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.

such as Escherichia coli. Optimized recombinant technologies facilitate purification, the study of proteins in isolation, the conception of a platform to modify and improve them, and the development of new applications. In the case of PhaZs, such applications include biosensors-such as time-temperature indicators (Anbukarasu et al., 2017) and pathogen detection platforms -and recycling of biodegradable polymers (Lee et al., 2018).
In this study, we established a platform for the rapid expression

| Bacterial strains and growth conditions
The bacterial strains used for isolation of the PhaZs, cloning and expression, as well as their growth medium and conditions, can be found in Table 1. Cell growth was monitored by measuring optical density of the cultures at 600 nm (OD 600 ) using a UV-Vis spectrophotometer (Biochrom, Ultrospec 50). Plating was performed on 1.5% w/v agar supplemented with the medium of interest and plates were incubated in a temperature-controlled incubator (Isotemp 500 Series, Fisher Scientific).

| PhaZ constructs
Genomic DNA was extracted from the PhaZ-producing strains (RNA/ DNA purification kit, Norgen Biotek for C. testosteroni, and GeneJET Genomic DNA Purification Kit, ThermoFisher Scientific for other strains). Inserts were obtained by amplifications of the mature phaZ genes (without signal peptides)-with primers (5′ to 3′ direction,

| Induction screening and His-tag verification
Starter cultures from single colonies of E. coli  or T7 Express lysY/I q E. coli containing the constructs were grown in 5 ml LB with corresponding antibiotics (Table 1b)  SF and insoluble fractions (IF) were then characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, based on the methods described by Laemmli (Laemmli, 1970 PhaZ expression levels on SDS-PAGE and activity on PHB plates: very high (++), high (+), medium (±), low (-), very low (--), and inactive/insoluble (---).
b Activity was monitored over 1 week of incubation.
The gels were then washed with Milli-Q water. Images of the gels were acquired under UV exposure (AlphaImager EC, Alpha Innotech) or with a regular camera. In the case of PhaZ Cte and Phaz Mal , the presence of the His-tag was verified through Western blot analysis by using mouse anti-His6 monoclonal antibody, and goat anti-mouse DyLight 488 secondary antibody (Life Sciences).  (Dawson, Elliott, Elliott, & Jones, 1986

| Comparison of rPhaZ activity
While PHB plates have been mostly used to screen for PHB-degrading bacteria (Jendrossek, 2005), activity from expressed PhaZs has also been estimated by clear zones on glass slides covered by PHB-agar mix (Briese et al., 1994;Jendrossek et al., 1993) (this test is limited to short-term incubations due to agar drying-unless a humidity chamber is used for incubation-but is advantageous for preliminary assessment and when only small volumes of sample are available).
In this study, an easy-to-use method using PHB plates was used to compare PhaZ activity at various pH and temperatures and provided semiquantitative assessments of activity based on the diameter of degradation halos formed (Figure 2). At 37°C, degradation was observed on the first day of incubation for all rPhaZs tested, while longer incubation periods were required at 15°C. PhaZ Mal showed the highest activity at 15°C (halo observed after 1 day) compared with the other rPhaZs (halos observed after 6 days), which could be explained by its marine origin (Green, Bowman, Smith, Gutierrez, & Bolch, 2006). All enzymes were rendered inactive at pH 4.3 (no halos discernable) but PhaZ Csp and PhaZ Rsp retained significant activity at pH 5.4, which can render these enzymes useful for applications under conditions below neutral or alkaline pH. This is important as most PhaZs identified to date display optimal activity at higher pH and inactivity at low pH. Also, this is consistent with their broad pH working ranges (PhaZ Csp is stable when stored at pH 5.0-8.0 (Kasuya, Inoue, Yamada, & Doi, 1995) with optimum activity at pH 7.5 (Tanio et al., 1982), and the optimum pH range of PhaZ Rsp is 5.0-6.0 ).
These results could be confirmed and semiquantified by comparing the rate of change of the degradation halos under the different conditions tested (Figure 2 (Hiraishi et al., 2006).

| CON CLUS IONS
This study presents a streamlined platform for the rapid production of rPhaZs. Five active PHB-degrading extracellular PhaZs

CO N FLI C T O F I NTE R E S T
None declared.

AUTH O R CO NTR I B UTI O N
Diana Martínez Tobón contributed equally to conceptualization, took the lead in formal analysis, investigation, methodology, writing-original draft, and writing-review and editing. Brennan Waters supported in investigation and methodology. Anastasia Elias contributed equally to conceptualization, funding acquisition, project administration, supervision, and writing-review and editing, and supported in methodology. Dominic Sauvageau contributed equally to conceptualization, funding acquisition, project administration, supervision, writing-review and editing, and supported in formal analysis, methodology, and writing-original draft.

E TH I C S S TATEM ENT
None required.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated in this study are available from the corresponding author upon reasonable request.