基于磷脂酶水解圈定向筛选高效聚对苯二甲酸乙二醇酯降解酶

Screening of efficient PET-degrading enzymes based on hydrolysis circle of phospholipase

摘要:

【目的】目前自然环境中聚对苯二甲酸乙二醇酯(polyethylene terephthalate, PET)废弃物的积累严重威胁生态健康,因此PET的降解问题已成为全球性的热点问题。生物酶法降解PET技术以其绿色环保而备受关注,但天然PET降解酶的催化活性普遍偏低,亟待进一步定向改造。现阶段定向进化为快速提高PET降解酶催化性能提供了可能,其中筛选方法是成功获得高性能突变体的关键所在。本研究旨在提出一种新型高效灵敏的筛选方法并应用于褐色喜热裂孢菌(Thermobifida fusca)来源角质酶Tfu-0883的定向改造,以期快速获得PET降解活性提高的突变体。【方法】基于易错PCR构建突变体文库,涂布于卵黄磷脂平板,以水解圈的大小作为筛选指标获得PET降解活性提高的突变体;对突变体进行酶学定性并筛选出潜在的分子改造位点,最终获得高性能突变体。【结果】从卵黄磷脂平板中挑取水解圈直径最大的单菌落,即突变体H10(N2D/D94H/A149E),其PET降解能力是野生型的1.5倍,最适温度与pH分别为60℃和8.0。突变体H10中第2位和第149位氨基酸残基远离底物结合凹槽,其突变会导致酶蛋白稳定性下降;第94位氨基酸残基则位于底物结合凹槽附近,由负电荷氨基酸Asp突变为正电荷氨基酸His,有利于吸附在带负电荷的PET表面,是突变体H10降解能力提升的关键因素;随后将野生型的第94位氨基酸残基Asp分别突变为His及同为正电荷且空间位阻更小的Lys和Arg,突变体D94H、D94K和D94R对PET降解能力均有提升,其中,突变体D94K降解PET能力是野生型的3.6倍。【结论】本研究基于磷脂酶水解圈构建了一种新的PET降解酶定向筛选方法,以此获得了降解活性提高的突变体,并证实角质酶Tfu-0883第94位氨基酸残基位点具有提升其PET降解活性的潜在能力。

关键词:定向进化;PET降解;水解圈;热稳定性;分子改造

Abstract:

【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 of Thermobifida 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 °C 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.

Key words:directed evolution;PET degradation;hydrolysis circle;thermostability;molecular modification