Center for Nanomaterials and Devices, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto, 606-8585, Japan,
Appl Microbiol Biotechnol. 2014 Dec;98(24):10053-64. doi: 10.1007/s00253-014-5860-y. Epub 2014 Jun 15.
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.
目前仅报道了两种聚对苯二甲酸乙二醇酯（PET）降解酶，其生化降解 PET 的机制仍不清楚。为了鉴定一种新型的 PET 降解酶，从嗜热菌 S. viridis AHK190 中克隆了一个假定的角质酶基因（cut190），并在大肠杆菌 Rosetta-gami B（DE3）中表达。突变分析表明，将丝氨酸 226 突变为脯氨酸，将精氨酸 228 突变为丝氨酸，可获得最高的活性和热稳定性。钙离子增强了野生型和突变型 Cut190 的酶活性和热稳定性。圆二色性表明钙离子改变了 Cut190 的三级结构（S226P/R228S），在 20%甘油存在下，该结构在 65-75°C 和 pH6.5-8.0 时具有最佳活性。该酶在 pH5-9 的范围内稳定，在 65°C 下稳定 24 小时，在 70°C 下孵育 1 小时后仍保持 40%的活性。Cut190（S226P/R228S）能有效地水解各种脂肪族和脂肪族-芳香族聚酯薄膜。此外，该酶能在 60°C 以上降解 PET 薄膜。因此，Cut190 是一种新型的报道的 PET 降解酶，具有在聚酯降解、单体回收和 PET 表面改性等领域的工业应用潜力。因此，Cut190 将是阐明 PET 降解、Ca（2+）激活和稳定的分子机制的有用工具。
