Production, purification and spectral properties of metal-dependent beta-lactamases of Bacillus cereus.
New methods for the production of consistently high levels of metal-dependent beta-lactamases (beta-lactamhydrolase, EC 3.5.2.6) from strains 569/H/9 and 5/B/6 of Bacillus cereus are described which have significant advantages over those reported previously. For example, these techniques do not require a fermentor with pH-stat capabilities. We also describe rapid very-high-yield purification schemes for the metal-dependent beta-lactamases from these strains, employing high-performance ultrafiltration (HPUF) and mass ion exchange techniques. Furthermore, we have developed improved methods for the removal of the active site Zn(II) and reconstitution of the beta-lactamase enzymatic activity with Co(II), which result in higher recovery of the original activity than previously reported. In order to characterize the purified beta-lactamases II of B. cereus 569/H/9 and 5/B/6 we have examined the molecular weights, and steady state kinetic parameters of Zn(II) enzymes, and the electronic and EPR spectra of the Co(II)-reconstituted enzymes. EPR spectra of CO(II)-reconstituted beta-lactamase from B. cereus 5/B/6 have not been previously reported.
Myers
J L
JL
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock 79409.
Shaw
R W
RW
D001409:Bacillus cereus / Q000201:enzymology* / Q000254:growth & development; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D007700:Kinetics; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism
D001618:beta-Lactamases