Cloning and Characterization New Gly-Asp-Ser-Leu Esterase/Lipase from Pseudoxanthomonas taiwanensis AL17
Abstract
A thermostable Gly-Asp-Ser-Leu (GDSL) family esterase encoding gene, namely ITBGDSL_1, was directly cloned from the genomic DNA of Pseudoxanthomonas taiwanensis AL17. Homological analysis revealed that the enzyme contained all conserved regions of GDSL family esterase despite a low homology to other GDSL enzymes. Recombinant fusion hexahistidine-tagged ITBGDSL_1 was overexpressed and purified. Biochemical properties were characterised. ITBGDSL_1 having maximal activity to pNP C2. The enzyme was observed to have the highest activity at 55 °C. ITBGDSL_1 exhibited the highest activity at pH 8. Different from other thermostable GDSLs, ITBGDSL_1 showed stability up to 60 h following incubation at optimal temperature. The activity of ITBGDSL_1 preferred more polar solvents and was inhibited by Ethylenediaminetetraacetic acid (EDTA), Phenylmethylsulfonylfluoride (PMSF), β-mercaptoethanol, and surfactants. Furthermore, the activity was influenced in the presence of metal ions. Monovalent metal ions, such as Na+ and K+, deactivated the enzyme; meanwhile, most divalent metal ions activated it. Unlike the other GDSL enzymes, the activity was inhibited by the presence of Cu2+ or Zn2+ ions; the activity of ITBGDSL_1 was increased. This was confirmed by the orientation change at his loop. ITBGDSL_1 is a new member of GDSL family esterase and may employ a distinct catalytic mechanism.