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.
Highlights
The study of extracellular polyhydroxybutyrate (PHB) depolymerases (PhaZs) produced by a variety of microorganisms [1,2,3] remains an important and evolving research area
– Pre-Print – Buffer exchange was done prior to assays in which imidazole and glycerol caused interference with Amicon Ultra 0.5-mL filters or dialysis (Slide-A-Lyzer MINI Dialysis Devices, 20K MWCO, Thermo Scientific). 2.5 PHB plates Recombinant His-tagged versions of PhaZs (rPhaZ) activity comparison Rapid PHB degradation assays were performed by dispensing 100 μl of soluble fractions in cylindrical wells made in double-layer mineral medium/agar plates containing PHB (medium 474: 20 ml first layer — mineral medium with agar (0.016 g/ ml) — and 10 ml second layer — mineral medium with agar supplemented with 0.66 ml of sterile PHB suspension)
Since PhaZCte was classified as insoluble, and the other PhaZs had scores (0.657– 0.765) near the PROSO II threshold for solubility of 0.6, insolubility was considered a potential drawback for production of rPhaZs
Summary
The study of extracellular polyhydroxybutyrate (PHB) depolymerases (PhaZs) produced by a variety of microorganisms [1,2,3] remains an important and evolving research area. Purification of rPhaZs has been performed: several PhaZs from P. lemoignei (PhaZ1–PhaZ5) [12,13], PhaZ7 and related mutants [14]), Pseudomonas stuzeri [15], Alcaligenes faecalis AE122 [16], Marinobacter sp. (formerly Alcaligenes faecalis T1) and related mutants [20,21,22,23,24] These studies each required the development of specific methods for heterologous expression of specific PhaZs. In addition, in many cases affinity tags were not employed, requiring significant additional steps for purification [12,13,16,21,22,23]. – Pre-Print – strategies was successfully employed for expression, purification, and preliminary comparison of degradation performance under different conditions
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