Please use this identifier to cite or link to this item: http://hdl.handle.net/10397/63781
Title: Recombinant Enterobacter cloacae P99 Amp Cbeta-lactamase and its mutants : overexpression in Bacillus subtilis, purification and characterization
Authors: Tsang, MW
Leung, YC 
Issue Date: 2005
Publisher: Wiley-Blackwell
Source: FEBS journal, 2005, v. 272, no. Suppl. 1, p. 518-519 How to cite?
Journal: FEBS journal 
Abstract: With the recent rapid spread of Amp C beta-lactamase-mediatedantibiotic resistance, studies on structure–function relationship ofAmp C beta-lactamase is becoming an important task in the clin-ical and pharmacological aspects for drug improvement andrational inhibitor design. In our study, new strategies have devel-oped in expressing recombinant Enterobacter cloacae P99 AmpC beta-lactamase in Bacillus subtilis. With the utilization of athermo-inducible phi 105 MU331 system, functionally activeAmp C beta-lactamase was successfully expressed in Bacillus sub-tilis, either intracellularly with N-terminal histidine-tag or extra-cellularly as a native enzyme. Our results indicated that higherenzyme expression level was accomplished by producing theenzyme as an intracellular histidine-tagged protein rather than asa native extracellular protein. Moreover, with the use of theintracellular histidine-tagged expression strategy, more than 95%pure histidine-tagged P99 wild-type, K315C and Y150C mutantswere obtained by a single step of nickel affinity chromatographywith a yield of 29 mg/l of culture, 6 mg/l of culture and 66 mg/lof culture respectively. In addition, the roles of the conserved res-idues, K315 and Y150, in the Amp C beta-lactamase were exam-ined. The observation of a drastic impairment of the catalyticefficiency of the two mutants on nitrocefin, a cephalosporin-type beta-lactam antibiotic, was consistent with the proposed catalyticroles of these two residues. On the contrary, as revealed by thefar-UV CD analysis, replacement of cysteine at positions 315 and150 did not cause any significant structural perturbation of theenzyme. This implied that these two residues are not involved inthe structural architecture of the Amp C enzyme.
URI: http://hdl.handle.net/10397/63781
ISSN: 1742-464X
EISSN: 1742-4658
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