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Wei Sheng Wu Xue Bao. 2009 Feb;49(2):191-7.

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.

Author information

1
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. wangjj@sun.im.ac.cn

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.

PMID:
19445174
[Indexed for MEDLINE]

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