GDSL esterase Xcc_est from Xanthomonas campestris pv. campestris 8004 and its esterase domain: gene expression in Escherichia coli，refolding and characterization

Abstract:

Abstract: [Objective] To characterize the GDSL (glycine, asparticacid, serine and leucine motif in protein sequence) esterase Xcc_est from Xanthomonas campestris pv. campestris (Xcc) 8004. [Methods] Xcc_est gene and different domains of Xcc_est gene were PCR amplified and expressed in Escherichia coli, the HIS-Tagged fusion proteins were purified by Ni-NTA chromatography. [Results] The optimum pH and temperature of partly purified Xcc_est were pH 8.0 and 52 °C when pNPB (4- nitrophenylbutyrate) was used as substrate. The Km and Vmax value of Xcc_est and the passenger domain (Xcc_estN1-334) for pNPB were 47.6 ± 4.6 μmol/L, 67.6 ± 7.8 U/mg and 469.4 ± 9.8 μmol/L, 2.5 ±0.9 U/mg respectively. Inclusion bodies of mature domain Xcc_est (Xcc_estN26-606) could be refolded but inclusion bodies of the passenger domain (Xcc_estN26-334) could not be refolded. Refolded mature domain had broad substrate spectrum and showed higher stability than Xcc_est when stored at 25 °C. [Conclusions] To some extend, refolded Xcc_estN26-606 is a candidate for biotransformation application.