Characterization of an intracellular poly(3-hydroxyalkanoate) depolymerase from the soil bacterium, Pseudomonas putida LS46
Introduction
Polyhydroxyalkanoate (PHA) polymers have emerged as a potential alternative to petrochemical-based conventional plastics due to their high biodegradability, chemical diversity, their manufacture from renewable carbon resources, and release of nonpolluting products after degradation [1]. PHAs are synthesized and accumulated by many prokaryotic microorganisms as storage compounds for carbon and energy when non-carboneous nutrients (e.g., nitrogen or phosphorus) are limiting. The accumulation of these polymers facilitates enhanced survival under environmental stress conditions. Based on the chain length of the incorporated 3-hydroxy fatty acids, PHAs are grouped into short chain length (scl-)PHAs, which consist of subunits with carbon-chains of 3–5 C-atoms, and medium chain length (mcl-)PHAs, which consist of subunits containing 6-14 C-atoms.
The biodegradation of PHA by depolymerases has attracted much attention in recent years for the production of enantiopure (R)-3-hydroxyalkanoic acids (RHAs), i.e., the monomeric, dimeric and/or oligomeric units of the polymer [2,46]. RHAs have gained popularity as potential antibacterial, antiviral, synthons for organic synthesis and biofuels [3,50]. It has been used for the synthesis of a class of antibiotics called macrolides [4]. PHA depolymerases are classified as intracellular and extracellular PHA depolymerases based on their mode of action towards substrates. Intracellular PHA depolymerases degrade the PHA granules that are deposited as carbon reservoir within the bacterial cell. PHA granules consist of PHA polymers with a surface layer of proteins and phospholipids. Extracellular PHA depolymerases are secreted from the bacterial cells to hydrolyze and degrade extracellular PHA granules, which lack a surface layer and are partially crystalline. These are further grouped into four families based on the substrate specificity: intracellular PHA depolymerases are labelled as “nPHAscl” and “nPHAmcl” depolymerases; extracellular PHA depolymerases are labelled as “dPHAscl” and “dPHAmcl depolymerases” [5]. dPHAscl depolymerases have been reported from Acidovorax sp. TP4 [6], Bacillus sp. strain NRRL B-14911 [7], Streptomyces ascomycinicus [8], Burkholderia cepacia DP1 [9] and Paucimonas lemoigei [10]. dPHAmcl depolymerases have been identified in the obligated predator Bdellovibrio bacteriovorus HD100 [11], in the Actinobacteria Streptomyces roseolus SL3 [12] and Streptomyces venezuelae SO1 [13]. While intracellular scl-PHA depolymerases have been studied in Hydrogenomonas H 16 [14], Alcaligenes eutrophus [15], Hydrogenophaga pseudoflava [16], Ralstonia eutropha H16 [17], Paracoccus denitrificans [18], Azospirillum brasilense [19], Rhodospirillum rubrum [20], Bacillus thuringiensis [21] and Bacillus megaterium [22]. nPHAmcl depolymerases have been found in P. oleovorans [23], P. putida KT2442 [24] and P. chlororaphis PA23 [25].
Based on the location and physical state of the PHA polymer, degradation process could either occur in amorphous state or denatured state. Apparently, the nPHA gets metabolized by intracellular PHA depolymerases releasing HA monomers and thus, act as exo-hydrolases as reported in the case of in vivo intracellular polymer degradation by the PHA depolymerase of Pseudomonas putida KT2442 [24], whereas dPHA being crystalline gets converted into HA dimers as shown by the endo-acting depolymerases from P. fluorescens GK13 [26,27] and B. bacteriovorus HD100 [11] or oligomers recorded in Ralstonia pickettii T1 [28], Acidovora sp. SA1 [28] and Ralstonia eutropha H16 [29].
Technological advancement has increased the growth rate in the worldwide production of high value added PHAs and their biomedical applications. However, limited studies have been performed on improving the yield of enantiomerically pure RHAs using PHA depolymerases, and the biomedical application of PHA monomers and dimers as antimicrobials [3,30]. In this respect, genetic engineering of the PHA degradation enzymes can be used as a powerful tool to increase the yield of RHAs for biomedical application. Further, PHA degrading microorganisms, especially mcl-PHA degraders are present in low abundance in the environment, and therefore, limited information is available on the properties of these polymer-degrading enzymes. We have investigated the biophysical and biochemical properties of the recombinant PHA depolymerase enzyme, PhaZLS46 from Pseudomonas putida LS46 and its applicability in the degradation of PHA polymers for the production of value-added R-hydroxyalkanoic acids. We also evaluated the antibacterial potential of the extracted RHAs.
Section snippets
Bacterial strains and plasmids
Pseudomonas putida LS46, isolated from a local wastewater treatment plant in Winnipeg, Manitoba, Canada (International Depository Authority of Canada Accession Number 181110-03) [31] was used for the production of PHA polymers. Eschericila coli BL21 was used for testing the antibacterial potential of the hydroxyalkanoic acids obtained by degradation of PHA. Escherichia coli DH5α and E. coli BL21 (DE3) were used as host strain for plasmid construction and protein expression, respectively.
Cloning, expression and purification of PhaZLS46
Pseudomonas putida LS46 has been well characterized for its ability to synthesize high concentrations of mcl-PHAs when cultured with different carbon sources [31]. The PHA synthesis forms a part of the central metabolic pathway in P. putida [31]. The PHA synthesis operon in P. putida comprises two PHA synthase genes (phaC1 and phaC2) flanked by a PHA depolymerase gene (phaZ), and three regulatory genes (phaD, phaF and phaI) encoding a transcriptional activator of pha genes [36]. The
Conclusions
The recombinant mcl-PHA depolymerase of Pseudomonas putida LS46 is significantly different compared to the known mcl-PHA depolymerases in terms of the substrate specificity. The ability of the P. putida LS46 (PhaZLS46) intracellular depolymerase to hydrolyze wide-range of substrates from PHA polymers, pNP-alkanoates as well as petrochemical based polymers (PES, PCL), makes it an excellent candidate biocatalyst for environmental, industrial, and medical applications. PhaZLS46 is suitable for
CRediT authorship contribution statement
Nisha Mohanan: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing - original draft, Visualization. Parveen K. Sharma: Methodology. David B. Levin: Conceptualization, Resources, Writing - review & editing, Supervision, Project administration, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors gratefully acknowledge financial assistance from the Natural Sciences and Engineering Research Council of Canada (NSERC) through an NSERC Discovery grant (RGPIN-04945-2017) held by DBL. Thanks are also due to the Science and Engineering Research Board, Department of Science and Technology, Government of India, for awarding overseas postdoctoral fellowship (Grant no. SB/OS/PDF-006/2016-17) to NM. The authors also wish to thank Ms. Emy Komatsu, a Technician of the Chemistry Department
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