Characterization of an intracellular poly(3-hydroxyalkanoate) depolymerase from the soil bacterium, Pseudomonas putida LS46. (May 2020)

• Record Type: Journal Article
• Authors: Mohanan, Nisha; Sharma, Parveen K.; Levin, David B.
• Is Part Of: Polymer degradation and stability. Volume 175(2020)
• Publication Date: 2020-05
• Subjects: Polyhydroxyalkanoate (PHA) polymers -- mcl-PHAs -- PHA depolymerase -- Lipase -- Esterase -- R-hydroxyalkanoic acids; Polymers -- Deterioration -- Periodicals; Stabilizing agents -- Periodicals; Polymères -- Dégradation -- Périodiques; Stabilisants -- Périodiques; 668.9
• DOI: 10.1016/j.polymdegradstab.2020.109127
• Languages: English
• ISSNs: 0141-3910

Abstract

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 Gx1 Sx2 G 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. 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 .