A novel Ca2+-activated, thermostabilized polyesterase capable of hydrolyzing polyethylene terephthalate from Saccharomonospora viridis AHK190

Abstract

Only two polyethylene glycol terephthalate (PET)-degrading enzymes have been reported, and their mechanism for the biochemical degradation of PET remains unclear. To identify a novel PET-degrading enzyme, a putative cutinase gene (cut190) was cloned from the thermophile Saccharomonospora viridis AHK190 and expressed in Escherichia coli Rosetta-gami B (DE3). Mutational analysis indicated that substitution of Ser226 with Pro and Arg228 with Ser yielded the highest activity and thermostability. The Ca(2+) ion enhanced the enzyme activity and thermostability of the wild-type and mutant Cut190. Circular dichroism suggested that the Ca(2+) changes the tertiary structure of the Cut190 (S226P/R228S), which has optimal activity at 65-75 °C and pH 6.5-8.0 in the presence of 20 % glycerol. The enzyme was stable over a pH range of 5-9 and at temperatures up to 65 °C for 24 h with 40 % activity remaining after incubation for 1 h at 70 °C. The Cut190 (S226P/R228S) efficiently hydrolyzed various aliphatic and aliphatic-co-aromatic polyester films. Furthermore, the enzyme degraded the PET film above 60 °C. Therefore, Cut190 is the novel-reported PET-degrading enzyme with the potential for industrial applications in polyester degradation, monomer recycling, and PET surface modification. Thus, the Cut190 will be a useful tool to elucidate the molecular mechanisms of the PET degradation, Ca(2+) activation, and stabilization.

Influence of surface charge, binding site residues and glycosylation on Thielavia terrestris cutinase biochemical characteristics

“…We also reported that for polyethylene terephthalate (PET) hydrolysis and polyvinyl acetate deacetylation, the engineered cutinase from Humicola insolens (HiC) developed by Novozymes has higher catalytic efficiency than wt-FsC and a cutinase from Pseudomonas mendocina (PmC) (Ronkvist et al 2009a, b). Cutinases from thermophilic bacteria such as Thermobifida fusca ( T m = 70 °C) (Roth et al 2014), Thermobifida alba ( T m = 70 °C) (Hu et al 2010) and Saccharomonospora viridis ( T m = 70 °C) (Kawai et al 2014) are relatively more thermostable than the fungal cutinases FsC, PmC, AoC and HiC. Furthermore, these thermophilic bacteria derived cutinases and are known to have PET hydrolysis activity.…”

Discovery of Polyesterases from Moss-Associated Microorganisms

“…EstC5 shows the typical triad Ser-Asp-His, while EstC9 may utilize glutamic acid (Ser-Glu-His) instead of aspartic acid. To compare the affiliations of the metagenome esterases with those of previously characterized polyesterases with documented activity toward PBAT, the following enzymes were included in the phylogenetic analysis: esterases Cbotu_EstA, Cbotu_EstB (Clostridium botulinum) (20), and Chath_Est1 (Clostridium hathewayi) (21), lipase Pfl1 (Pelosinus fermentans) (44), and cutinases Cut190 (Saccharomonosporas viridis) (19) and Thc_Cut1 (Thermobifida cellulosilytica) (45). These six known PBAT hydrolases share the same catalytic triad Ser-Asp-His (or Ser-Glu-His, in the case of Chath_Est1).…”

“…and from fungi (e.g., Fusarium solani, Humicola insolens, Aspergillus oryzae, and Rhizopus spp.) (6,11,(16)(17)(18)(19)(20)(21). While searching for new microorganisms, Yoshida et al recently discovered the novel bacterium Ideonella sakaiensis, which contains two PET hydrolases and is capable of utilizing PET as an energy and carbon source (22).…”

Recycling of poly(ethylene terephthalate) – A review focusing on chemical methods

“…They found that lipase is capable of fully converting DGTP to terephthalic acid (TPA), whereas the degradation of PET to TPA was negligible. Finally, Kawai et al [130] used saccharomonospora viridis cutinase polyesterase for degradation of PET at reaction conditions of 63°C and 3 days. With this enzyme it was possible to obtain 10-27% of TPA form PET degradation.…”

“…Generally, PET was incubated in the enzymatic solution at temperatures between 30 to 60°C for a period of time ranging from 3 to 14 days. Residual PET and solution were separated for product characterization [130][131][132][133]. As shown in Table 1, different enzymes such as saccharomonospora viridis cutinase polyesterase, thermobifidia fusca hydrolase, cutinase and lipase were applied for PET degradation.…”