Heat‐Induced Translocation of Cytoplasmic β‐Galactosidase across Inner Membrane of Escherichiacoli

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Abstract
The behaviors of heat‐induced translocation of cytoplasmic β‐galactosidase to periplasm across the inner membrane of Escherichia coli cells were investigated in order to apply such phenomena to the process for production and separation of intracellular biomolecules. The heat stress was found to induce translocation of cytoplasmic β‐galactosidase (β‐gal) together with reduction of the amounts of intracellular soluble proteins and formation of their inactive aggregates. The translocation of β‐gal was then analyzed using (a) the location factor of β‐gal (LFG), which meant enzyme location in the cells and could be determined from the kinetic analysis of enzyme release process, and (b) the percentage of β‐gal activity in periplasm after solublizing the outer membrane of E. coli cells by lysozyme/EDTA treatment. The LFG values were maximized when cells were stressed at the temperature of 42–47 °C. From the results on the surface properties of both β‐gal and cell membrane under the heat stress, it is suggested that (1) the conformational change of cytoplasmic oligomeric β‐gal to the partially dissociated and/or unfolded state with higher local hydrophobicity, (2) the increase in membrane fluidity of inner membrane, (3) the enhancement of hydrophobic interaction between lipid and protein, and (4) the inhibition of its translocation by GroEL restabilizing the proteins could underlie the heat‐induced translocation of β‐gal across the inner membrane. The possibility to apply the heat‐induced translocation of β‐gal for the enhancement of the target selectivity at the process upstream is finally presented.

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