A modified osmotic shock involving pretreatment of the cells with divalent cation (Ca2+ or Mg2+) for periplasmic release of a recombinant creatinase from Escherichia coli was proposed. The pretreatment enhanced the release of lipopolysaccharide (LPS) when the cells were subsequently treated with ethylenediaminetetraacetate (EDTA), thus increased the permeability of the outer membrane. Based on the data of cell disruption by sonication, the standard osmotic shock resulted in a 60% recovery of creatinase and a 3.9-fold purification. With Ca-pretreatment, the efficiency of osmotic shock could be improved to 75% recovery and 4.5-fold purification. The pretreatment involved incubation of the cells with 5 mM Ca2+ for 5 min, which was accompanied by an increased EDTA concentration (from 0.5 to 5 mM) in the subsequent osmotic shock. Mg-pretreatment could function similarly, but was less effective. When the cells were pretreated simultaneously with Ca 2+ and Mg2+, no additive effect on the periplasmic release was found, indicating that the binding sites on LPS are common to both cations. A possible adsorption isotherm for the binding of the cations to LPS was postulated. In addition, a protocol capable of dealing with suspensions of increased cell concentration was suggested.
All Science Journal Classification (ASJC) codes
- Environmental Engineering
- Biomedical Engineering