title: Glycine-aspartic_ acid-serine-leucine esterase Xcc_ est from Xanthomonas campestris pv. campestris 8004 and its esterase domain: gene expression in Escherichia coli, refolding and characterization.
abstract: OBJECTIVE
  To characterize the GDSL (glycine, aspartic acid, 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 8.0 and 52 degrees 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 mol/L, 67.6 +/- 7.8 U/mg and 469.4 +/- 9.8 mol/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 degrees C.
  
  
  CONCLUSIONS
  Refolded Xcc_ estN26-606 can be a candidate for biotransformation application.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. wangjj@sun.im.ac.cn
authors.forename: Jianjun
authors.initials: J
authors.lastname: Wang
authors.forename: Liu
authors.initials: L
authors.lastname: Yang
authors.forename: Yanping
authors.initials: Y
authors.lastname: Cao
authors.forename: Guojun
authors.initials: G
authors.lastname: Zheng
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D004950:Esterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D016959:Xanthomonas campestris / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D004950:Esterases