An engineered PET depolymerase to break down and recycle plastic bottles

Tournier, V.; Topham, C. M.; Gilles, A.; David, B.; Folgoas, C.; Moya-Leclair, E.; Kamionka, E.; Desrousseaux, M.-L.; Texier, H.; Gavalda, S.; Cot, M.; Guémard, E.; Dalibey, M.; Nomme, J.; Cioci, G.; Barbe, S.; Chateau, M.; André, I.; Duquesne, S.; Marty, A.

doi:10.1038/s41586-020-2149-4

Article
Published: 08 April 2020

An engineered PET depolymerase to break down and recycle plastic bottles
一种工程化 PET 解聚酶，用于分解和回收塑料瓶

V. Tournier¹^na1,
C. M. Topham ORCID: orcid.org/0000-0002-9147-0184¹^na1,
A. Gilles¹,
B. David¹,
C. Folgoas¹,
E. Moya-Leclair¹,
E. Kamionka¹,
M.-L. Desrousseaux¹,
H. Texier¹,
S. Gavalda¹,
M. Cot²,
E. Guémard³,
M. Dalibey³,
J. Nomme¹,
G. Cioci¹,
S. Barbe¹,
M. Chateau³,
I. André ORCID: orcid.org/0000-0001-6280-4109¹,
S. Duquesne¹ &
…
A. Marty^1,3

Nature 自然 volume 580, pages 216–219 (2020)Cite this article
216–219 (2020) 引用这篇文章

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Abstract 抽象的

Present estimates suggest that of the 359 million tons of plastics produced annually worldwide¹, 150–200 million tons accumulate in landfill or in the natural environment². Poly(ethylene terephthalate) (PET) is the most abundant polyester plastic, with almost 70 million tons manufactured annually worldwide for use in textiles and packaging³. The main recycling process for PET, via thermomechanical means, results in a loss of mechanical properties⁴. Consequently, de novo synthesis is preferred and PET waste continues to accumulate. With a high ratio of aromatic terephthalate units—which reduce chain mobility—PET is a polyester that is extremely difficult to hydrolyse⁵. Several PET hydrolase enzymes have been reported, but show limited productivity^6,7. Here we describe an improved PET hydrolase that ultimately achieves, over 10 hours, a minimum of 90 per cent PET depolymerization into monomers, with a productivity of 16.7 grams of terephthalate per litre per hour (200 grams per kilogram of PET suspension, with an enzyme concentration of 3 milligrams per gram of PET). This highly efficient, optimized enzyme outperforms all PET hydrolases reported so far, including an enzyme^8,9 from the bacterium Ideonella sakaiensis strain 201-F6 (even assisted by a secondary enzyme¹⁰) and related improved variants^11,12,13,14 that have attracted recent interest. We also show that biologically recycled PET exhibiting the same properties as petrochemical PET can be produced from enzymatically depolymerized PET waste, before being processed into bottles, thereby contributing towards the concept of a circular PET economy.
目前的估计表明，全球每年生产 3.59 亿吨塑料 ¹ ，其中 150-2 亿吨积累在垃圾填埋场或自然环境中 ² 。聚对苯二甲酸乙二醇酯 (PET) 是最丰富的聚酯塑料，全球每年生产近 7000 万吨用于纺织品和包装 ³ 。 PET 的主要回收过程通过热机械方式，导致机械性能损失 ⁴ 。因此，从头合成是首选，PET 废物不断积累。 PET 具有高比例的芳香族对苯二甲酸酯单元（会降低链的流动性），是一种极难水解的聚酯 ⁵ 。已经报道了几种PET水解酶，但其生产力有限 ^6,7 。在这里，我们描述了一种改进的 PET 水解酶，最终在 10 小时内将至少 90% 的 PET 解聚成单体，生产率为每升每小时 16.7 克对苯二甲酸酯（每公斤 PET 悬浮液 200 克，使用酶浓度为每克 PET 3 毫克）。这种高效、优化的酶优于迄今为止报道的所有 PET 水解酶，包括来自细菌 Ideonella sakaiensis 菌株 201-F6 的酶 ^8,9 （甚至在辅助酶 ¹⁰ 的帮助下）和相关的最近引起人们兴趣的改进变体 ^11,12,13,14 。我们还表明，在加工成瓶子之前，可以通过酶解聚 PET 废物生产具有与石化 PET 相同特性的生物回收 PET，从而有助于实现循环 PET 经济的概念。

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Machine learning-aided engineering of hydrolases for PET depolymerization
用于 PET 解聚的水解酶的机器学习辅助工程

Article 27 April 2022
文章 2022 年 4 月 27 日

Discovery and mechanism-guided engineering of BHET hydrolases for improved PET recycling and upcycling
BHET 水解酶的发现和机制引导工程可改善 PET 回收和升级循环

Article Open access 文章开放获取 13 July 2023 2023 年 7 月 13 日

Discovery and rational engineering of PET hydrolase with both mesophilic and thermophilic PET hydrolase properties
具有中温和高温 PET 水解酶特性的 PET 水解酶的发现和合理工程

Article Open access 文章开放获取 28 July 2023 2023 年 7 月 28 日

Main 主要的

Given that crystalline PET has been found to be subject to only limited depolymerization by cutinase enzymes^8,15,16,17, we used a commercially available amorphous PET (Gf-PET, from the supplier Goodfellow) to compare the activity of several enzymes reported previously to hydrolyse PET in their optimal conditions. These enzymes were Thermobifida fusca hydrolases 1 and 2 (BTA1 and BTA2), Fusarium solani pisi cutinase (FsC), Ideonella sakaiensis PETase (Is-PETase) and leaf-branch compost cutinase (LCC) (Extended Data Fig. 1a, b). LCC outperformed all other enzymes tested, reaching an initial PET-specific depolymerization rate of 93.2 mg_TAeq_. h⁻¹ mg_enzyme⁻¹ at 65 °C (Fig. 1a; ‘TAeq.’ refers to equivalents of terephthalic acid, as detailed in Supplementary Method 1). We found LCC to be at least 33 times more efficient than any other enzyme tested (Fig. 1a and Extended Data Fig. 1b), while also demonstrating the highest thermostability, with a determined melting temperature of 84.7 °C (Extended Data Fig. 1a). This depolymerization performance of LCC at 65 °C was slightly lower when using bottle-grade PET (Pf-PET) as a substrate (Fig. 1a and Extended Data Fig. 1b), with an initial PET-specific depolymerization rate of 81.9 mg_TAeq. h⁻¹ mg_enzyme⁻¹. While approaching the glass transition temperature, known to maximize PET chain mobility and enzymatic depolymerization^18,19, the LCC reaction stopped after 3 days at 65 °C with a Pf-PET conversion level of only 31% (Fig. 1b). We observed no inhibition by the depolymerization products ethylene glycol and terephthalic acid at the concentrations that would be obtained at 100% Pf-PET depolymerization (Extended Data Fig. 2a), and Pf-PET crystallinity was unchanged after 6 days at 65 °C (10% crystallinity, as measured by differential scanning calorimetry). Moreover, the initial kinetics could be restored by adding LCC to the stopped reaction (Extended Data Fig. 2b), implying that the thermostability of LCC was most likely to be the limiting factor, notwithstanding its high melting temperature when free in solution. To optimize depolymerization yields, we sought to improve both the activity and the thermostability of LCC through enzyme engineering.
鉴于已发现结晶 PET 只能通过角质酶 ^8,15,16,17 进行有限的解聚，我们使用市售的无定形 PET（Gf-PET，来自供应商 Goodfellow）来比较所报告的几种酶的活性之前在最佳条件下水解 PET。这些酶是 Thermobifida fusca 水解酶 1 和 2（BTA1 和 BTA2）、Fusarium solani pisi 角质酶（FsC）、Ideonella sakaiensis PETase（Is-PETase）和叶枝堆肥角质酶（LCC）（扩展数据图 1a、b）。 LCC 的性能优于所有其他测试的酶，在 65 ℃ 时达到 93.2 mg _TAeq h ⁻¹ mg _enzyme ⁻¹ 的初始 PET 特异性解聚率°C（图 1a；“TAeq.”是指对苯二甲酸的当量，如补充方法 1 中详述）。我们发现 LCC 的效率至少比任何其他测试酶高 33 倍（图 1a 和扩展数据图 1b），同时还表现出最高的热稳定性，确定的熔化温度为 84.7 °C（扩展数据图 1a））。当使用瓶级PET（Pf-PET）作为基材时，LCC在65℃下的解聚性能略低（图1a和扩展数据图1b），初始PET特异性解聚率为81.9 mg < b5>小时 ⁻¹ 毫克 _enzyme ⁻¹ 。当接近玻璃化转变温度时，已知可最大化 PET 链流动性和酶解聚 ^18,19 ，LCC 反应在 65 °C 3 天后停止，Pf-PET 转化水平仅为 31%（图 1b））。我们观察到解聚产物乙二醇和对苯二甲酸在 100% Pf-PET 解聚时获得的浓度没有抑制作用（扩展数据图 1）。 2a)，Pf-PET 结晶度在 65 °C 下 6 天后没有变化（10% 结晶度，通过差示扫描量热法测量）。此外，通过将LCC添加到停止的反应中可以恢复初始动力学（扩展数据图2b），这意味着LCC的热稳定性很可能是限制因素，尽管其在溶液中游离时的熔化温度很高。为了优化解聚产率，我们试图通过酶工程提高 LCC 的活性和热稳定性。

Fig. 1: LCC outperformed all other evaluated PET hydrolases during PET-depolymerization assays.
图 1：在 PET 解聚测定中，LCC 优于所有其他评估的 PET 水解酶。

To identify amino acid residues for mutagenesis to improve catalytic activity, we used molecular docking and enzyme contact-surface analysis to investigate the mode of binding of a model substrate, 2-HE(MHET)₃, in the active site of wild-type LCC (Protein Data Bank (PDB; https://www.rcsb.org) ID 4EB0). We found that the substrate binds in an elongated and predominantly hydrophobic groove (Fig. 2a) present in all X-ray structures of prokaryotic cutinases reported to date^20,21,22. In total, we identified 15 amino acid residues in the first contact shell, of which we chose 11 for targeted mutagenesis (Extended Data Table 1). We subjected these 11 positions to site-specific saturation mutagenesis to generate all possible 209 variants. Most variants showed impaired depolymerization of Pf-PET: more than 25% of the variants exhibited less than 1% specific activity by comparison with wild-type LCC, and more than 75% exhibited less than 48% specific activity (Extended Data Fig. 3a). Variants with 75% or more activity compared with wild-type LCC were purified and analysed individually for Pf-PET depolymerization specific activity and determination of the melting temperature by differential scanning fluorimetry (DSF). Of 25 such variants, we selected variants I and W of the F243 position for further analysis, as they showed improved activity versus wild-type LCC (Fig. 2b); we also highlighted variants T96M, Y127G, N246D and N246M, which exhibited an increased melting temperature, although no improvement in Pf-PET depolymerization activity (Extended Data Fig. 3b).
为了鉴定用于诱变以提高催化活性的氨基酸残基，我们使用分子对接和酶接触表面分析来研究模型底物 2-HE(MHET) ₃ 在活性中的结合模式野生型 LCC 位点（蛋白质数据库（PDB；https://www.rcsb.org）ID 4EB0）。我们发现底物结合在细长且主要疏水的凹槽中（图2a），该凹槽存在于迄今为止报道的所有原核角质酶的X射线结构中 ^20,21,22 。总共，我们在第一个接触壳中鉴定了 15 个氨基酸残基，其中我们选择了 11 个进行靶向诱变（扩展数据表 1）。我们对这 11 个位置进行位点特异性饱和诱变，以生成所有可能的 209 种变体。大多数变体显示 Pf-PET 解聚受损：与野生型 LCC 相比，超过 25% 的变体表现出低于 1% 的比活性，超过 75% 的变体表现出低于 48% 的比活性（扩展数据图 3a））。与野生型 LCC 相比，具有 75% 或更高活性的变体被纯化并单独分析 Pf-PET 解聚比活性，并通过差示扫描荧光法 (DSF) 测定熔解温度。在 25 个此类变体中，我们选择了 F243 位点的变体 I 和 W 进行进一步分析，因为它们与野生型 LCC 相比显示出更高的活性（图 2b）；我们还重点介绍了变体 T96M、Y127G、N246D 和 N246M，它们表现出较高的熔化温度，但 Pf-PET 解聚活性没有改善（扩展数据图 3b）。

**Fig. 2: Improvement of the PET-depolymerization specific activity of LCC after mutagenesis by saturation of the residues in contact with a 2-HE(MHET)₃ substrate.**

To improve the thermostability of LCC, we sought divalent-metal-binding sites known to increase enzyme thermal stability in PET hydrolases^23,24. No such metal-ion-binding site could be directly observed in the 1.5 Å X-ray structure of LCC²⁵. However, such a site—formed by the side chains of three acidic amino acid residues—can be identified at topologically equivalent positions in the X-ray structures of three cutinase homologues (Fig. 3a). The corresponding residue triplet in LCC comprises two acidic residues (E208 and D238) and the neutral S283 (Fig. 3a). DSF experiments confirmed that LCC was thermally stabilized by the addition of calcium ions (the melting temperature increased by 9.3 °C on addition of 35 mM CaCl₂), in accordance with previous results²⁵ (Fig. 3b). However, in order to reduce reaction costs and the need for extensive downstream purification, we preferred to avoid salts and additives. We adopted the alternative strategy of replacing the divalent-metal-binding site with a disulfide bridge²⁶. The Cα–Cα (4.5 Å) and Cβ–Cβ (4.4 Å) interatomic separation distances for the D238 and S283 residue pair in the LCC X-ray structure were compatible with the engineering of a disulfide bridge²⁷. A D238C/S283C variant was produced and disulfide-bond formation verified by DSF (Extended Data Fig. 4), allowing thermal stabilization of LCC without dependence on calcium ions (Fig. 3b). This variant exhibited a melting temperature of 94.5 °C (9.8 °C higher than that of wild-type LCC), with a decrease in activity of only 28% (Extended Data Fig. 3b).

**Fig. 3: Improvement of LCC thermostability by addition of a disulfide bridge.**

We added the two mutations leading to the highest specific activity to the new thermostable variant. The resulting F243I/D238C/S283C (ICC) and F243W/D238C/S283C (WCC) variants restored the activity of the D238C/S283C variant to at least wild-type LCC levels (122% and 98%, respectively), with melting temperatures that were 6.2 °C and 10.1 °C higher, respectively (Extended Data Fig. 3b). Subsequently, we added T96M, Y127G, N246D or N246M mutations—shown to improve thermostability—to ICC and WCC, generating eight new variants. After comparative analysis of specific activities and melting temperatures (Extended Data Fig. 3b), we selected variants F243I/D238C/S283C/Y127G (ICCG), F243I/D238C/S283C/N246M (ICCM), F243W/D238C/S283C/Y127G (WCCG) and F243W/D238C/S283C/N246M (WCCM), as they retained specific activity that was similar to or higher than that of wild-type LCC, with improved melting temperatures ranging from +9.3 °C to +13.4 °C.
我们添加了两个突变，导致新的热稳定变体具有最高的比活性。由此产生的 F243I/D238C/S283C (ICC) 和 F243W/D238C/S283C (WCC) 变体将 D238C/S283C 变体的活性恢复到至少野生型 LCC 水平（分别为 122% 和 98%），且具有解链温度分别高出 6.2 °C 和 10.1 °C（扩展数据图 3b）。随后，我们向 ICC 和 WCC 添加了 T96M、Y127G、N246D 或 N246M 突变（显示可提高热稳定性），产生了 8 个新变体。经过比活性和熔解温度的比较分析（扩展数据图3b），我们选择了变体F243I/D238C/S283C/Y127G（ICCG）、F243I/D238C/S283C/N246M（ICCM）、F243W/D238C/S283C/Y127G（ WCCG) 和 F243W/D238C/S283C/N246M (WCCM)，因为它们保留了与野生型 LCC 相似或更高的比活性，并且熔解温度提高了 +9.3 °C 至 +13.4 °C。

To enable a scale-up of the process, we evaluated these four quadruple variants (ICCG, ICCM, WCCG and WCCM) in bioreactor conditions and using post-consumer coloured-flake PET waste (PcW-PET). This PcW-PET is the under-valorized residue remaining after the ultimate sorting that leads to clear PET waste (the latter being used for the thermomechanical process of PET recycling). We raised the concentration of PcW-PET to 200 g kg⁻¹ of the total weight of the reaction volume (PET plus liquid phase) in order to maximize reactor productivity. We applied pretreatment technologies (extrusion and micronization) that are widely used by plastic industries^28,29 to amorphize and increase the exchange surface of PcW-PET, performing this pretreatment on a ton-scale here. We raised the temperature to 72 °C in order to maximize kinetic turnover (Fig. 4), 75 °C being detrimental to PET conversion (Extended Data Fig. 5a) owing to rapid recrystallization of PcW-PET. Indeed, 24.7% crystallinity was reached in 9 h at 72 °C, whereas it took only 6 h to reach its maximal 37.5% at 75 °C (Extended Data Fig. 6). The best conversion level was obtained with ICCG and WCCG, achieving 82% and 85% conversion in 20 h and 15 h respectively (Fig. 4a). Wild-type LCC, which shows reduced thermostability compared with the ICCG and WCCG variants (resulting in a rapid decrease in reaction kinetics after 2 h), reached only 53% conversion in 20 h. The remaining 47% of PcW-PET had a high level of crystallinity, estimated at 28.9%, making an immediate reuse for PET depolymerization unsuitable.
为了扩大该工艺的规模，我们在生物反应器条件下并使用消费后彩色片状 PET 废料 (PcW-PET) 评估了这四种四元变体（ICCG、ICCM、WCCG 和 WCCM）。这种 PcW-PET 是最终分选后剩余的价值不足的残渣，可产生清澈的 PET 废物（后者用于 PET 回收的热机械过程）。我们将 PcW-PET 的浓度提高到反应体积总重量（PET 加液相）的 200 g kg ⁻¹ ，以最大限度地提高反应器生产率。我们应用塑料行业广泛使用的预处理技术（挤出和微粉化） ^28,29 来非晶化并增加 PcW-PET 的交换表面，在这里以吨级进行这种预处理。我们将温度提高到 72 °C，以最大限度地提高动力学周转（图 4），由于 PcW-PET 的快速重结晶，75 °C 不利于 PET 转化（扩展数据图 5a）。事实上，在 72 °C 下 9 小时内就达到了 24.7% 结晶度，而在 75 °C 下仅用了 6 小时就达到了最大 37.5%（扩展数据图 6）。 ICCG 和 WCCG 获得了最佳转化水平，分别在 20 小时和 15 小时内实现了 82% 和 85% 的转化（图 4a）。与 ICCG 和 WCCG 变体相比，野生型 LCC 表现出较低的热稳定性（导致 2 小时后反应动力学迅速下降），在 20 小时内仅达到 53% 的转化率。其余 47% 的 PcW-PET 结晶度较高，估计为 28.9%，因此不适合立即再用于 PET 解聚。

Fig. 4: Improved performance of LCC variants in enzymatic depolymerization of post-consumer PET waste.
图 4：LCC 变体在消费后 PET 废物的酶解聚中的性能得到改善。

As a ratio of 3 milligrams of enzyme per gram of PET appeared to maximize PcW-PET depolymerization (Extended Data Fig. 5b), we compared ICCG and WCCG at this ratio. Under these conditions, 90% depolymerization was obtained for both WCCG and ICCG variants in 10.5 h and 9.3 h, respectively (Fig. 4b). For the ICCG variant, a higher initial rate was observed (Extended Data Fig. 5c), and we determined a maximum specific space-time-yield of 70.1 g_TA l⁻¹ h⁻¹ g_enzyme⁻¹ (27.9 g_TA l⁻¹ h⁻¹ g_enzyme⁻¹ over the course of the reaction), corresponding to a maximum productivity of 42.1 g_TA l⁻¹ h⁻¹ (16.7 g_TA l⁻¹ h⁻¹ over the course of the reaction). This mean productivity was 98-fold higher than the productivity reported previously by using TfCut2 with amorphous PET¹⁷. Notably, it is also considerably higher than the productivity reported during degradation of starch³⁰ (4 g l⁻¹ h⁻¹) or cotton³¹ (0.31 g l⁻¹ h⁻¹ ). To enable the scientific community to use this enzyme as a reference, we assayed its performance with commercially available Gf-PET in laboratory-scale conditions (as described in Supplementary Method 1); the initial rate was 105.6 ± 3.9 mg_TAeq. h⁻¹ mg_enzyme⁻¹. To gain better insight into the structural effects of the mutations, we combined X-ray crystallography (Extended Data Table 2 and Extended Data Fig. 7) and molecular-dynamics simulations of the enzymes. In the free state (Extended Data Fig. 8a), no substantial difference was observed between parental LCC and ICCG. Conversely, molecular-dynamics simulations of the ICCG variant in complex with the 2-HE(MHET)₃ model substrate (Extended Data Fig. 8b, c) revealed that the mutations introduced in ICCG facilitate the catalytically productive binding of 2-HE(MHET)₃ compared with parental LCC (Extended Data Fig. 8b, c). Accordingly, molecular mechanics/generalized Born surface area (MM/GBSA) calculations predicted an increased affinity of ICCG towards 2-HE(MHET)₃ (ΔΔG = −1.37 kcal mol⁻¹).
由于每克 PET 3 毫克酶的比例似乎可以最大程度地实现 PcW-PET 解聚（扩展数据图 5b），因此我们在此比例下比较了 ICCG 和 WCCG。在这些条件下，WCCG 和 ICCG 变体分别在 10.5 小时和 9.3 小时内获得 90% 解聚（图 4b）。对于 ICCG 变体，观察到较高的初始速率（扩展数据图 5c），我们确定最大比时空产量为 70.1 g _TA l ⁻¹ h < b2> g _enzyme ⁻¹ (27.9 g _TA l ⁻¹ h ⁻¹ g _enzyme ⁻¹ 在反应过程中），对应的最大生产率为 42.1 g _TA l ⁻¹ h ⁻¹ （反应过程中 16.7 g _TA l ⁻¹ h ⁻¹ ）。该平均生产率比之前报道的使用 TfCut2 与非晶 PET ¹⁷ 的生产率高 98 倍。值得注意的是，它也大大高于淀粉 ³⁰ （4 g l ⁻¹ h ⁻¹ ）或棉花 ³¹ 降解过程中报告的生产率(0.31 g l ⁻¹ h ⁻¹ )。为了使科学界能够使用这种酶作为参考，我们在实验室规模的条件下使用市售的 Gf-PET 测定了其性能（如补充方法 1 中所述）；初始速率为 105.6 ± 3.9 mg _TAeq. h ⁻¹ mg _enzyme ⁻¹ 。为了更好地了解突变的结构效应，我们结合了 X 射线晶体学（扩展数据表 2 和扩展数据图 7）和酶的分子动力学模拟。在游离状态下（扩展数据图8a），亲代LCC和ICCG之间没有观察到显着差异。相反，ICCG 变体与 2-HE(MHET) ₃ 模型底物复合物的分子动力学模拟（扩展数据图 8b、c）表明，ICCG 中引入的突变促进了催化生产结合2-HE(MHET) ₃ 与亲代 LCC 相比（扩展数据图 8b、c）。因此，分子力学/广义玻恩表面积 (MM/GBSA) 计算预测 ICCG 对 2-HE(MHET) ₃ 的亲和力增加 (ΔΔG = -1.37 kcal mol ⁻¹ ) 。

As a compromise between productivity and enzyme cost, we used the optimized ICCG variant at 2 mg g_PET⁻¹ in a 150-l pilot-scale depolymerization at a very high PcW-PET content (200 g kg⁻¹). We calculate that the cost of enzyme needed to recycle 1 ton of PET represents approximately 4% of the ton-price of the virgin PET, with a production cost of US$25 per kilogram of protein estimated using the cost of production of a cellulase in Trichoderma reesei ($5–$23 per kilogram of protein³²).

As a first step of process development, we recycled only the terephthalic acid that represents the main part of the PET in weight (1 ton of PET waste leads to 863 kg of terephthalic acid). Using industrially relevant processes (namely discolouration by activated carbon, commonly used in large-scale processes³³, and crystallization, the final step for the chemical production of terephthalic acid³⁴), we purified terephthalic acid monomers to a level higher than 99.8%, with an American Public Health Association (APHA) colour number of 2.9. During this process, 0.6 kg of sodium sulfate was produced per kg of recycled PET. With our new process, the recycling of 100,000 tons of PET per year will produce around 60,000 tons of sodium sulfate per year, representing 0.28% of the world market of sodium sulfate (21.5 million tons per year, especially in the detergent, paper and glass industries, with a 2.9% annual growth rate)³⁵. We used the terephthalic acid produced here to synthesize virgin PET. During PET synthesis (in the polycondensation step), the levels of diethylene glycol, antimony and phosphorous and the value of carboxylic end groups were in the range of the bottle-grade PET norm (2.2%, 177 ppm, 14 ppm, 23 mg_KOH g⁻¹, respectively). At the end of the synthesis process (during solid-state polymerization), the evaluated number average molecular weight (36,650 g mol⁻¹) and intrinsic viscosity (0.75 dl g⁻¹) of the newly made PET were similar to those of PET synthesized using petrochemical terephthalic acid (evaluated number average molecular weight 38,390 g mol⁻¹ and intrinsic viscosity 0.78 dl g⁻¹). Bottles blown from this PET had similar mechanical properties (a displacement of 2.9 mm at a maximal top-load force of 176 N) to those of commercial PET bottles (a displacement of 3.0 mm at a maximal top-load force of 181 N). Moreover, their excellent lightness values of 87.5% are better than those of the minimal standard for PET bottles (greater than 85%).
作为工艺开发的第一步，我们仅回收了占 PET 重量主要部分的对苯二甲酸（1 吨 PET 废料可产生 863 公斤对苯二甲酸）。使用工业相关工艺（即大规模工艺中常用的活性炭变色 ³³ ，以及结晶，对苯二甲酸化学生产的最后一步 ³⁴ ），我们纯化了对苯二甲酸单体含量高于 99.8%，美国公共卫生协会 (APHA) 色度值为 2.9。在此过程中，每公斤回收PET产生0.6公斤硫酸钠。采用我们的新工艺，每年回收 10 万吨 PET 将产生约 6 万吨硫酸钠，占世界硫酸钠市场（每年 2150 万吨，特别是在洗涤剂、纸张和玻璃领域）的 0.28%行业，年增长率为 2.9%） ³⁵ 。我们使用这里生产的对苯二甲酸来合成原生 PET。在 PET 合成过程中（缩聚步骤），二甘醇、锑和磷的含量以及羧基端基值均在瓶级 PET 标准范围内（2.2%、177 ppm、14 ppm、23 mg < b3> g ⁻¹ ，分别）。在合成过程结束时（固态聚合期间），评估的数均分子量（36,650 g mol ⁻¹ ）和特性粘度（0.75 dl g ⁻¹ ）新制备的PET与石化对苯二甲酸合成的PET相似（评估数均分子量为38,390 g mol ⁻¹ ，特性粘度为0.78 dl g ⁻¹ ）。用这种 PET 吹制的瓶子具有相似的机械性能（位移为 2.9 毫米（最大顶部负载力 176 N 时）与商用 PET 瓶的位移（最大顶部负载力 181 N 时位移 3.0 毫米）。此外，其87.5%的优异亮度值优于PET瓶的最低标准（大于85%）。

Here, using computer-aided enzyme engineering, we have improved enzyme-catalysed PET depolymerization to 90% conversion in less than 10 h, with a mean productivity of 16.7 g_TA l⁻¹ h⁻¹. We have used the resulting purified terephthalic acid monomers to synthesize PET, which was ultimately blown into bottles, closing the loop of the circular economy. With the urgent global need to address the issue of plastic disposal—and with many governments, national and international agencies and manufacturers committing to sustainability goals and the idea of a circular economy—the enzymatic processing of PET waste described here may help to meet such goals.
在这里，利用计算机辅助酶工程，我们将酶催化的 PET 解聚在不到 10 小时的时间内提高到 90% 的转化率，平均生产率为 16.7 g _TA l ⁻¹ h ⁻¹ 。我们使用所得的纯化对苯二甲酸单体来合成 PET，最终将其吹制成瓶子，形成循环经济的闭环。随着全球迫切需要解决塑料处置问题，以及许多政府、国家和国际机构和制造商致力于可持续发展目标和循环经济理念，本文描述的 PET 废物酶处理可能有助于实现这些目标。

Reporting summary 报告摘要

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.
有关研究设计的更多信息，请参阅本文链接的《自然研究报告摘要》。

Data availability 数据可用性

The authors declare that all data supporting the findings of this study are available within the article, its Extended Data, its Source Data or from the corresponding authors upon reasonable request. The atomic coordinates and structure factors of the reported structures have been deposited in the Protein Data Bank under accession codes 6THS for LCC-S165A and 6THT for ICCG-S165A.
作者声明，支持本研究结果的所有数据均可在文章、其扩展数据、源数据中获得，或根据合理要求从相应作者处获得。所报告结构的原子坐标和结构因子已存储在蛋白质数据库中，LCC-S165A 的登录码为 6THS，ICCG-S165A 的登录码为 6THT。

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Acknowledgements

We thank the ICEO facility of the Toulouse Biotechnology Institute (TBI), which is part of the Integrated Screening Platform of Toulouse (PICT, IBiSA), for providing access to ultrahigh-performance liquid chromatography (UHPLC) and protein-purification equipment; and Toulouse White Biotechnology (TWB, UMS INRAE 1337/UMS CNRS 3582) for providing access to Minibio bioreactors. We acknowledge Carbios (Saint-Beauzire, France), CRITT Bio-Industries (Toulouse, France), Pivert (Venette, France) and Leitat Technological Center (Barcelona, Spain) for their contribution to the purification of the terephthalic acid and for PET and bottle production. We also thank the Structural Biophysics Team of the Institute of Pharmacology and Structural Biology (IPBS, Toulouse, France) for access to the crystallization facility, as well as the ALBA (Barcelona, Spain) and ESRF (Grenoble, France) synchrotrons for data collection. We also acknowledge the use of High-Performance Computing resources on the Occigen (CINES, Montpellier, France) and Curie (TGCC, Paris-Saclay, France) supercomputers as well as on the Computing Mesocenter of Région Midi-Pyrénées (CALMIP, Toulouse, France). This study was supported by Truffle Capital (P. Pouletty) and a grant-in-aid for scientific research (THANAPLAST project, OSEO ISI contract number I 1206040W).

Author information

These authors contributed equally: V. Tournier, C. M. Topham

Authors and Affiliations

TBI, Université de Toulouse, CNRS, INRAE, INSA, Toulouse, France
V. Tournier, C. M. Topham, A. Gilles, B. David, C. Folgoas, E. Moya-Leclair, E. Kamionka, M.-L. Desrousseaux, H. Texier, S. Gavalda, J. Nomme, G. Cioci, S. Barbe, I. André, S. Duquesne & A. Marty
CRITT Bio-Industries, INSA, Toulouse, France
M. Cot
Carbios, Biopôle Clermont Limagne, Saint-Beauzire, France
E. Guémard, M. Dalibey, M. Chateau & A. Marty

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V. Tournier
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C. M. Topham
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A. Gilles
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B. David
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C. Folgoas
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H. Texier
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S. Gavalda
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M. Cot
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I. André
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Contributions

I.A., S.D., M. Chateau, V.T. and A.M. designed and directed the research. This work was further conceptualized by G.C., C.M.T., B.D. and J.N. In investigation and validation, E.M.-L., A.G., V.T. and H.T. performed enzyme engineering, purification and variant kinetic analysis. I.A., C.M.T., B.D., S.B. and C.F. performed molecular modelling. S.G. and J.N. carried out structural and physical characterization of variants. M.-L.D., M. Cot, E.G. and E.K. carried out reactions in Minibio reactors. M.D. characterized PET powders. E.G., M.D., M. Chateau and M. Cot developed the scheme for purifying terephthalic acid and supervised the production of PET and bottles. M. Chateau, A.M., I.A., V.T. and S.D. wrote the original draft. All authors reviewed and accepted the manuscript.

Corresponding authors

Correspondence to I. André, S. Duquesne or A. Marty.

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Competing interests

E.G., M.D., M. Chateau and A.M. are employees of Carbios. V.T. has been an employee of Carbios since January 2019. C.M.T., H.T., V.T., M.-L.D., S.D., I.A., S.B. and A.M. have filed patents WO 2018/011284 and WO 2018/011281 for ‘Novel esterases and uses thereof’. H.T., M.-L.D., S.D., A.M., M.D. and M. Chateau have filed patent WO 2017/198786, ‘A process for degrading plastic products’, for protection of part of the work described herein. Confidentiality agreements prevent them from disclosing any newly submitted declaration of invention. All other authors declare no competing interests.

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Peer review information Nature thanks Peter Rem and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 LCC outperformed all other evaluated PET hydrolases during PET-depolymerization assays.

a, Enzymes used here that are reported to hydrolyse PET^{8,16,23,36,37}. Melting temperatures (T_m) were assessed by DSF; values correspond to means ± s.d. (n = 3). b, Hydrolysis of amorphous Gf-PET using equimolar amounts of purified Is-PETase, FsC, BTA-hydrolase 1, BTA-hydrolase 2 or LCC (6.9 nmol_protein g_PET⁻¹ and 2 g_PET l_buffer⁻¹) in their respective buffers at various temperatures. The highest specific activities obtained are in bold. The specific activity of LCC towards Pf-PET is also shown. Means ± s.d. (n = 3) are indicated; n.d., not determined.

Extended Data Fig. 2 The thermostability of LCC at 65 °C is the limiting factor in PET depolymerization.

a, Depolymerization of PET by wild-type LCC is not affected by the products of hydrolysis. The graphs compare the kinetics of Pf-PET depolymerization at 65 °C with or without further addition of ethylene glycol (EG; calculated yields (percentages) are obtained from the quantity of terephthalic acid equivalents (TA_eq.) released during the reaction), and with or without further addition of TA (calculated yields obtained from the quantity of EG released during the reaction). The amount of EG or TA added at reaction initiation corresponds to the quantity of products released with 100% PET depolymerization. Each symbol shows a mean ± s.d. (n = 3). b, The thermostability of LCC is a limiting factor for the PET-depolymerization yield. No change is observed upon adding 100 mg amorphized Pf-PET after 3 days of reaction at 65 °C (empty diamond) compared with the kinetics of Pf-PET depolymerization by wild-type LCC (filled diamond). However, adding 0.69 nmol of wild-type LCC after 3 days of reaction at 65 °C restarts the previously stopped assay (filled circle) with an identical specific activity to that determined originally (shown in the table). Each symbol represents a mean ± s.d. (n = 3); n.a., not applicable; n.d., not determined.

Extended Data Fig. 3 Comparative analysis of the LCC variants generated here.

a, Boxplot distribution of the saturation results. We produced 209 variants by semi-purification, and determined their respective percentage of specific activity with respect to the wild-type LCC, measured in the same Pf-PET-depolymerization conditions. Q1 and Q3 correspond to the first and third quartiles of the distribution, respectively. The median value is shown as a red line. b, Comparison of specific activities and melting temperatures of wild-type LCC and the variants used here. Experiments were performed through preparative production of enzymes followed by a Pf-PET-depolymerization assay (see Supplementary Method 1). Means ± s.d. (n = 3) are shown.

Extended Data Fig. 4 Formation of an additional disulfide bridge within the D238C/S283C variant of LCC.

First derivatives were calculated from DSF thermal denaturation curves for wild-type LCC (solid line) and the D238C/S283C variant (dashed line) in the presence of 0 mM, 1 mM or 100 mM dithiothreitol (DTT). First-derivative peaks correspond to protein melting temperatures. Each curve is representative of a triplicate test. The increasing concentrations of DTT reduced disulfide bridges, resulting in proteins with reduced thermostability. Grey highlighting represents populations with 0, 1 or 2 formed disulfide bridges. Low DTT concentrations (blue curves) were not sufficient to completely reduce all disulfide bridges, resulting in a mixed population of proteins with intermediate melting temperatures. RFU, relative fluorescence units.

Extended Data Fig. 5 Comparative analysis of the kinetics of PcW-PET depolymerization in Minibio bioreactors by the LCC variants generated here.
扩展数据图 5 通过此处生成的 LCC 变体对 Minibio 生物反应器中的 PcW-PET 解聚动力学进行比较分析。

a, Effect of temperature on PcW-PET depolymerization by the LCC variant F243I/D238C/S283C/Y127G at 72 °C or 75 °C, with 1 mg_enzyme g_PET⁻¹. b, Effect of enzyme concentration on PcW-PET depolymerization by the LCC variant F243I/D238C/S283C/Y127G at 72 °C with 1 mg_enzyme g_PET⁻¹, 2 mg_enzyme g_PET⁻¹ or 3 mg_enzyme g_PET⁻¹. Percentages of PET depolymerization were calculated on the basis of NaOH consumption. c, Comparison of wild-type LCC and variants in assays of PcW-PET depolymerization in Minibio bioreactors. The first two columns show the parameters used during PcW-PET depolymerization (temperature and enzyme concentration). The next four columns show the calculated depolymerization yields (after 24 h), based on either NaOH consumption, EG produced, TA_eq. produced, or the weight of residual PET. The last column represents the calculated initial rate of the reaction, based on NaOH consumption.
a, 在 72 °C 或 75 °C、1 mg _enzyme g _PET ⁻¹ 。 b, 酶浓度对LCC变体F243I/D238C/S283C/Y127G在72°C下1 mg _enzyme g _PET ⁻¹ 克 _PET ⁻¹ 或3毫克 _enzyme 克 _PET ⁻¹ .根据 NaOH 消耗量计算 PET 解聚百分比。 c，Minibio生物反应器中PcW-PET解聚测定中野生型LCC和变异体的比较。前两列显示 PcW-PET 解聚过程中使用的参数（温度和酶浓度）。接下来的四列显示了计算的解聚产率（24 小时后），基于 NaOH 消耗、产生的 EG、产生的 TA _eq. 或残留 PET 的重量。最后一列表示基于 NaOH 消耗计算出的反应初始速率。

Extended Data Fig. 6 Kinetics of PET crystallization.
扩展数据图 6 PET 结晶动力学。

Evolution of the crystallinity level of PET (from coloured PcW-PET) at 65 °C, 70 °C, 72 °C and 75 °C.
PET（来自有色 PcW-PET）在 65°C、70°C、72°C 和 75°C 下结晶度的演变。

Extended Data Fig. 7 Overall structures of the catalytic inactive LCC variant S165A and the catalytic inactive F243I/D238C/S283C/Y127G variant S165A.
扩展数据图7催化失活LCC变体S165A和催化失活F243I/D238C/S283C/Y127G变体S165A的总体结构。

a, Left, wild-type LCC (PDB ID 4EB0; green) and the catalytic inactive variant S165A (cyan) are superimposed (with a root mean square deviation (RMSD) of 0.25 Å over 214 Cα atoms). Catalytic residues are represented as magenta rods. The close-up on the right focuses on the catalytic serine (S165) and neighbouring residues. Also shown is an unbiased composite omit map (grey mesh, 2F_o − F_c) contoured at 2.0σ around residues 164–166. The S165A mutation does not structurally affect the folding of the protein around this position. Moreover, the inactivated enzyme is more liable to crystallize and generate better-quality crystals, so we introduced the S165A mutation to our most efficient LCC variant, namely F243I/D238C/S283C/Y127G (ICCG). b, Wild-type LCC (PDB ID 4EB0; green) and the catalytic inactive S165A mutant of the ICCG variant (tan) are superimposed (RMSD = 0.27 Å over 220 Cα atoms). Catalytic (magenta) and mutated (tan) residues are represented as rods. Close-ups show the different mutations and their surrounding residues. Residues from wild-type LCC are represented as thinner rods by comparison with the ICCG variant. Unbiased composite omit maps (grey mesh, 2F_o − F_c) contoured at 1.5σ are shown. None of the introduced Y127G, S165A, F243I or D238C–S283C (engineered disulfide bridge) mutations affected the overall structure of LCC. The asterisked close-up shows that alternative conformations were observed for the cysteine residues, labelled as conformers a and b.
a，左图，野生型 LCC（PDB ID 4EB0；绿色）和催化失活变体 S165A（青色）叠加（214 个 Cα 原子的均方根偏差 (RMSD) 为 0.25 Å）。催化残留物表示为洋红色棒。右侧的特写镜头集中于催化丝氨酸 (S165) 和邻近的残基。还显示了一个无偏复合省略图（灰色网格，2F _o - F _c ），其轮廓在残基 164-166 周围为 2.0σ。 S165A 突变在结构上不会影响该位置周围的蛋白质折叠。此外，失活的酶更容易结晶并产生质量更好的晶体，因此我们将 S165A 突变引入我们最有效的 LCC 变体，即 F243I/D238C/S283C/Y127G (ICCG)。 b，野生型 LCC（PDB ID 4EB0；绿色）和 ICCG 变体（棕褐色）的催化失活 S165A 突变体叠加（RMSD = 0.27 Å，超过 220 个 Cα 原子）。催化（洋红色）和突变（棕褐色）残基表示为棒。特写镜头显示了不同的突变及其周围的残基。与 ICCG 变体相比，野生型 LCC 的残基表示为较细的棒。显示了轮廓为 1.5σ 的无偏复合省略图（灰色网格，2F _o - F _c ）。引入的 Y127G、S165A、F243I 或 D238C-S283C（工程二硫桥）突变均不会影响 LCC 的整体结构。带星号的特写显示观察到半胱氨酸残基的替代构象，标记为构象异构体 a 和 b。

Extended Data Fig. 8 Analysis of 30-ns molecular-dynamics simulations carried out with wild-type LCC (blue) and the ICCG variant (red).

a, Comparison of protein backbone flexibility using average root mean square fluctuations (RMSF) of Cα atoms calculated per residue along molecular-dynamics simulations of enzymes in the apo conformation. The RMSF is linked to the crystallographic B-factor (B) as follows: $RMSF = \sqrt (\frac{3 B}{8 π^{2}})$ . Red arrows, β-strands; black rectangles, α-helices; yellow rectangles, loops in the X-ray crystal structure of the wild-type LCC (PDB ID 4EB0); dashed lines, positions of catalytic residues. b, Monitoring of key catalytic interatomic distances that characterize the catalytic events occurring during molecular-dynamics simulations of enzymes in complex with the model substrate 2-HE(MHET)₃. At the right is a representation of the catalytic triad (residues S165 (Ser 165), H242 (His 242) and D210 (Asp 210)) and 2-HE(MHET)₃, highlighting three relevant interatomic distances (d1, d2, d3). The three graphs show the distributions of these three distances over the first 30 ns of molecular-dynamics simulations of wild-type and ICCG LCC in complex with 2-HE(MHET)₃ (represented as histograms and Gaussian kernel densities), starting from the same initial conformation. Red arrows show changes occurring during the nucleophilic attack of the catalytic serine on the substrate reactive centre; dashed blue lines show hydrogen bonds that assist the catalytic mechanism. The graphs highlight the favoured catalytically productive state adopted by 2-HE(MHET)₃ in variant ICCG. Substantial changes are observed for d1 and d2. Whereas ICCG mainly sampled conformations near the catalytically productive state (average d1 is approximately 3.2 Å; average d2 is approximately 2.8 Å), the wild-type LCC showed a pronounced bimodal distribution with the major conformational population centred on higher distance values, indicating less efficient catalysis. Overall, along the first 30 ns of simulations of these enzymes in complex with 2-HE(MHET)₃, the average distance separating the substrate cleavage site from the catalytic serine (S165) hydroxyl oxygen was substantially shorter in ICCG than in parental LCC, suggesting that formation of the covalent intermediate during catalysis would be facilitated. c, Occurrence of key hydrogen bonds (HBs) between pairs of catalytic residues. The third and fourth columns show the proportion of snapshots in which an HB interaction is observed between the pairs of catalytic residues S165/H242 and H242/D210 during the first 30 ns of simulations. The higher occurrence of HBs in the ICCG simulation between the S165 hydroxyl oxygen and the catalytic H242 ε nitrogen could assist in the abstraction of the S165 hydroxyl hydrogen by H242, and thus enhance the catalytic performance of this variant.

Extended Data Table 1 Sequence analysis of eight prokaryotic cutinase homologues of known crystal structure

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Extended Data Table 2 Data collection and refinement statistics

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Supplementary information

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Supplementary Method 1 | Materials, methods and associated references from the study.

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Supplementary Method 2 | List of nucleotide sequences and expressed amino acid sequences of the genes used in this study. Genetic code specifically used to generate protein variants of LCC is provided.

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Tournier, V., Topham, C.M., Gilles, A. et al. An engineered PET depolymerase to break down and recycle plastic bottles. Nature 580, 216–219 (2020). https://doi.org/10.1038/s41586-020-2149-4

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Received: 27 June 2019
Accepted: 19 February 2020
Published: 08 April 2020
Issue Date: 09 April 2020
DOI: https://doi.org/10.1038/s41586-020-2149-4

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