Thermostable esterase from Thermoanaerobacter tengcongensis: high-level expression, purification and characterization.
The lipA gene encoding a thermostable esterase was cloned from Thermoanaerobacter tengcongensis and over-expressed in Escherichia coli. The recombinant esterase, with a molecular mass of approx. 43 kDa determined by SDS-PAGE, was purified to homogeneity through Sephadex G-100 gel filtration. The purified enzyme actively hydrolyzed tributyrin but not olive oil. Maximum activity was observed on p-nitrophenyl (NP)-propionate (C3) and p-NP-butyrate (C4), with little activity towards p-NP-palmitate (C16). The esterase was optimally active at 70 degrees C (over 15 min) and at pH 9. It is highly thermostable, with a residual activity greater than 80% after incubation at 50 degrees C for more than 10 h. The activity was not inhibited by 5 mM EDTA and PMSF, indicating the esterase is not a metalloenzyme and may contain a specific structure around the catalytic serine residue. In addition, it was stable for 1 h at 37 degrees C in 1% CHAPS and Triton X-100 but not stable in 1% Tween 20 or SDS.
D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000145:classification / Q000235:genetics*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013696:Temperature
D001426:Bacterial Proteins; C079999:LipA protein, Bacteria; D011994:Recombinant Proteins