Abstract:[Objective] To investigate the feasibility of substituting lactose for isopropyl-β-D-1-thiogalactopyranoside (IPTG) in inducing efficient expression of 3-ketosteroid-Δ1-dehydrogenase (KstD) in Escherichia coli (E. coli） BL21(DE3). [Method] Upon the established IPTG-induced protocol for obtaining high yields of soluble and catalytically active recombinant protein SZD-KstD, the lactose induction parameters under shake-flask culture conditions were optimized. Subsequently, high-density fermentation was conducted in a 15 L bioreactor. Critical process parameters influencing expression of the recombinant protein SZD-KstD were evaluated, including addition time, lactose induction method, induction duration, and carbon/nitrogen feeding protocols. [Result] The maximum amounts of SZD-KstD and bacteria were obtained under the induction with lactose at the final concentration of 0.2 mmol/L. Since lactose can be used as a carbon source at the same time, adding lactose in three batches outperformed adding it in one batch. Under lactose induction conditions, SZD-KstD accounted for 39.58% of total soluble protein content, which was not significantly different from that under IPTG induction. [Conclusion] Lactose, as a cheap and efficient inducer, can replace IPTG for the large-scale fermentation of KstD, which solves a major problem for the preparation of steroid drugs by bioenzyme method and provides a reference for the production of other recombinant proteins.
