Extracellular production of cloned α-amylase by Escherichia coli

Overexpression of Bacillus stearothermophilus gene coding for thermostable α-amylase in Escherichia coli was shown to cause outer-membrane damage leading to extracellular location of periplasmic proteins. Prolonged high expression of the α-amylase gene under lacZpo control eventually also lysed cells. Surprisingly, expression controlled by the p l promoter of phage λ allowed specific release of periplasmic proteins into the growth medium without total cell lysis. Accumulation of α-amylase in the growth medium continued for at least 24 h under λ p l control, whereas β-lactamase activity ceased to increase beyond the exponential growth phase. The extent of outer membrane damage caused by α-amylase expression was monitored by following growth kinetics in the presence of lysozyme and by electron microscopy of the cells. Supplementing growth medium with Mg 2+ restored the normal growth kinetics. It is suggested that periplasmic protein release caused by α-amylase overexpression is a stress response of the cell. A role for induced autolytic activity of the cell as a final effector of protein release is also proposed.