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

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.