Purification of metallo-B-lactamase CcrA from Bacteroides fragilis with salting-out method



Document title: Purification of metallo-B-lactamase CcrA from Bacteroides fragilis with salting-out method
Journal: Brazilian archives of biology and technology
Database: PERIÓDICA
System number: 000366259
ISSN: 1516-8913
Authors: 1
1
1
1
Institutions: 1Northwest University, College of Chemistry and Materials Science, Xi'an, Shaanxi. China
Year:
Season: Sep-Oct
Volumen: 56
Number: 5
Pages: 811-816
Country: Brasil
Language: Inglés
Document type: Artículo
Approach: Experimental, analítico
English abstract A simple and effective salting-out method was developed for the purification of the metallo-β-lactamase CcrA from Bacteroides fragilis based on the plasmid pMSZ02, in which the crude protein secreted into growth medium was precipitated by 80% sulfate saturation of the medium and purified with Q-Sepharose to offer pure CcrA with yield of 20.1 mg per litter medium. The dependence of the amount of protein precipitation on sulfate saturation was investigated, which showed that more than 80% sulfate saturation resulted the maximum protein precipitated. The purified CcrA was evaluated by steady-state kinetics using penicillin G and cephalothin V as substrates, which showed the Kmvalues of 68±2 and 17±2 µM and Kcatvalues of 63±1 and 102±3 S-1, respectively. The comparison with the data of the protein from literature method showed that the salting-out method was viable, and it could be useful for the purification of other proteins secreted into growth medium
Disciplines: Biología,
Química
Keyword: Bacterias,
Bioquímica,
Microbiología,
Bacteroides fragilis,
Metalo-beta-lactamasa,
Purificación,
Precipitación salina
Keyword: Biology,
Chemistry,
Bacteria,
Biochemistry,
Microbiology,
Bacteroides fragilis,
Metallo-betha-lactamase,
Purification,
Salting out process
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