Expression of His-tagged NADPH-dependent acetoacetyl-CoA reductase in recombinant Escherichia coli BL-21(DE3)

Abstract

Poly-3-hydroxybutyrate (P(3HB)), a member of the polyhydroxyalkanoate (PHA) family, is a biodegradable polyester with diverse industrial applications. NADPH-dependent acetoacetyl-CoA reductase (phaB) is the enzyme which plays an essential role in P(3HB) synthesis by catalyzing the conversion of the intermediates. The expression of phaB enzyme using the recombinant Escherichia coli BL-21(DE3) and the purification of the synthesized enzyme were studied. The pET-B3 plasmid harbouring the phaB gene derived from Ralstonia eutropha H16, was driven by the lac promoter in E.coli BL-21(DE3). The enzyme was expressed with different induction time, temperatures and cell age. Results showed that the cell age of 4h, induction time of 12h at 37°C were identified as the optimal conditions for the enzyme reductase expression. A specific activity of 0.151 Umg-1 protein and total protein concentration of 0.518mgmg-1 of dry cell weight (DCW) were attained. Affinity chromatography was performed to purify the His-tagged phaB enzyme, in which enhanced the specific activity (14.44 Umg-1) and purification fold (38-fold), despite relative low yield (44.6%) of the enzyme was obtained. The purified phaB showed an optimal enzyme activity at 30°C and pH 8.0. The findings provide an alternative for the synthesis of the reductase enzyme which can be used in the industrial-scale production of the biodegradable polymers.