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High-level production of recombinant His-tagged rhamnulose 1-phosphate aldolase in Escherichia coli
|Authors:||Vidal, Luis; Durany, Olga; Suau, T.; Ferrer, Pau; Benaiges, M. D.; Caminal, Glòria|
|Citation:||Journal of Chemical Technology and Biotechnology 78(11): 1171-1179 (2003)|
|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 126.96.36.199), 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--D-thiogalactoside) IPTG for optimal induction was estimated in 18-20 ºmol 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 ºC. 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.|
|Description:||9 pages, 8 figures, 2 tables.-- Printed version published Nov 2003.|
|Publisher version (URL):||http://dx.doi.org/10.1002/jctb.909|
|Appears in Collections:||(IQAC) Artículos|
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