Novel extracellular medium-chain-length polyhydroxyalkanoate depolymerase from Streptomyces exfoliatus K10 DSMZ 41693: a promising biocatalyst for the efficient degradation of natural and functionalized mcl-PHAs

Abstract

Cloning and biochemical characterization of a novel extracellular medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase from Streptomyces exfoliatus K10 DSMZ 41693 are described. The primary structure of the depolymerase (PhaZSex2) includes the lipase consensus sequence (serine-histidine-aspartic acid) which is known for serine hydrolases. Secondary structure analysis shows 7.9 % α-helix, 43.9 % β-sheet, 19.4 % β-turns, and 31.2 % random coil, suggesting that this enzyme belongs to the α/β hydrolase fold family, in agreement with other PHA depolymerases and lipases. The enzyme was efficiently produced as an extracellular active form in Rhodococcus and purified by two consecutive hydrophobic chromatographic steps. Matrix-assisted laser desorption-time-of-flight (MALDI-TOF) analysis of the purified enzyme revealed a monomer of 27.6 kDa with a midpoint transition temperature of 44.2 °C. Remarkably, the activity is significantly enhanced by low concentrations of nonionic and anionic detergents and thermal stability is improved by the presence of 10 % glycerol. PhaZSex2 is an endo-exohydrolase that cleaves both large and small PHA molecules, producing (R)-3-hydroxyoctanoic acid monomers as the main reaction product. Markedly, PhaZSex2 is able to degrade functionalized polymers containing thioester groups in the side chain (PHACOS), releasing functional thioester-based monomers and oligomers demonstrating the potentiality of this novel biocatalyst for the industrial production of enantiopure (R)-3-hydroxyalkanoic acids.

Influence of Depolymerases and Lipases on the Degradation of Polyhydroxyalkanoates Determined in Langmuir Degradation Studies

“…[ 23 ] However, these results are not surprising given that enzymatic degradation is dependent on the affinity of the enzyme for the substrate, and PhaZ sex2 has a strong affinity for PHOHHx. [ 39 ] Nonetheless, the considerable activity of PhaZ sex2 toward PHB with small side‐chain‐length is a new insight from this study. The rate constants obtained from the fit‐curves at pH = 10 in Figure 2A were k = 0.013 min −1 for PHOHHx and k = 0.003 min −1 for PHB, assuming the longest water‐soluble fragment was a dimer in both cases.…”

“…Enzyme expression was achieved in Rhodococcus sp T104 (pENVO), grown in 2 × YTG (yeast extract/bactotriptone/NaCl) medium supplemented with glucose (5 g L −1 ), at 30 °C for 72 h. Recombinant PhaZ Sex2 was purified by using affinity chromatography using a HiTrap Butyl HP sepharose column (GE Healthcare). [ 39 ] Lipases from Pseudomonas cepacia (LPC) , Rhizopus oryzae (LRO), Mucor miehei (LMM), Candida antarctica (LCA), proteinase K from (Tritirachium album) , esterase from porcine liver and lipase from porcine pancreas (containing other hydrolases, such as esterases, amylases and protease) were purchased from Sigma‐Aldrich (Darmstadt, Germany). The enzymatic activities and other technical information provided by the manufacturer are listed in Table S1 in the Supporting Information.…”

Biocatalytic potential of Streptomyces spp. isolates from rhizosphere of plants and mycorrhizosphere of fungi

“…On the other hand, PHA depolymerase was the least detected enzyme activity amongst 118 Streptomyces isolates. PHA depolymerase degrades extracellular PHA in the environment, so the released oligomers and monomers can be used as a source of carbon and energy .…”

Review of recent advances in the biodegradability of polyhydroxyalkanoate (PHA) bioplastics and their composites

“…184 However, these enzymes do not function on medium and long chain PHAs effectively. Research availability of medium chain length depolymerases is minimal with very few species isolated with the particular enzymes, 185 which is further reflected by the few poly (3-hydroxyoctanoate-co-3-hydroxydecanoate) degraders seen in Streptomyces. 186 This may be why medium chain length polymers such as PHBO with 10% HO content only show between 88-95% biodegradation in anaerobic and aerobic environments.…”