Violaxanthin conversion by recombinant diatom and plant de-epoxidases, expressed in Escherichia coli - comparative analysis.

Violaxanthin conversion by recombinant diatom and plant de-epoxidases, expressed in Escherichia coli - comparative analysis. Acta Biochim Pol. 2019 Jul 07;: Authors: Olchawa-Pajor M, Bojko M, Strzałka W, Strzałka K, Latowski D Abstract The purpose of this research was to obtain recombinant violaxanthin de-epoxidases (VDEs) from two species. The first one was VDE of Arabidopsis thaliana (L.) Heynh. (WT Columbia strain) (AtVDE) which in vivo catalyzes conversion of violaxanthin (Vx) to zeaxanthin (Zx) via anteraxanthin (Ax). The second one was VDE of Phaeodactylum tricornutum Bohlin, 1897 (CCAP 1055/1 strain) (PtVDE) which is responsible for de-epoxidation of diadinoxanthin (Ddx) to diatoxanthin (Dtx). As the first step of our experiments, open reading frames coding for studied enzymes were amplified and subsequently cloned into pET-15b plasmid. For recombinant proteins production Escherichia coli Origami b strain was used. The molecular weight of the produced enzymes were estimated approximately at 45kDa and 50kDa for AtVDE and PtVDE, respectively. Both enzymes, purified under native conditions by immobilized metal affinity chromatography, displayed comparable activity in assay mixture and converted up to 90% Vx in 10 min in two steps enzymatic de-epoxidation, irrespective of enzyme origin. No statistically significant differences were observed when kinetics of the reactions catalyzed by these enzymes were compared. Putative role of...
Source: Acta Biochim Pol - Category: Biochemistry Authors: Tags: Acta Biochim Pol Source Type: research