Main 主要的

Given that crystalline PET has been found to be subject to only limited depolymerization by cutinase enzymes8,15,16,17, 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 mgTAeq. h−1 mgenzyme−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 mgTAeq. h−1 mgenzyme−1. While approaching the glass transition temperature, known to maximize PET chain mobility and enzymatic depolymerization18,19, 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。
figure 1

a, Comparison of the specific hydrolysis activity towards amorphous Gf-PET by: Is-PETase or FsC in 50 mM glycine NaOH buffer, pH 9, at 40 °C; BTA-hydrolase 1 or BTA-hydrolase 2 (BTA1 and BTA2 respectively) in 1 M potassium phosphate buffer, pH 8, at 65 °C; and LCC in 100 mM potassium phosphate buffer, pH 8, at 65 °C. The hydrolysis of Pf-PET by LCC in 100 mM potassium phosphate buffer, pH 8, at 65 °C is also shown. Equimolar amounts of purified proteins were used (6.9 nmolprotein gPET−1 and 2 gPET lbuffer−1). Means ± s.d. (n = 3) are shown. b, Detailed hydrolysis kinetics for Pf-PET depolymerization by LCC, as described in a. Each filled symbol represents the mean ± s.d. (n = 3).
a,通过 Is-PETase 或 FsC 在 50 mM 甘氨酸 NaOH 缓冲液(pH 9)、40 °C 下对无定形 Gf-PET 的比水解活性进行比较; BTA-水解酶 1 或 BTA-水解酶 2(分别为 BTA1 和 BTA2),溶于 1 M 磷酸钾缓冲液,pH 8,65 °C;和 LCC,溶于 100 mM 磷酸钾缓冲液,pH 8,65 °C。还显示了 LCC 在 100 mM 磷酸钾缓冲液(pH 8)、65 °C 下对 Pf-PET 的水解。使用等摩尔量的纯化蛋白质(6.9 nmol protein g PET −1 和 2 g PET l buffer −1 )。平均值±标准差(n = 3) 显示。 b,通过 LCC 进行 Pf-PET 解聚的详细水解动力学,如 a 中所述。每个实心符号代表平均值±s.d。 (n = 3)。

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 date20,21,22. 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.
figure 2

a, Structural model of 2-HE(MHET)3 (coloured stick model) docked in wild-type LCC (grey ribbon). The putative substrate-binding site of LCC can be subdivided into three subsites (−2, −1, +1), each in contact with the MHET units numbered relative to the scissile ester bond (red triangles). Amino acids in the first contact shell of LCC are shown as grey rods. Catalytic residues are in magenta. b, Calculated percentage improvement in specific activity of Pf-PET depolymerization by the F243I and F243W variants compared with wild-type LCC at 65 °C (6.9 nmolprotein gPET−1 and 2 gPET lbuffer−1). Means ± s.d. (n = 3) are shown; *P < 0.025; **P < 0.005 (one-sided t-test).

To improve the thermostability of LCC, we sought divalent-metal-binding sites known to increase enzyme thermal stability in PET hydrolases23,24. 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.
figure 3

a, The main figure shows locations of putative sites that coordinate divalent metal ions in the crystal structures of identified PET hydrolases. On wild-type LCC (ribbon), catalytic residues (S165, D210 and H242) and the C-terminal disulfide bond (C275–C292) are shown as blue rods. Divalent metal ions are shown as green spheres. In the upper inset, residues that bind metal ions in Thermobifida alba Est119 (PDB ID 3WYN) are shown as purple sticks, with the residues indicated with an asterisk; metal-binding residues in Thermobifida cellulosilytica Thc Cut1 (PDB ID 5LUI) are shown as yellow sticks, with the residues underlined in black; and the metal-binding sites in Saccharomonospora viridis Cut190 variants (PDB ID 4WFJ and 5ZNO) are shown as grey sticks, with residue names underlined in red. The left panels show a putative calcium-binding site formed by E208, D238 and S283 in LCC (with calcium shown as a blue sphere), and the disulfide bond (yellow) introduced here. b, Assessment of melting temperature (Tm) by DSF for both wild-type LCC and the D238C/S283C variant in the presence of increasing concentrations of CaCl2. Curves represent the optimal fit to the data, and each symbol represents the mean ± s.d. (n = 3).
a,主图显示了在已鉴定的PET水解酶的晶体结构中协调二价金属离子的假定位点的位置。在野生型 LCC(色带)上,催化残基(S165、D210 和 H242)和 C 末端二硫键(C275–C292)显示为蓝色棒。二价金属离子显示为绿色球体。在上插图中,Thermobifida alba Est119 (PDB ID 3WYN) 中与金属离子结合的残基显示为紫色棒,其中残基用星号表示; Thermobifida cellulosilytica Thc Cut1 (PDB ID 5LUI) 中的金属结合残基显示为黄色棒,残基用黑色下划线显示; Saccharomonospora viridis Cut190 变体(PDB ID 4WFJ 和 5ZNO)中的金属结合位点显示为灰色棒,残基名称用红色下划线显示。左图显示了 LCC 中由 E208、D238 和 S283 形成的推定钙结合位点(钙显示为蓝色球体),以及此处引入的二硫键(黄色)。 b,在存在增加浓度的 CaCl 2 的情况下,通过 DSF 对野生型 LCC 和 D238C/S283C 变体的解链温度 (T m ) 进行评估。曲线代表数据的最佳拟合,每个符号代表平均值±s.d。 (n = 3)。

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 industries28,29 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。
figure 4

Assays of PcW-PET (200 g kg−1) depolymerization were carried out in Minibio bioreactors at pH 8 and 72 °C. a, Comparison of PcW-PET depolymerization kinetics at 1 mgenzyme gPET−1 for the WCCG, ICCG, WCCM and ICCM variants and wild-type LCC. b, Comparison of PcW-PET depolymerization kinetics at 3 mgenzyme gPET−1 for the WCCG and ICCG variants. PET-depolymerization percentages were calculated on the basis of NaOH consumption.
PcW-PET (200 g kg −1 ) 解聚分析在 Minibio 生物反应器中在 pH 8 和 72 °C 下进行。 a,WCCG、ICCG、WCCM 和 ICCM 变体以及野生型 LCC 在 1 mg enzyme g PET −1 浓度下的 PcW-PET 解聚动力学比较。 b,WCCG 和 ICCG 变体在 3 mg enzyme g PET −1 浓度下的 PcW-PET 解聚动力学比较。 PET 解聚百分比是根据 NaOH 消耗量计算的。

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 gTA l−1 h−1 genzyme−1 (27.9 gTA l−1 h−1 genzyme−1 over the course of the reaction), corresponding to a maximum productivity of 42.1 gTA l−1 h−1  (16.7 gTA 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 mgTAeq. h−1 mgenzyme−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 gPET−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 mgKOH 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 gTA 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》。