Strains, metagenomics library construction, and screening lipolytic activity

E. coli DH5α and BL21(DE3) were used for all cloning and expression experiments, respectively. A soil sample (35°52′N, 127°3′E) was collected from the Korea Expressway Corporation Arboretum in Jeonju City, South Korea. Soil DNA was prepared by directed DNA extraction and purification as previously described (Kim et al. 2007). The metagenomic cosmid library was constructed according to the method of Yun et al. (2005). Two-step DNA purification was applied to remove humic compounds present in the soil DNAs using pulsed-field gel electrophoresis (PFGE) (CHEF, BioRad). To remove humic compounds in DNA extracted from soil sample, 1 % low melting point agarose was prepared and crude DNA was fractionated by PFGE under a 4 V cm−1 electrical field at 14 °C for 12 h. A gel containing 100–190 kb of DNA was purified by agarase (1 U per 100 mg slice, Takara, Japan). The isolated DNA was partially digested by Sau3AI (0.05 U μl−1 of DNA, 37 °C for 1 h), and then the digested DNA was fractionated by PFGE. A gen containing approximately 40-kb lengths DNA was again purified by agarase. The 40-kb DNA was ligated into a pSuperCosI (Stratagene, La Jolla, CA) and packaged using MaxPlax Lambda Packaging Extracts (Epicentre, Madison, WI). For screening esterase activity, the transformants were plated on Luria–Bertani (LB) agar plates with chloramphenicol (12.5 μg ml−1) and tributyrin (1 %). After incubation at 37 °C for 1 day, the plates were subsequently incubated at 4 °C for a week. Candidate colonies were selected based on the presence of clear zone on the plate.

Subcloning and sequence analysis

Positive colony (pCosSTR1) with a clear halo was selected on the plate. To identify the gene encoding esterase active, the subclone library was constructed by method as described by Jeon et al. (2011). The transformants with esterase activity were selected by the presence of the clear halo zone on LB agar plates containing 100 μg ml−1 ampicillin and 1 % tributyrin. DNA sequencing of the subclone with esterase activity was performed with an ABI3100 (PE Applied Biosystems, Foster City, CA, USA) and assembled using the Vector NTI suite 7 software package (InforMax, North Bethesda, MD, USA). The open reading frames (ORFs) and sequence homology searches in the complete assembled sequence were analysed by the ORF finder and BlastX search provided by the National Center for Biotechnology Information (NCBI) (Altschul et al. 1997).

Multiple alignment and phylogenetic analysis

Multiple sequence alignments were carried out by the ClustalW program (Thompson et al. 1994) for the protein sequences. To compare the amino acid sequences of EstSTR1 and EstU1, we performed sequence alignment using the Expresso program on the T-Coffee Server (Notredame et al. 2000), found at the ExPASy web site (http://tcoffee.vital-it.ch/apps/tcoffee/index.html). Expresso is able to combine sequence information with protein structural information. Molecular Evolutionary Genetics Analysis 4.1 software (MEGA, version 4.1) (Tamura et al. 2007) was used to make the phylogenetic tree using a neighbour-joining method (Saitou and Nei 1987).

Expression and purification of recombinant EstSTR1

The estSTR1 gene was amplified by PCR the following primers: STR1-F (5′-GAGACCC CATATG AGCACCGGGATCGAAATTCAAG-3′) and STR1-R (5′-CTAT CTCGAG GCTGTTCGGCAGGCAGCGATAC-3′). NdeI and XhoI sites for cloning are underlined. The PCR product (1173 bp) for the estSTR1 gene was cloned into pET-24a (+) vector containing the T7 polymerase promoter system (Novagen, Madison, Wisconsin, USA), and then the recombinant plasmid was introduced into E. coli BL21(DE3) cells. When E. coli BL21(DE3) cell harbouring pET-24a(+)/His 6 -EstSTR1 reached approximately 0.6 at 600 nm, 1 mM IPTG (isopropyl β-d-1-thiogalactopyranoside) was added to induce expression. The cells were harvested by centrifugation (5000×g, 4 °C, 20 min) after induction at 25 °C for 10 h, resuspended in 10 mL of buffer A (50 mM Tris–HCl pH 8.0, 10 % glycerol, 100 mM KCl). The solution was vortexed, and then sonicated for 20 min. To obtain the soluble protein, the crude lysate disrupted by sonication was centrifuged (15,000×g, 4 °C, 60 min). To purify EstSTR1 with the His 6 tag, the soluble proteins were loaded onto a column of TALON® metal affinity resin (BD Biosciences Clontech, Palo Alto, CA, USA) and washed with buffer B (50 mM Tris–HCl pH 8.0, 10 mM imidazole, 10 % glycerol, 100 mM KCl). The bound EstSTR1 was eluted with buffer C (50 mM Tris–HCl pH 8.0, 300 mM imidazole, 10 % glycerol, 100 mM KCl). For further purification, size exclusion chromatography was performed. Eluted sample was purified using Superdex-75 (16/60) column (GE Healthcare, Piscataway, NJ, USA) equilibrated and run using buffer D (150 mM NaCl, 20 mM Tris–HCl pH, 7.8).

Characterizations of EstSTR1 for pNP esters

Enzyme activity was determined by colorimetric method measuring released p-nitrophenol from p-nitrophenyl (pNP) esters (Sigma, St. Louis, MO, USA) at 405 nm. Esterase activity was measured by reaction mixture with 1 mM p-nitrophenyl esters in 50 mM Tris–HCl (pH 8.0) containing 1 % (v/v) acetonitrile at 35 °C. The substrate specificity of enzyme was determined in the presence of 1 mM of p-nitrophenyl esters with different aliphatic side chains: acetate (C2), butyrate (C4), hexanoate (C6), octanoate (C8), decanoate (C10), laurate (C12), myristate (C14), palmitate (C16), and stearate (C18). The optimum temperature of enzyme was determined at temperatures ranging from 5 to 70 °C using p-nitrophenyl butyrate as a substrate in 50 mM Tris–HCl buffer (pH 8.0). For optimization of the pH of enzyme, enzyme activity was measured for a pH range of 4.0–10.0. The buffers used were 50 mM sodium acetate (pH 4.0–6.0), 50 mM sodium phosphate (pH 6.0–7.5), 50 mM Tris–HCl (pH 7.5–8.5), and 50 mM CHES (pH 8.5–10.0).

β-Lactamase assay of EstSTR1

Antibiotics (cephalothin, cefoxitin, cefotaxime, and cefepime) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The chemical structures of cephalosporins (cephalothin, cefoxitin, cefotaxime, and cefepime) are shown in the Additional file 1: Figure S1. The hydrolysing activity of EstSTR1 for cephalosporins was measured by the paper disc method as previously described (Jeon et al. 2011). The enzyme (330 μM) was incubated with antibiotic substrates (3 mM cephalothin, 1 mM cefoxitin, 1 mM cefotaxime, and 1 mM cefepime) in 50 mM Tris–HCl (pH 8.0) for 2 h at 35 °C, and then reaction mixtures were loaded onto small paper discs. After 8 h incubation at 37 °C, the diameters of the inhibition zones around the small paper discs were recorded. For comparison of β-lactam hydrolysing activity, a negative control containing antibiotics without enzyme and a positive control containing antibiotics and the CMY-10, a plasmid-encoded class C extended-spectrum β-lactamase (ESBL) from Enterobacter aerogenes (Kim et al. 2006), were used.

Determination of kinetic parameters

The kinetic parameters (k cat and K m ) of EstSRT1 were obtained by measuring the absorbance variation using the molecular extinction coefficient of each substrate: p-nitrophenyl butyrate (Δε 405 = 13,500 M−1 cm−1), cephalothin (Δε 262 = −7660 M−1 cm−1), cefotaxime (Δε 264 = −7250 M−1 cm−1), and cefepime (Δε 267 = −9120 M−1 cm−1). The assay for p-nitrophenyl butyrate was conducted in 50 mM CHES (pH 9.0) containing (approximately 2.43 nM) and substrates (10–600 μM). The assays for β-lactam substrates were conducted in 10 mM MES buffers (pH 6.8) with enzymes (396 μM), substrates (10–500 μM), and bovine serum albumin (20 μg ml−1). Steady-state kinetic constants were determined by fitting the initial rates (in triplicate) directly to the Henri-Michaelis–Menten equation using non-linear regression with the program DYNAFIT (Kuzmic 1996). When K m values for β-lactam substrates were too low to be determined, the values were determined as competitive inhibition constants, K i , in the presence of a reporter substrate (100 μM cephalothin), and K i values were calculated as previously described (Galleni and Frere 1988; De Meester et al. 1987).

Site-directed mutagenesis of EstSTR1

A site-directed change to alanine (S71A) was made using the Stratagene Quik Change kit (La Jolla, CA, USA). The primers (S71A-F and S71A-R) designed to introduce the S71A substitution were as follows: S71A-F (5′-CGCTCATCAATACCTAT GCG ACCACCAAGGGCATGG-3′) and S71A-R (5′-CCATGCCCTTGGTGGT CGC ATAGGTATTGATGAGCG-3′). The sequence corresponding to the mutated codons are underlined. The catalytic activity of the variant was tested and compared with that of the wild-type enzyme.

Nucleotide sequence accession number

The nucleotide sequence of EstSTR1 has been deposited in the GenBank database under the accession number KJ530984.