The effect of secondary reactions on DHAP-dependent aldolase stereoselective synthesis yields is reported. The fuculose-1-phosphate aldolase catalyzed synthesis between DHAP and Cbz-S-alaninal has been chosen as case study. It has been demonstrated that DHAP is not only chemically degraded in the reaction medium, but also enzymatically. The last reaction has been shown to take place when type II aldolases are used as biocatalysts. In order to minimize the effect of non-desired reactions, temperature reduction has been shown to be favorable, and operation at 4 degrees C has been chosen as appropriate. On the other hand, the fed-batch addition of DHAP also increased the synthesis yields and, combined with low temperature, led to almost quantitative conversion.

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.

No abstract

No abstract