Streamlined production, purification, and characterization of recombinant extracellular polyhydroxybutyrate depolymerases

Abstract

Heterologous production of extracellular polyhydroxybutyrate (PHB) depolymerases (PhaZs) has been of interest for over 30 years, but implementation is sometimes difficult and can limit the scope of research. With the constant development of tools to improve recombinant protein production in Escherichia coli, we propose a method that takes characteristics of PhaZs from different bacterial strains into account. Recombinant His‐tagged versions of PhaZs (rPhaZ) from Comamonas testosteroni 31A, Cupriavidus sp. T1, Marinobacter algicola DG893, Pseudomonas stutzeri, and Ralstonia sp. were successfully produced with varying expression, solubility, and purity levels. PhaZs from C. testosteroni and P. stutzeri were more amenable to heterologous expression in all aspects; however, using the E. coli Rosetta‐gami B(DE3) expression strain and establishing optimal conditions for expression and purification (variation of IPTG concentration and use of size exclusion columns) helped circumvent low expression and purity for the other PhaZs. Degradation activity of the rPhaZs was compared using a simple PHB plate‐based method, adapted to test for various pH and temperatures. rPhaZ from M. algicola presented the highest activity at 15°C, and rPhaZs from Cupriavidus sp. T1 and Ralstonia sp. had the highest activity at pH 5.4. The methods proposed herein can be used to test the production of soluble recombinant PhaZs and to perform preliminary evaluation for applications that require PHB degradation.

Three-Dimensional Printed and Biocompatible Conductive Composites Comprised of Polyhydroxybutyrate and Multiwalled Carbon Nanotubes

“…In recent years, PHB has become increasingly utilized in medical applications due to its good biological performance, thermal stability, and thermoplastic properties. Although it has been shown that PHB can be enzymatically degraded in the environment by various types of bacteria, , it has also been shown to be relatively stable in the body, undergoing a mass loss of only 0–1.6 wt % during 6 months of implantation (depending on its chemical formulation) . For some applications, electrical conductivity would be a benefit.…”

Novel Production Methods of Polyhydroxyalkanoates and Their Innovative Uses in Biomedicine and Industry

“…Molecular approaches, such as RBS optimization, promoter/repressor optimization, gene deletion, or expression-weakening methods, have been developed in non-model organisms [ 48 ]. For instance, different PHA may be obtained by weakening the beta-oxidation cycle in Pseudomonas putida and Pseudomonas entomophila [ 49 ]. Other strategies include the downregulation of branch products, controlling oxygen availability, regulation of NADH/NAD+ ratios, increasing oxygen availability, and regulation of the PHA molecular weight and granules, forming larger PHA granules and regulating PHA molecular weight also help to enhance PHA production.…”