Kawai_2014_Appl.Microbiol.Biotechnol_98_10053

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 Ca2+ ion enhanced the enzyme activity and thermostability of the wild-type and mutant Cut190. Circular dichroism suggested that the Ca2+ changes the tertiary structure of the Cut190 (S226P/R228S), which has optimal activity at 65-75 degrees 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 degrees C for 24 h with 40 % activity remaining after incubation for 1 h at 70 degrees C. The Cut190 (S226P/R228S) efficiently hydrolyzed various aliphatic and aliphatic-co-aromatic polyester films. Furthermore, the enzyme degraded the PET film above 60 degrees 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, Ca2+ activation, and stabilization.