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[Objective] Since the accumulation of polyethylene terephthalate (PET) waste causes a major threat to the health of the natural environment, the degradation of PET has become a global hot issue. Enzymatic degradation of PET has garnered considerable attention because of its eco-friendly properties. However, due to the low catalytic activity, natural PET-degrading enzymes remain to be modified according to specific needs. Directed evolution enables the rapidly enhancement of the catalytic activities of PET-degrading enzymes, in which screening methods are the key for obtaining high-performance mutants. This study develops a novel, efficient, and sensitive screening method and applies it to direct modification ofThermobifida fusca cutinase Tfu-0883 to obtain the mutants with improved PET-degrading activity. [Methods] A mutant library constructed by error-prone PCR was coated on phospholipid plates. The mutant with improved PET-degrading activity was screened out based on the size of the hydrolytic circle. The enzymatic properties of the mutant were determined, and the rational modification sites were identified. Finally, a forward mutant was obtained. [Results] The single colony with the largest hydrolysis circle, mutant H10 (N2D/D94H/A149E), was selected from the phospholipid plate, with the PET-degrading activity 1.5 times that of the wild type. The mutant H10 exhibited the best performance at 60 ℃ and pH 8.0. The residues at positions 2 and 149 in the mutant H10 were distantly located from the substrate-binding groove, and any mutation in the residues would result in decreased enzyme stability. The residue at position 94 was situated near the substrate-binding groove, where it underwent a change from negatively charged Asp to positively charged His. This alteration facilitated adsorption onto the negatively charged PET surface and played a crucial role in enhancing the degradation ability of mutant H10. With the wild type as a template, the 94th amino acid residue was mutated to His, Lys, and Arg, which possess positive charges but exhibit reduced steric hindrance. The mutants D94H, D94K, and D94R all exhibited enhanced PET-degrading ability. Notably, among these mutants, D94K demonstrated a 3.6-fold higher rate of PET degradation than the wild type. [Conclusion] We developed a method for screening PET-degrading enzymes based on the phospholipase cycle and obtained the mutants with enhanced PET-degrading activity. The 94th residue of the cutinase Tfu-0883 is demonstrated as the first to possess the potential for enhancing the PET-degrading activity.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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