High-level production of recombinant His-tagged rhamnulose 1-phosphate aldolase in Escherichia coli

An 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 IZhuA 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-beta-D-thiogalactoside) IPTG for optimal induction was estimated in 18-20 mumol 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 37degreesC. 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. (C) 2003 Society of Chemical Industry.