A10].

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References [1] Joseph, B., Ramteke, P. W., Thomas, G. (2008) Cold active microbial lipases: Some hot issues and recent developments. Biotechnol Adv 26, 457-470. [2] Gandhi, N. N. (1997) Applications of lipase. J Am Oil Chem Soc 74, 621-634. [3] Bernardo, K., Fleer, S., Pakulat, N., Krut, O., Hunger, F., Kronke, M. (2002) Identification of Staphylococcus aureus exotoxins by combined sodium dodecyl sulfate gel electrophoresis and matrix-assisted laser desorption/ ionization-time of flight mass spectrometry. Proteomics 2, 740-746. [4] Rollof, J., Normark, S. (1992) In vivo processing of Staphylococcus aureus lipase. J Bacteriol 174, 1844-1847. [5] Rollof, J., Braconier, J. H., Soderstrom, C., Nilsson-Ehle, P. (1988) Interference of Staphylococcus aureus lipase with human granulocyte function. Eur J Clin Microbiol Infect Dis 7, 505-510. [6] Sibbald, M. J., Ziebandt, A. K., Engelmann, S., Hecker, M., de Jong, A., Harmsen, H. J., Raangs, G. C., Stokroos, I., Arends, J. P., Dubois, J. Y., van Dijl, J. M. (2006) Mapping the pathways to staphylococcal pathogenesis by comparative secretomics. Microbiology and molecular biology reviews : MMBR 70, 755-788. [7] Gordon, R. J., Lowy, F. D. (2008) Pathogenesis of methicillin-resistant Staphylococcus aureus infection. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 46 Suppl 5, S350-359. [8] Schleifer, F. G. T. B. K.-H., in Eugene Rosenberg, E. S., Fabiano Thompson, Stephen Lory, Edward F. DeLong Ed. (2006) The Genera Staphylococcus and Macrococcus, pp. 5– 75. [9] Falugi, F., Kim, H. K., Missiakas, D. M., Schneewind, O. (2013) Role of Protein A in the Evasion of Host Adaptive Immune Responses by Staphylococcus aureus. mBio 4. [10] Sydnor, E. R., Perl, T. M. (2011) Hospital epidemiology and infection control in acute-care settings. Clin Microbiol Rev 24, 141-173. [11] Jabra-Rizk, M. A., Meiller, T. F., James, C. E., Shirtliff, M. E. (2006) Effect of farnesol on Staphylococcus aureus biofilm formation and antimicrobial susceptibility. Antimicrob Agents Chemother 50, 1463-1469. [12] Le Marechal, C., Seyffert, N., Jardin, J., Hernandez, D., Jan, G., Rault, L., Azevedo, V., Francois, P., Schrenzel, J., van de Guchte, M., Even, S., Berkova, N., Thiery, R., Fitzgerald, J. R., Vautor, E., Le Loir, Y. (2011) Molecular basis of virulence in Staphylococcus aureus mastitis. Plos One 6, e27354. [13] Leitner, G., Krifucks, O., Glickman, A., Younis, A., Saran, A. (2003) Staphylococcus aureus strains isolated from bovine mastitis: virulence, antibody production and protection from challenge in a mouse model. FEMS Immunol Med Microbiol 35, 99-106. [14] Cristina Bogni, L. O., Claudia Raspanti, José Giraudo, Alejandro Larriestra, Elina Reinoso, Mirta Lasagno, Mirian Ferrari, Edith Ducrós, Cecilia Frigerio, Susana Bettera,Matías Pellegrino, Ignacio Frola, Silvana Dieser and Claudina Vissio, in MéndezVilas, A. Ed. (2011) War against mastitis: Current concepts on controlling bovine mastitis pathogens [15] Hu, C., Xiong, N., Zhang, Y., Rayner, S., Chen, S. (2012) Functional characterization of lipase in the pathogenesis of Staphylococcus aureus. Biochem Biophys Res Commun 419, 617-620. [16] Turkyılmaz S, Y. O., Oryasın E, Kaynarca S, Bozdogan B (2010) Molecular Identification of Bacteria Isolated from Dairy Herds with Mastitis. Kafkas Univ Vet Fak Derg 16 1025-1032. [17] Jain, A., Agarwal, A. (2009) Biofilm production, a marker of pathogenic potential of colonizing and commensal staphylococci. J Microbiol Methods 76, 88-92. This article is protected by copyright. All rights reserved.

17

[18] Kloos, W. E., Bannerman,T.L., in Murry, R. P., Baron,E.J., Pfaller,M.A., Tenover,M.C., Yolken, R.H. Ed. (1999) Staphylococcus and Micrococcus, American society of Microbiology, 7th ed, pp. 262–282. . [19] CLSI, (2007) Performance standards for antimicrobial disc susceptibility tests; approved standard-9th ed. , Wayne. [20] Kouker, G., Jaeger, K. E. (1987) Specific and sensitive plate assay for bacterial lipases. Appl Environ Microbiol 53, 211-213. [21] Sambrook, J., Russell, D. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press. [22] Thompson, J. D., Higgins, D. G., Gibson, T. J. (1994) Clustal-W - Improving the Sensitivity of Progressive Multiple Sequence Alignment through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice. Nucleic Acids Res 22, 46734680. [23] Studier, F. W. (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41, 207-234. [24] Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. [25] Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-254. [26] Gill, S. C., von Hippel, P. H. (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182, 319-326. [27] Inoue, Y., Shiraishi, A., Hada, T., Hamashima, H., Shimada, J. (2004) The Antibacterial Effects of Myrcene on Staphylococcus aureus and Its Role in the Essential Oil of the Tea Tree (Melaleuca alternifolia). Natural medicines 58, 10-14. [28] Mosbah, H., Sayari, A., Mejdoub, H., Dhouib, H., Gargouri, Y. (2005) Biochemical and molecular characterization of Staphylococcus xylosus lipase. Biochim Biophys Acta 1723, 282-291. [29] Togashi, N., Inoue, Y., Hamashima, H., Takano, A. (2008) Effects of two terpene alcohols on the antibacterial activity and the mode of action of farnesol against Staphylococcus aureus. Molecules 13, 3069-3076. [30] Shallcross, L. J., Fragaszy, E., Johnson, A. M., Hayward, A. C. (2013) The role of the Panton-Valentine leucocidin toxin in staphylococcal disease: a systematic review and metaanalysis. The Lancet. Infectious diseases 13, 43-54. [31] Otto, M. (2010) Basis of Virulence in Community-Associated Methicillin-Resistant Staphylococcus aureus. Annual Review of Microbiology 64, 143-162. [32] Honeyman, A., Friedman, Herman, Bendinelli, Mauro (2002) Staphylococcus aureus Infection and Disease. [33] Zhu, Y., Weiss, E. C., Otto, M., Fey, P. D., Smeltzer, M. S., Somerville, G. A. (2007) Staphylococcus aureus biofilm metabolism and the influence of arginine on polysaccharide intercellular adhesin synthesis, biofilm formation, and pathogenesis. Infect Immun 75, 42194226. [34] Pablo, G., Hammons, A., Bradley, S., Fulton, J. E., Jr. (1974) Characteristics of the extracellular lipases from Corynebacterium acnes and Staphylococcus epidermidis. J Invest Dermatol 63, 231-238. [35] Weld, J. T., O'Leary, W. M. (1963) Events Associated with Development of Lipid Plaques on Plasma Agar. Nature 199, 510-511. [36] Gould, S. W., Chadwick, M., Cuschieri, P., Easmon, S., Richardson, A. C., Price, R. G., Fielder, M. D. (2009) The evaluation of novel chromogenic substrates for the detection of

This article is protected by copyright. All rights reserved.

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lipolytic activity in clinical isolates of Staphylococcus aureus and MRSA from two European study groups. Fems Microbiol Lett 297, 10-16. [37] Qiao, N.-n., Gao, Q., Zhao, Q., Wang, D.-p., Chen, Y.-p., Zhao, W.-y., Yu, C.-y., pp. 1-3. [38] Stehr, F., Kretschmar, M., Kroger, C., Hube, B., Schafer, W. (2003) Microbial lipases as virulence factors. J Mol Catal B-Enzym 22, 347-355. [39] Ryding, U., Renneberg, J., Rollof, J., Christensson, B. (1992) Antibody response to Staphylococcus aureus whole cell, lipase and staphylolysin in patients with S. aureus infections. FEMS Microbiol Immunol 4, 105-110. [40] Cadieux, B., Vijayakumaran, V., Bernards, M. A., McGavin, M. J., Heinrichs, D. E. (2014) Role of lipase, from community-associated methicillin-resistant Staphylococcus aureus strain USA300, in hydrolyzing triglycerides into growth inhibitory free fatty acids. J Bacteriol, 10.1128/JB.02044-14. [41] Brehm-Stecher, B., Johnson, E. (2003) Sensitization of Staphylococcus aureus and Escherichia coli to antibiotics by the sesquiterpenoids nerolidol, farnesol, bisabolol, and apritone. Antimicrob Agents Chemother 47, 3357 - 3360. [42] Inoue, Y., Shiraishi, A., Hada, T., Hirose, K., Hamashima, H., Shimada, J. (2004) The antibacterial effects of terpene alcohols on Staphylococcus aureus and their mode of action. FEMS Microbiol Lett 237, 325 - 331. [43] Gomes, F. I., Teixeira, P., Azeredo, J., Oliveira, R. (2009) Effect of farnesol on planktonic and biofilm cells of Staphylococcus epidermidis. Curr Microbiol 59, 118-122. [44] Kaneko, M., Togashi, N., Hamashima, H., Hirohara, M., Inoue, Y. (2011) Effect of farnesol on mevalonate pathway of Staphylococcus aureus. J Antibiot 64, 547-549. [45] Nikoleit, K., Rosenstein, R., Verheij, H. M., Gotz, F. (1995) Comparative biochemical and molecular analysis of the Staphylococcus hyicus, Staphylococcus aureus and a hybrid lipase. Indication for a C-terminal phospholipase domain. Eur J Biochem 228, 732738. [46] Oh, B., Kim, H., Lee, J., Kang, S., Oh, T. (1999) Staphylococcus haemolyticus lipase: biochemical properties, substrate specificity and gene cloning. Fems Microbiol Lett 179, 385392. [47] Sayari, A., Agrebi, N., Jaoua, S., Gargouri, Y. (2001) Biochemical and molecular characterization of Staphylococcus simulans lipase. Biochimie 83, 863-871. [48] Jung, W.-H. K., Hyung-Kwoun ; Lee, Chan-Yong ; Oh, Tae-Kwang (2002) Biochemical properties and substrate specificity of lipase from Staphylococcus aureus B56. Journal of microbiology and biotechnology 12, 25-30. [49] Lima, V. M. G., Krieger, N., Mitchell, D. A., Fontana, J. D. (2004) Activity and stability of a crude lipase from Penicillium aurantiogriseum in aqueous media and organic solvents. Biochem Eng J 18, 65-71. [50] Talon, R., Dublet, N., Montel, M. C., Cantonnet, M. (1995) Purification and characterization of extracellular Staphylococcus warneri lipase. Curr Microbiol 30, 11-16. [51] Simons, J. W., Gotz, F., Egmond, M. R., Verheij, H. M. (1998) Biochemical properties of staphylococcal (phospho)lipases. Chem Phys Lipids 93, 27-37. [52] Rosenstein, R., Götz, F. (2000) Staphylococcal lipases: Biochemical and molecular characterization. Biochimie 82, 1005-1014. [53] Simons, J. W., Adams, H., Cox, R. C., Dekker, N., Gotz, F., Slotboom, A. J., Verheij, H. M. (1996) The lipase from Staphylococcus aureus. Expression in Escherichia coli, largescale purification and comparison of substrate specificity to Staphylococcus hyicus lipase. Eur J Biochem 242, 760-769.

This article is protected by copyright. All rights reserved.

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[54] Kuroda, M., Nagasaki, S., Ito, R., Ohta, T. (2007) Sesquiterpene farnesol as a competitive inhibitor of lipase activity of Staphylococcus aureus. Fems Microbiol Lett 273, 28-34.

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Fig. 1 SDS–PAGE analysis of whole cell extracts of a culture of E. coli BL21(DE3) pSalip35 following auto-induction with Studier medium. The arrow indicates the position of the recombinant protein. Lane M molecular mass standards (molecular masses in kDa are indicated on the left); lane 1 cell extracts of autoinduced E. coli BL21(DE3) pET14b after 16 h (control); lane 2 cell extract of autoinduced E. coli BL21(DE3) pSalip35 4h; lane 3 cell extract of auto-induced E. coli BL21(DE3) pSalip35 after 11h; lane 4 cell extract of auto-induced E. coli BL21(DE3) pSalip35 after 16 h; lane 5 insoluble fraction of E. coli BL21(DE3) pSalip35; lane 6 cell free extract; lane 7 flow through (wash fraction of the cell free extract with 10 mmol l−1 imidazole); lane 8-9 purified His6-tagged Salip35 eluted from Ni-affinity column with 200 and 400 mmol l−1 imidazole, respectively.

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Fig. 2 MALDI-TOF MS analysis of His6-tagged Salip35. The experimentally determined mass along with the predicted masses are highlighted by arrows for the singly charged (M+) and double charged (M)2+ species. Mass for the singly monomer was found to be 46246 Da.

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Fig. 3 Activity-pH profile of Salip35 profiles were determined using (●) sodium phosphate (pH 6.5–8.0), (○) Tris-HCl (pH 7.0–9.0) and (▲) sodium bicarbonate (pH 9.2–10.0) buffers. The specific activity was measured using pNPP as substrate and expressed as μmolmin−1mg−1 active enzyme and the relative specific activity is expressed as the percentage of the maximum specific activity obtained under the experimental conditions.

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Fig. 4 (A) Activity-temperature profile of Salip35 catalysed hydrolysis of pNPP. Each point is the mean of triplicate assays. (B) Thermal stability of Salip35. The enzyme was in 50 mmol l−1 Tris-HCl (pH 7.0) at a concentration of 0.55 mg mL-1, and incubated for 0–60 min at the temperatures indicated. (■) 25 °C, (●) 30 °C, (▲) 35 °C, () 45 °C, () 50 °C, ( ) 55 °C. The remaining residual activity was determined at 25°C using pNPP activity assay and expressed as a percentage of activity obtained without heat pretreatment of the enzyme. Each point is the mean of triplicate assays.

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Fig. 5 (A) Effects of farnesol on the growth of S. aureus (isolate 35). S. aureus was inoculated into BHI broth medium that contained no farnesol (■) or 5 (▲), 10(), 50 (●), and 300 (◄) μmol l−1 farnesol. (B) Inhibitory effect of farnesol on Salip35 lipase activity. Lineweaver – Burk plots were generated from the initial velocity of Salip35 and the indicated concentrations of pNPP; no farnesol (▲) 0.57 mmol l−1 farnesol (●), 1.14 mmol l−1 farnesol(■).

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Fig. 6 Pylogenetic tree based on deduced amino acids sequences of Salip35and other staphylococcal lipases including S. xylosus (AAU88142), S. aureus NCTC8530 (AAA26634), S. aureus (PS54C) ( P10335), S. aureus B56 (AAK29127), S. aureus (ABW37176), S. epidermidis 9 (AAC67547), S. epidermidis RP62A (AAC38597), S. epidermidis 9 (AAA19729), S. haemolyticus L62 (AF096928), S. hyicus (P04635), 2HIH A Chain A, Crystal Structure Of S. hyicus (SHyL-2HIH), S. simulans (CAC83747 or Q84EK3), S. saprophyticus (AAT34964. 1 ), S. warneri 863 (Q9F0R0), S. xylosus DSM 20266 (AF208229. 1)

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Table 1. Kinetic parameters of recombinant SaLip35. pNPA: p-nitrophenyl acetate, pNPB: pnitrophenyl butyrate, pNPC: p-nitrophenyl caprylate, pNPL: p-nitrophenyl laurate, pNPM; pnitrophenyl myristate, pNPP: p-nitrophenyl palmitate.

Substrate

kcat (s-1)

KM (mM)

pNPA

31.98±2.17

4.15±0.62

7.70

pNPB

82.93±6.63

1.35±0.21

61.25

pNPC

28.11±0.54

0.11±0.01

247.40

pNPL

58.97±1.66

0.12±0.01

512.33

pNPM

43.83±1.34

0.08±0.01

579.02

pNPP

118.39±2.73

0.12±0.02

973.62

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kcat/KM (s-1mM-1)

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A new lipase as a pharmaceutical target for battling infections caused by Staphylococcus aureus: Gene cloning and biochemical characterization.

Staphylococcus aureus lipases along with other cell-wall-associated proteins and enzymes (i.e., catalase, coagulase, protease, hyaluronidase, and β-la...
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