AbstractAn expression system based on Escherichia coli and the T5 promoter allowed the overproduction of a his‐tagged rhamnulose‐1‐phosphate aldolase (RhuA; EC 4.1.2.19), an enzyme with applications in the production of deoxyazasugars and deoxysugars compounds. Shake flask and bioreactor cultivation with E coli M15 (pQErham) were performed under different media and inducing conditions for RhuA expression. A Defined Medium (DM) with glucose as carbon source gave a high volumetric and enzyme productivity (3460 AU dm−3 and 288 AU dm−3 h−1 respectively) compared with Luria–Bertoni (LB) medium (2292 AU dm− 3 and 255 AU dm−3 h−1). The minimum quantity of (isopropyl‐β‐D‐thiogalactoside) IPTG for optimal induction was estimated in 18–20 µmol IPTG gDCW−1. The highest volumetric production of RhuA (8333 AU dm−3) was obtained when IPTG was added in the late log‐phase. No significant differences were found in specific RhuA activity for induction temperatures of 30 and 37 °C. An effective two‐step purification process comprising affinity chromatography and gel permeation has been developed (overall recovery 66.5%). These studies provide the basis for the further development of an integrated process for recombinant RhuA production suitable for biotransformation applications. Copyright © 2003 Society of Chemical Industry