R Kuboi — Page 2 | PubMed Health

PubMedDate not supplied

Utilization of cell response under heat, chemical, and combined stresses for selective recovery of cytoplasmic beta-galactosidase from Escherichia coli cells.

The effective bioseparation process was developed by exploiting the cell response under heat, chemical, and combined stresses for selective recovery of cytoplasmic beta-galactosidase from Escherichia coli cells. At the observed optimal condition for heat stress (45 degrees C, 30 min) and that for chemical stress (20 mM Triton X-100, 5 mM EDTA, 30 min), E. coli cells were exposed to these stresses either simultaneously or sequentially. The operational sequence highly affected the yield and selectivity of the target recovery. Exposing the cells to the sequential stress (heating at 45 degrees C for more than 30 min after Triton X-100/EDTA treatment for 30 min) was found to be most effective for selective recovery of target beta-galactosidase among possible sequences.

Biotechnology↗

PubMedDate not supplied

Conformationally changed cytochrome c-mediated fusion of enzyme- and substrate-containing liposomes.

The fusion between enzyme-containing liposomes and substrate-containing liposomes was studied, utilizing conformationally altered cytochrome c as fusion mediator under stress conditions. The liposomes were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and liposome aggregation and subsequent liposome fusion were induced by the addition of cytochrome c, which was partially denatured by 0.5 M guanidinium hydrochloride (GuHCl). In the presence of 0.5 M GuHCl, cytochrome c was found to have a significantly large local hydrophobicity which was determined with the aqueous two-phase partitioning method. Under these conditions, cytochrome c could efficiently bind to POPC bilayer membranes as quantitatively evaluated by immobilized liposome chromatography (ILC). The retardation of cytochrome c treated with 0, 0.5, and 1 M GuHCl on ILC could be correlated with the corresponding local hydrophobicity of cytochrome c. The enzymatic reaction triggered by liposome fusion involved the proteolytic enzyme alpha-chymotrypsin and its substrate succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Suc-AAPF-pNA), which were separately trapped in POPC liposomes. Addition of partially denatured cytochrome c (most likely in the molten globule state) to the mixture of enzyme- and substrate-containing liposomes resulted in the release of one of the hydrolysis products, p-nitroaniline, to the outer phase of the fused liposomes, indicating that the enzymatic reaction occurred during the liposome fusion process. Such a coupled fusion-reaction system may have specific advantages over the conventional fusion analysis and may find application as drug delivery system.

Cell Membrane Permeability↗