2.3) programmes (Altschul et al., 1990). ORFs were identified using the National Centre for Biotechnology Information (NCBI) ORF finder tool. In order to clone FE hydrolase gene into pET29a(+) with NdeI and XhoI, overlapping PCR was carried out
to eliminate the XhoI site at 386 bp of the deduced ORF. The forward primer F1: 5′-CATATGGCGAACATCGAAGGCGTA-3′ learn more overlapped an NdeI site (underlined) at the initiation site for esterase gene, the reverse primer R1: 5′-CTCGAGTCAACTCAAAGCGTCGTAGGC-3′ overlapped an XhoI site (underlined) after the termination codon. They were used to amplify the complete sequence of FE hydrolase gene. The forward primer F2: 5′-GTTCACCCTGGAGAACATTTG-3′ and the reverse primer R2: 5′-CAAATGTTCTGGAGGGTGAAC-3′ were a pair of overlapping complementary primers, which were synthesized to obtain the structural genes without XhoI. And they would bind to the structural gene of the FE hydrolase gene at the 377–398 bp. The complete amplified fragment of the FE hydrolase gene was about 1.14 kb length. It was purified by PCR GSK1120212 cell line purification kit, digested with NdeI and XhoI, ligated with NdeI–XhoI-digested pET-29a(+), and then transformed into E. coli BL21(DE3) competent cells. The recombinant
plasmid was designated pET-29a-feh. E. coli BL21(DE3) harbouring pET-29a-feh was grown to OD600 nm = 0.5–0.6 in LB medium containing 50 mg L−1 kanamycin at 37 °C and the feh gene expression was induced by adding 0.2 mmol L−1 IPTG (isopropyl-β-D-thiogalacto-pyranoside)
for 24 h at 18 °C. The crude enzyme extract of E. coli BL21(DE3) was prepared by ultrasonic disruption. Zymogram analysis of the crude enzyme extract was carried out according to the procedure described previously. The activity of the crude enzyme extract was determined according to the enzyme assay of CFE of strain T1. The molecular mass of FE hydrolase was determined by SDS-PAGE. FE and its metabolites in the cultures were extracted with an equal volume of dichloromethane after the whole culture had been acidified to pH 2.0 by the addition of 10% HCl. Extracts were then dried over anhydrous Na2SO4. The treated extracts were examined at 200–350 nm with an ultraviolet spectrophotometer (UV-2450, Shimadzu). Next, 1 mL of extract Resminostat was evaporated at room temperature. Residual material was re-dissolved in an equal volume of methanol. All samples were immediately analysed by HPLC (RID-10A, Shimadzu). The mobile phase was 100% methanol, and the flow rate was 1.0 mL min−1. The separation column (Inertsil ODS-SP, 4.6 × 250 mm) was filled with Kromasil 100-5C18 and the injection volume was 20 μL. The metabolites of FE were analysed by HPLC/MS (Agilent Technologies, LC/MSD VL). The metabolites were ionised by negative polarity electro-spray. The nucleotide sequences of the Rhodococcus sp.