Screening of the esterase gene from a compost metagenomic fosmid library
Metagenomic DNA from compost soil was extracted as previously described [27], and it was used to construct a metagenomic library using the CopyControl™ fosmid library production kit (Epicentre, USA), according to the manufacturer's instructions. Activity-based esterase/lipase screening was performed to identify recombinant clones with lipase activity. The transformants were spread on Luria-Bertani (LB) agar plates containing chloramphenicol (25 μg/mL), 1% (v/v) tributyrin (C4) as a substrate, and 0.5% (w/v) gum arabic. Active colonies producing clear halos on the plates were selected, and the lipolytic genes were sequenced from the fosmids of active colonies by a random shotgun sequencing method. The open reading frame (ORF) of the esterase was compared with reference sequences that were retrieved from protein and nucleotide databases on the Entrez server at the NCBI (http://www.ncbi.nlm.nih.gov/sites/entrez/). Sequence similarity searches were performed using the BLAST 2.0 program, and multiple alignments were conducted with highly similar sequences with the CLUSTAL W program. A phylogenetic tree was constructed with the MEGALIGN program (DNA STAR Inc., USA).
Cloning and overexpression of the estCS2 gene
The putative esterase gene was amplified by PCR with 2 primers (5'-GGGCATATG ATGCGAGCCGAGTTGC-3' and 5'-GCGCTCGAG TGATTTTTGGGGATCT-3'; the underlined sequences indicate the Nde I and Xho I recognition sites, respectively). PCR products were digested with Nde I and Xho I, and then, they were ligated into the pET-22b(+) vector (Novagen, Germany) that had been digested with the same restriction enzymes. The recombinant DNA was transformed into E. coli BL21(DE3).
Transformants were cultured in LB liquid medium containing ampicillin (100 μg/mL) at 37°C. When the optical density of the culture reached an absorbance of around 0.6 at 600 nm, 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) was added for the induction of protein expression, and the transformants were incubated for additional 18 h at 21°C. The cells were harvested by centrifugation at 7,000 rpm at 4°C for 10 min and then resuspended in binding buffer (50 mM Tris-HCl at pH 8.0, containing 300 mM NaCl). The resuspended cells were disrupted by sonication, and the crude cell lysate was centrifuged at 16,000 rpm for 30 min; the supernatant, which constitutes the cell extract, was withdrawn. The cell extract was applied to a column containing Ni-NTA resin (QIAGEN GmbH, Germany). After washing with 25 column volumes of the binding buffer containing 2 mM imidazole, bound proteins were eluted with binding buffer containing 250 mM imidazole. Eluted fractions were concentrated by centrifugation at 500 × g at 4°C. Concentrated proteins were loaded onto a gel filtration column, Superdex 200 10/300 GL (GE Healthcare, USA), equilibrated with 50 mM Tris-HCl buffer (pH 8.0) containing 300 mM NaCl, and separation was conducted at a flow rate of 0.5 mL/min on a BioLogic DuoFlow Chromatography System (Bio-Rad Laboratories, USA). The resulting protein fractions were analyzed by SDS-PAGE in 10% polyacrylamide gels.
N-terminal amino acid sequence analysis
N-terminal amino acid sequence analysis was performed on proteins that had been separated by SDS-PAGE and electrotransferred to PVDF membrane (Pro Blott; Applied Biosystems, USA). The gel was electroblotted at 50mA, for 60 min using freshly prepared 10 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) buffer at pH 10 and 10% (v/v) methanol (HPLC grade) in distilled water as a blotting buffer. Subsequently, the gel was stained with Coomassie brilliant blue to confirm effective transfer. After blotting, selected protein bands were cut from the membrane, and N-terminal sequence analysis was carried out using an automatic protein sequencer (Applied Biosystems, USA).
Plate assay for degradation of polyurethane and triglyceride
Activity of the EstCS2, to degrade polyurethane as substrate, was tested on an indicator plate. The polyurethane esterase indicator plate was prepared with a sonicated emulsion of 0.5% poly DEGA in 20 mM Tris-HCl at pH 8.0 [16]. The lipolytic activity indicator plate contained emulsified tributyrin. A crude cell extract of E.coli BL21(DE3)/pET22b, and purified EstCS2, were transferred into holes in the plates, and each plate was incubated for 5 h at ambient temperature.
Characterization of EstCS2
Substrate preference toward p-nitrophenyl esters (C2-C18) was determined enzymatically by measuring the amount of p-nitrophenol released by hydrolysis. Absorbance was continuously measured at 405 nm for 4 min, using a DU800 spectrophotometer (Beckman, USA). As a standard assay solution, a mixture (1:4:95, by vol) of 10 mM p-nitrophenyl caprylate (C8), ethanol, and 100 mM GTA buffer was used. One unit of enzyme activity was defined as the amount of enzyme needed to release 1 μmol of p-nitrophenol per min at 25°C. Protein concentration was determined according to the method of Bradford (Bio-Rad Protein Assay), using bovine serum albumin as the standard. Substrate specificities toward various triacylglycerols were measured by titrating free fatty acid released by substrate hydrolysis. Substrates were emulsified in a solution containing 10 mM NaCl, 1 mM CaCl2, and 0.5% (w/v) gum arabic solution. The substrate emulsion (30 mL) was adjusted to pH 8.0 with 10 mM NaOH, and the reaction was initiated by adding purified enzyme. The pH of the reaction was recorded by a pH-stat (842 Titrando; Metrohm, USA), equipped with thermostat and stirrer, at 25°C for 6 min. One unit of esterase activity was defined as the amount of enzyme that liberates 1 μmol of fatty acid per min.
The optimum pH for enzyme activity was determined at 25°C in 100 mM GTA buffer, in the pH range 4.0-11.0. pH stability of the esterase was determined by incubating the enzyme in 100 mM GTA buffer (from pH 4-11) for 16 h at 4°C, and residual activity was measured at 25°C. To determine the optimum reaction temperature, the reaction mixture was incubated for 10 min at various temperatures (10-80°C). Thermostability of the esterase was determined by preincubation for 1 h, over a temperature range of 10-80°C, in 100 mM GTA buffer (pH 9.0); subsequently, residual activity was measured at 25°C.
To measure the effects of detergents on the esterase activity, aliquots of purified enzyme, mixed with various detergents and metal ions, were incubated in 100 mM GTA buffer (pH 9.0) at 30°C for 1 h. After incubation, residual activity was measured under standard assay conditions.
The effect of several inhibitors (at 1 mM, 5 mM, and 10 mM), such as PMSF, dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA), and 2-mercaptoethanol were tested by incubation with the enzyme for 1 h at 30°C. The stability of the enzyme in the presence of organic solvents was tested with 10%, 20%, and 50% (w/v) of methanol, ethanol, 2-propanol, 1-butanol, acetonitrile, dimethyl sulfoxide (DMSO), acetone, and dimethylformamide (DMF) by incubation of the enzyme for 1 h at 30°C in various organic solvent dilutions.
Hydrolysis of linalyl acetate and ketoprofen ethyl ether by EstCS2
The reaction mixture, containing rac-linalyl acetate (15 mg/mL) and the purified protein (10 mg/mL) in 50 mM Tris-HCl (pH 8.0), was agitated on a rotary shaker (200 rpm) at ambient temperature. The reaction was stopped by the addition of 4 volumes of ethanol, and insoluble material was removed by centrifugation. Samples were periodically withdrawn and analyzed by TLC using Merck silica gel 60 F254, and petroleum ether:ethyl acetate (5:1) as an eluent. Compounds were visualized by spraying with a solution of 5 g vanillin/L concentrated H2SO4 and subsequently heating.
(R/S)-Ketoprofen alkyl ester was synthesized by a general method for esterification. (R/S)-Ketoprofen (12 g) was solubilized in 100 mL of 100% ethanol in a round-bottom flask. H2SO4 was then added as a catalyst for esterification. The mixture was refluxed for 6 h at 70°C, with stirring, for the synthesis of ketoprofen ethyl ester. Sodium acetate (0.5 g) was added to quench the catalyst, the residual alcohol was removed by vacuum evaporation, and the esterified material was washed 3 times with aqueous 1 M NaHCO3 to remove unreacted ketoprofen, sulfuric acid, and ethanol [28]. The hydrolytic activity, using the (R/S)-ketoprofen ethyl ester as substrate, was determined at 30°C for 24 h with the purified enzyme (6 mg/mL) in a reaction mixture containing 50 mM Tris-HCl (pH 8.0). The reaction was stopped by the addition of 1 volume of ethanol, and insoluble material was removed by centrifugation. The resulting solution was then analyzed for conversion yield and enantioselectivity by high performance liquid chromatography (HPLC). The concentrations of (R/S)-ketoprofen ethyl ester, (R)-ketoprofen and (S)-ketoprofen were determined using an HPLC system (Hitachi, Japan). The column used was a chiral compound analytical column (Chirex Phase 3005; Phenomenex). Methanol containing 30 mM ammonium acetate was used as the mobile phase. The column was run at a constant flow rate (1.5 mL/min), and the eluent was monitored spectrophotometrically at 254 nm. One unit of enzyme activity was defined as the amount of enzyme producing 1 μmol of ketoprofen per min under the specified conditions.
Nucleotide sequence accession number
The nucleotide sequence data of the esterase gene (estCS2) has been deposited in Gen Bank, under the accession number GU256649.
