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

A rapid, efficient approach for the expression of active recombinant extracellular polyhydroxybutyrate (PHB) depolymerases originating from various bacterial strains. It enables the characterization of new PHB depolymerases and the direct comparison of activity between enzymes and conditions; as an example, the activity of five PHB depolymerases is evaluated and compared at different pH and temperatures. This approach can accelerate the investigation and development of applications based on the degradation of biopolymers. AbstractHeterologous 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 inEscherichia coli, we propose a method that takes characteristics of PhaZs from different bacterial strains into account. Recombinant His ‐tagged versions of PhaZs (rPhaZ) fromComamonas testosteroni 31A,Cupriavidus sp. T1, Marinobacter algicola DG893,Pseudomonas stutzeri, andRalstonia sp. were successfully produced with varying expression, solubility, and purity levels. PhaZs fromC.  testosteroni andP.  stutzeri were more amenable to heterologous expression in all aspects; however, using theE.  coli Rosetta ‐gami B(DE3) expression strain and establishing optimal conditions for expression and purification (variation of IPTG concentration and use of size excl...
Source: MicrobiologyOpen - Category: Microbiology Authors: Tags: ORIGINAL ARTICLE Source Type: research