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

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 4 h, induction time of 12 h at 37 degrees C were identified as the optimal conditions for the enzyme reductase expression. A specific activity of 0.151 U mg(-1) protein and total protein concentration of 0.518 mg mg(-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 U mg(-1)) and purification fold (38-fold), despite relative low yield (4 4.6%) of the enzyme was obtained. The purified phaB showed an optimal enzyme activity at 30 degrees 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.(c) 2023, The Society for Biotechnology, Japan. All rights reserved.

Automated summary

This study successfully expressed the key enzyme acetoacetyl-CoA reductase in the synthesis of poly-3-hydroxybutyrate using Escherichia coli and purified the enzyme. High enzyme activity and purification fold were obtained under optimal induction time, temperature, and cell age conditions. The findings provide an alternative method for industrial-scale production of biodegradable polymers.