Characterization of an intracellular poly(3-hydroxyalkanoate) depolymerase from the soil bacterium, Pseudomonas putida LS46

https://doi.org/10.1016/j.polymdegradstab.2020.109127Get rights and content

Highlights

  • The mcl-PHA depolymerase from P. putida LS46 (PhaZLS46) was characterized.

  • PhaZLS46 was significantly different compared to other known mcl-PHA depolymerases.

  • PhaZLS46 has a broad substrate specificity and acts as an exo-hydrolase.

  • Gel Permeation Chromatography confirmed degradation of a variety of polymers.

  • RHAs generated by PhaZLS46 had antibacterial action against E. coli.

Abstract

A gene encoding the intracellular PHA depolymerase of the saprotrophic soil bacterium, Pseudomonas putida LS46 was cloned and expressed in Escherichia coli. The gene has an open reading frame of 852 bp, encoding a protein of 283 amino acids with a predicted molecular mass of 31.5 kDa. The protein, PhaZLS46, has a α/β-hydrolase fold and a catalytic triad (serine-histidine-aspartic acid), which is found in all members of the lipase/esterase enzyme family. The catalytic serine is present in a Gx1Sx2G sequence motif, also known as lipase box, with the x1 and x2 positions occupied by valine101 and trypophan103, respectively. The purified recombinant enzyme was active optimally at 30 °C and pH 6.0, and displayed a broad-substrate specificity, with the ability to hydrolyze medium chain polyhydroxyalkanoates, as well as various para-nitrophenyl alkanoates. The enzyme also showed depolymerase activity against petroleum-based polymers, such as polyethylene succinate [PES] and poly(ϵ-caprolactone) [PCL], making it extremely useful for biodegradation. Our results suggest that PhaZLS46 from P. putida LS46 represents a new subgroup of intracellular mcl-PHA depolymerases. The degradation products of PhaZLS46 on different polymers were analyzed using GPC. The ESI-MS analysis revealed that PhaZLS46 belongs to exohydrolases capable of releasing monomers as major reaction products (R-hydroxyalkanoic acids, RHAs) upon PHA degradation. The extracted RHAs (3-hydroxyoctanoic acids) formed by the action of enzyme on PHO had improved antibacterial action against the tested strain (E. coli BL21), forming clear zones of growth inhibition on agar diffusion plates with the minimal inhibitory concentration value (MIC) of 4 mM.

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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