Large-scale production of thermostable alanine dehydrogenase from recombinant cells

We have cloned the thermostable alanine dehydrogenase gene from a thermophile, Bacillus stearothermophilus into Escherichia coli C600 with a vector plasmid pKK 223-3. The recombinant cells produced a large amount of the enzyme, which corresponded to about 10% of the total amount of cellular proteins. In order to prevent the cells from leaking to the environment, cell culture and disruption were consecutively carried out in the same fermentor equipped with a HEPA filter, which served as a biological barrier. The cells grown in the culture broth were treated with Triton X-100, lysozyme, and EDTA, whose final concentrations were 0.1%, 100 mg/l, and 10 mM, respectively, for 30 min, followed by heating at 60°C for 30 min. Almost all of the enzyme (about 98%) was found in the broth, whereas no living recombinant cells were found in the broth, by these treatments. Based on these we established a large-scale production system of thermostable enzyme from recombinant cells.