Improving the production of a thermostable amidase through optimising IPTG induction in a highly dense culture of recombinant Escherichia coli.

Journal Article


Journal Title

Biochemical Engineering Journal

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University of Cape Town

The production of a novel thermostable amidase (EC from Geobacillus pallidus RAPc8 using recombinant Escherichia coli BL21 (DE3) was investigated. Volumetric and specific enzyme activities were investigated in relation to inducer concentration in a batch process using a defined medium with glucose as the carbon source. While IPTG is routinely used to induce expression of genes under the control of lac promoter, the impact of high biomass concentration on IPTG induction has not been reported rigorously. In this study, biomass production was unaffected by IPTG concentration across the range 0–1000 M. Induction of recombinant protein expression by 400 M IPTG at late lag phase of growth (3rd hour) inhibited cell growth while induction at early exponential phase of growth (5th hour) gave a 3 fold increase in volumetric amidase activity compared to induction at mid exponential phase (8th hour). Protein production increased by a factor of two with IPTG addition, independent of IPTG concentration in the range of 40–1000 M. Amidase activity, measured on a volumetric basis and relative to protein and biomass concentrations, increased with increasing IPTG concentration up to 400 M. While inducer concentrations are typically reported on a volumetric basis, their mode of action is consistent with a biomass dependence. Analysis of the data across a range of biomass concentration confirmed that induction was a function of inducer concentration per unit biomass. The amidase enzyme was predominantly soluble and cytoplasmic with less than 3% retained within the cell debris.