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

✍ Scribed by L Vidal; O Durany; T Suau; P Ferrer; MD Benaiges; G Caminal

• Book ID: 102327500
• Publisher: Wiley (John Wiley & Sons)
• Year: 2003
• Tongue: English
• Weight: 190 KB
• Volume: 78
• Category: Article
• ISSN: 0268-2575
• DOI: 10.1002/jctb.909

✦ Synopsis

Abstract

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 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