Bacterial strains and plasmids
Escherichia coli EPI300™-T1R and pCC2FOS fosmid vector (CopyControl Fosmid Library Production Kit, Epicentre Biotechnologies, Madison, WI, USA) were used for constructing the metagenomic library. E. coli DH10B and the vectors pUC18 and pCR2.1 (Invitrogen Life Technologies, USA) were used for subcloning steps. E. coli BL21(DE3) and pET-28a(+) vector (Novagen, Madison, WI, USA) were used as the recombinant protein expression system.
Chemicals and enzymes
FideliTaq PCR Master Mix (USB, Cleveland, OH, USA) was used for DNA amplification. T4 DNA ligase, T4 DNA polymerase, Klenow fragment, T4 polynucleotide kinase, shrimp alkaline phosphatase (SAP), restriction enzymes and the protein molecular mass marker were purchased from Fermentas (Glen Burnie, MD, USA). The HiTrap Chelating HP column was purchased from GE Healthcare (Uppsala, Sweden). The nitrophenyl ester series, triacetin, tripropionin, tributyrin, trioctanoin, tridodecanoin, triolein, castor oil, BES buffer, (R)-glycidyl butyrate, (S)-glycidyl butyrate, resorufin butyrate and p-nitrophenol were purchased from Sigma-Aldrich (St. Louis, MO, USA). The natural oils for lipase analysis were commercial products purchased from a supermarket. All other chemicals used for lipase analysis were of analytical grade.
Sample collection and DNA extraction
Soil samples were collected from the banks of an anaerobic lagoon (GPS position, 24°56'11.11"S, 50° 7'27.06"W) of the wastewater treatment plant of a meat packing and dairy industry located in the state of Paraná, Brazil. The average soil temperature was 30°C and soil samples up to 3 cm of soil depth were aseptically stored for 24 h at ambient temperature and subjected to DNA extraction by an indirect lysis method , in which prokaryotic cells were separated from the sediment by low-speed centrifugation before cell lysis.
Metagenomic library construction and screening for lipolytic activity
Purified DNA samples were size-separated on a 0.7% low melting point (LMP) agarose gel in TAE buffer at 20 V/cm. DNA bands from 20 to 40 kb were excised from the gel and extracted by the phenol method . A metagenomic DNA library was constructed using the CopyControl Fosmid Library production kit, according to the manufacturer's protocol (Epicentre Biotechnologies, Madison, WI, USA). For activity screening, the transformed cells were plated onto modified Luria-Bertani (LB) agar plates (5 g/L peptone, 3 g/L yeast extract, 13 g/L bacteriological agar, 10 g/L gum arabic) containing 1% (v/v) emulsified tributyrin as substrate. Cells were grown at 37°C for four days and transformants with clear halos around individual colonies were chosen as possible lipase/esterase producing clones. The selected clones were transferred to 96-well microtiter plates with Terrific Broth and subjected to a second screening for lipolytic activity on the modified LB agar except that 1% (v/v) tricaprylin was used instead of tributyrin. Tricaprylin positive clones were transferred to 96-well microtiter plates and stored. True lipase producing clones were identified amongst the tricaprylin active clones by screening on modified LB agar containing 1% (v/v) triolein. Three clones that showed the strongest lipase activity (FosC12, FosE6 and FosH10) were further analyzed.
Subcloning and identification of the lipase gene
Sublibraries of the FosC12, FosE6 and FosH10 fosmids were constructed in pUC18 vector and subclones were screened for lipolytic activity on modified LB agar medium with 1% (v/v) triolein. The inserts of active subclones were sequenced on an ABI 377 Automated Sequencer (Applied Biosystems, USA) from both ends using DYEnamic ET Dye Terminator Kit (GE Life Sciences, USA). Sequence assembly and editing were performed with the CodonCode Aligner software (CodonCode Corporation, Dedham, MA, USA). The open reading frames (ORFs) were identified with the ORF Finder tool  and the amino acid sequences were compared with the non-redundant sequence database deposited at the NCBI using BLAST . Fosmids FosC12, FosE6 and FosH10 had the same lipase gene. Thus, further cloning steps were performed using the FosC12 fosmid and the lipase protein sequence is hereafter denominated LipC12 (lipC12 gene).
Lipase sequence analysis and phylogenetic tree construction
Prediction of transmembrane regions and signal peptide sequence were performed using the TMHMM 2.0  and SignalP 3.0 servers , respectively. Protein solubility prediction was carried out with the system SOSUI . The ProtParam tool was used to calculate the theoretical parameters of the protein . Multiple sequence alignment was performed with the ClustalW algorithm  in combination with Geneious software (Biomatters Ltd, Auckland, New Zealand). Phylogenetic analysis was carried out with the neighbor-joining method using MEGA version 5. Bootstrapping (10,000 replicates) was used to estimate the confidence levels of phylogenetic reconstructions . GeneDoc version 2.7 was used for displaying and highlighting the multiple sequence alignments.
Cloning of lipC12 gene
The forward primer LipC12.L (5'CAACGTCAAAGAGGTTATTC3') and the reverse primer LipC12.R (5'CGAGTGCTATCGTTCATTTA3') were used in a PCR reaction (FideliTaq PCR Master Mix, USB, USA) for the amplification of a 966 bp fragment. The amplified gene was first cloned into the pCR 2.1 vector (TA Cloning Kit, Invitrogen, USA) according to the manufacturer's recommendations and recombinant plasmids were transformed into E. coli DH10B competent cells by electroporation. White colonies were picked, plasmids were extracted  and their inserts were sequenced with M13 forward and reverse primers to confirm the absence of mutations in lipC12. Recombinant plasmid was then digested with NdeI (cut at lipC12 translation start codon) and BamHI. The insert was ligated into the pET28a(+) vector, which had been previously digested with the same restriction enzymes and dephosphorylated by SAP, yielding plasmid pET28a-lipC12, the insert of which was end-sequenced with the T7 promoter and terminator primers. Plasmid pET28a-lipC12 was then transformed into E. coli BL21(DE3) cells to express the recombinant (His)6-tagged LipC12 lipase.
Overexpression and purification of recombinant LipC12 lipase
E. coli BL21(DE3) cells carrying the pET28a-lipC12 plasmid were grown in 200 mL of LB medium at 37°C until an OD600 of 0.5 and induced by the addition of isopropyl-β-D thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. The induced culture was incubated for a further 16 h at 20°C before harvesting of the cells by centrifugation (10,000 × g for 5 min) at 4°C. The cell pellet was resuspended in 35 mL of lysis buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 10 mM imidazole, 10 mM β-mercaptoethanol, 10% (v/v) glycerol, 0.25% (w/v) Nonidet P-40) and disrupted by ultrasonication in an ice bath (10 cycles of 60 s pulses, 90 W, with 30 s intervals), using a SONICATOR® XL 2020 (Heat systems-Ultrasonics Inc., New Highway, Farmingdale, NY, USA). The crude extract was then centrifuged at 15,000 × g for 30 min at 4°C to pellet the cell debris. The supernatant containing the His-tagged protein was loaded onto a HiTrap Chelating HP column (GE Healthcare, USA), previously loaded with Ni2+ and equilibrated with lysis buffer, using an ÄKTAbasic chromatography system (GE Healthcare, USA). The column was washed with 5 volumes of the lysis buffer and further with 5 volumes of elution buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 10 mM imidazole, 10% (v/v) glycerol). The His-tagged protein was eluted with an increasing gradient of imidazole up to 500 mM in elution buffer. The elution of protein was monitored at 280 nm, protein fractions were analyzed by SDS-PAGE, pooled, dialyzed (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 mM CaCl2, 50% (v/v) glycerol) and stored at -24°C until use.
Protein content determination, electrophoresis and zymogram analyses
Protein content was determined using the Pierce BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA) with bovine serum albumin as the standard. Electrophoresis of protein samples was done with 12% (w/v) SDS-PAGE  and the gel was stained with Coomassie Brilliant Blue R-250 and destained with methanol/acetic-acid/water (5/1/4 v/v/v). Densitometric analysis of the stained SDS-PAGE gel was done using LabWorks Image Acquisition and Analysis Software 4.0 (UVP BioImaging Systems, Upland, CA, USA). Lipolytic activity of bands on the SDS-PAGE gel was detected using tributyrin or tricaprylin as substrate .
Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectra (MS) were acquired on a MALDI-TOF/TOF Autoflex II spectrometer (Bruker Daltonics, Bremen, Germany) in the reflector positive ion mode with an acceleration voltage of 20 kV, delay time of 150 ns and acquisition mass range 800-3200 Da. Spots were excised manually and in-gel digested with sequencing grade modified trypsin (Promega, Madison, USA) as described elsewhere . The sample was desalted using a ZipTipC18 pipette tip (Millipore Corporation, Bedford, MA, USA) and eluted directly onto the MALDI target plate using MALDI matrix (saturated solution of α-cyano-4-hydroxycinnamic acid in 50% (v/v) acetonitrile and 0.1% TFA). Mass profiles were identified by comparing the peptide masses obtained with in silico digestion of the His-tagged protein sequence using PeptideCutter and MS-Digest tools .
Spectrophotometric determinations of lipase activity using pNP substrates
Enzymatic activities were determined by following the amount of p-nitrophenol released from p-nitrophenyl ester at 410 nm  for at least 30 min at 30°C in a TECAN Infinite Series M200 microplate spectrophotometer (TECAN, Salzburg, Austria). Unless otherwise described, for the standard assay, the substrate solution was made by mixing a stock solution of 20 mM of p-nitrophenyl palmitate (pNPP) in acetonitrile/isopropanol (1/4 v/v) with an assay buffer containing Tris-HCl pH 7.5, CaCl2 and Triton X-100, under agitation in a water bath at 60°C, until the solution became transparent. Then 230 μL of the substrate solution was pipetted into a 96-well microtiter plate and the reaction was initiated by automatic addition (with the TECAN injector module) of 20 μL of the enzyme solution (50 mM Tris-HCl pH 7.5, 1 mM CaCl2) to a final concentration of 5 nM. The final volume of the reaction mixture (Tris-HCl 50 mM, pH 7.5, 1 mM CaCl2, 0.3% (v/v) Triton X-100, 5 nM enzyme, 1 mM pNPP, 4% (v/v) isopropanol, 1% (v/v) acetonitrile) was 250 μL. All experiments were performed in triplicate, the extinction coefficients of p-nitrophenol were determined under each reaction condition and the effect of nonenzymatic hydrolysis of substrates was subtracted. One unit of lipase activity was defined as 1 μmol of p-nitrophenol produced per minute. Linear regressions to determine initial reaction velocities and standard deviations of means were performed with Calc software from the OpenOffice.org package.
Substrate specificity for nitrophenyl esters was analyzed under standard conditions using p-nitrophenyl acetate (C2:0), p-nitrophenyl butyrate (C4:0), p-nitrophenyl valerate (C5:0), p-nitrophenyl caproate (C6:0), p-nitrophenyl decanoate (C10:0), p-nitrophenyl dodecanoate (C12:0), p-nitrophenyl myristate (C14:0) and p-nitrophenyl palmitate (C16:0).
The temperature giving maximum substrate conversion over a 1 h reaction time was determined by incubating the enzyme with p-nitrophenyl ester (2 nM enzyme, 10 mM p-nitrophenyl butyrate, MES 50 mM pH 6.0, 1 mM CaCl2, 0.3% (v/v) Triton X-100, 4% (v/v) isopropanol, 1% (v/v) acetonitrile) in a reaction volume of 50 μL at various temperatures within the range of 10°C to 60°C. In relation to the standard assay, p-nitrophenyl butyrate was used instead of pNPP to minimize substrate solubility variations with reaction temperature and the reaction pH was decreased to minimize substrate autohydrolysis, which occurs at higher temperatures. The reaction was carried out in a 96-well PCR plate on a Thermal Cycler (Eppendorf, Hamburg, Germany) running in gradient mode. After the reaction time, the plate was immediately chilled on ice and the enzymatic reaction was stopped by the addition of 100 μL of 0.1 M HCl. Then 125 μL was transferred to a 96-well microtiter plate, completed to 250 μL with deionized water and the amount of released p-nitrophenol was measured at 348 nm instead of 410 nm: the absorption of p-nitrophenol decreases at 410 nm as the pH decreases, due to changes in equilibrium between p-nitrophenol and p-nitrophenoxide, while 348 nm is the pH-independent isosbestic wavelength of p-nitrophenoxide and p-nitrophenol .
The effect of cations and anions on the enzymatic activity of LipC12 was also investigated. For analyses involving cations, the enzyme at 250 nM was previously incubated with 1 mM EDTA/50 mM Tris-HCl pH 7.5 for 1 h in order to remove cations from the enzyme. Then 20 μL of enzyme solution was added to each well of a 96-well microtiter plate, followed by 25 μL of cation solution and 205 μL of substrate solution (with 0.5 mM of EDTA) giving a reaction mixture (Tris-HCl 50 mM pH 7.5, 1 mM of a specific cation, 0.3% (v/v) Triton X-100, 20 nM enzyme, 1 mM pNPP, 0.5 mM EDTA, 4% (v/v) isopropanol, 1% (v/v) acetonitrile) that was monitored at 410 nm, as described above. The effect of cations on the enzymatic activity of LipC12 without previous cation depletion was also tested. For analyses involving anions, cation depletion with EDTA was not performed and a standard assay was carried out except that either 1 mM or 10 mM of a specific anion was added (all anions were added in the form of sodium salts).
Similarly, the effects of lipase inhibitors were investigated. Chelating agents such as ethylenediamine tetraacetic acid (EDTA) and ethylene glycol tetraacetic acid (EGTA) and modifying reagents such as phenylmethylsulfonyl fluoride (PMSF) and diethyl pyrocarbonate (DEPC) were used. The effects on LipC12 activity of commonly used detergents such as Tween 20, Tween 40, Tween 80, Triton X-100, Nonidet P-40 (NP40), sodium dodecyl sulfate (SDS), Sarkosyl NL (NLS) and hexadecyltrimethylammonium bromide (CTAB) were also investigated. The effect of gum arabic, commonly used as an emulsion stabilizer, was checked. In these cases, the activity measurements were done using the standard assay, except that the chemicals listed above were added to the wells before the addition of the substrate and enzyme solutions.
Thermostability was determined by measuring the residual activity after incubating 250 nM of the enzyme (50 mM Tris-HCl pH 7.5, 5 mM CaCl2) at various temperatures in the range of 0°C to 90°C for 1 h in 200 μL PCR Eppendorf tubes with mineral oil on top to prevent evaporation. The incubation was carried out using a volume of 50 μL on the Thermal Cycler (Eppendorf, Hamburg, Germany) running in gradient mode. After the incubation time, the tubes were chilled on ice and the residual activity was determined at 10 nM of enzyme in the reaction mixture.
Stability in organic solvents was determined by measuring the residual activity after incubation of the enzyme at 500 nM with 15% or 30% (v/v) organic solvents (200 mM Tris-HCl pH 8.0, 5 mM CaCl2) at 4°C for 48 h. In order to equalize the solvent concentration before the residual activity measurement, the solvent concentration of each solvent incubated at 15% (v/v) was corrected to 0.3% (v/v) during dilution of the enzyme (the enzyme was diluted 100-fold before performing the residual activity measurement).
Stability in high salt concentrations, up to 3.7 M, was investigated by incubating LipC12 at 785 nM (1 mM CaCl2, 50 mM Tris-HCl pH 7.5) for 24 h at 4°C. Several NaCl and KCl concentrations were tested. All salt concentrations were equalized to 37 mM during the 100-fold dilution of the enzyme prior to the residual activity measurement.
The pH stability was tested after incubation of the purified enzyme for 24 h at 4°C in different buffers. For a pH range of 3.0 to 11.0, the buffers used were 50 mM sodium acetate (pH 3.0 to 5.5), 50 mM MES (pH 5.5 to 7.0), 50 mM HEPES (pH 7.0 to 7.5), and 50 mM glycine (pH 7.5 to 11). The enzyme was diluted 100-fold in 50 mM HEPES pH 7.5 for the residual activity measurement under standard assay conditions.
Determination of lipase enantioselectivity was performed using the Quick E method [72
]. Substrate solutions were prepared using glycidyl butyrate enantiomers and resorufin butyrate was used as the reference substrate. A buffer/indicator solution was prepared by mixing 4-nitrophenol solution (1.2 mL of a 1.8 mM solution in 1.0 mM BES containing 0.33 mM Triton X-100, pH 7.2), BES buffer (3.3 mL of a 1.0 mM solution containing 0.33 mM Triton X-100, pH 7.2), and acetonitrile (65 μL). Substrate solution (35 μL of a 150 mM glycidyl butyrate enantiomer in acetonitrile) and resorufin ester solution (260 μL of 2.0 mM resorufin butyrate in acetonitrile) were added dropwise with continuous vortexing to form a clear emulsion that was stable for at least several hours. This solution was pipetted into a 96-well polystyrene microplate (100 μL/well). Lipase solution (5 μL in 5 mM BES) was added to each well and the microplate was placed in the microplate reader (TECAN Infinite Series M200 microplate spectrophotometer, TECAN, Salzburg, Austria) and shaken for 10 s. Then the decrease in absorbance at 404 nm and the increase in absorbance at 574 nm were followed for 30 min at 25°C. Blanks without enzyme were carried out for each substrate and data were collected in triplicate. Reaction rates for the reference substrate and each enantiomer were calculated using Eqs. (1) and (2), respectively, using the initial linear parts of the curves of absorbance versus time:
574/dt is the absorbance increase (at 574 nm) per minute, Δε574 is the difference in extinction coefficients at 574 nm for resorufin and resorufin butyrate (15,100 M-1 cm-1), l is the path length (0.3064 cm for a 105 μl reaction volume), V is the reaction volume in liters, dA
404/dt is the absorbance decrease (at 404 nm) per minute and Δε404 is the difference in extinction coefficients for the protonated and unprotonated forms of the 4-nitrophenol (17,300 M-1 cm-1).
The enantioselectivity was calculated from two measurements (one for each glycidyl butyrate enantiomer) as shown in Eq. (3):
where rateS represents the rate of hydrolysis of the (S)-glycidyl butyrate enantiomer and raterefS is the rate of hydrolysis of the reference substrate (resorufin butyrate) in the presence of the (S)-enantiomer. Analogous definitions apply for rateR and raterefR in terms of the (R)-enantiomer.
Titrimetric determinations of lipase activity using triacylglycerol substrates
The substrate emulsions consisted of triacylglycerol 67 mM, gum arabic 3% (w/v), CaCl2 2 mM, Tris-HCl 2.5 mM and NaCl 150 mM, dispersed in distilled water . The solution was emulsified with a handheld mixer (400 watts, Royal Philips Electronics) at high speed, initially for 10 min and then for an additional 2 min immediately before use. The free fatty acids released during the reaction were titrated automatically in a Metrohm 718 STAT Titrino potentiometric titrator (Metrohm, Herisau, Switzerland) with 0.05 M NaOH, for 5 min. The reactions were done in a glass vessel thermostated at 30°C containing 20 mL of substrate emulsion and 6.68 μg of purified enzyme added in 200 μL of solution buffer (Tris-HCl 2.5 mM pH 8, CaCl2 5 mM). The titration point was set to pH 8.5. All measurements were performed in triplicate and chemical hydrolysis of the substrate was subtracted. One unit (U) of enzymatic activity was defined as the release of 1 μmol of fatty acid per minute.
The pH optimum for LipC12 was determined using tributyrin as a substrate . The substrate emulsion and reaction conditions were the same as described above except that the setpoint pH was set at different values. Corrections were made for autohydrolysis and for the partial dissociation of butanoic acid assuming a pKa of 4.57.
Nucleotide sequence accession number
The LipC12 nucleotide sequence reported here is available in the GenBank database under the accession number JF417979.