An expression system based on Escherichia coli and the T5 promoter allowed the overproduction of a his-tagged rhamnulose-1-phosphate aldolase (RhuA; EC 188.8.131.52), an enzyme with applications in the production of deoxyazasugars and deoxysugars compounds. Shake flask and bioreactor cultivation with E coli Ml 5 (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-3h-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. © 2003 Society of Chemical Industry.
- E coli
- Recombinant aldolase