提升未预处理聚对苯二甲酸乙二酯的酶法降解效率

doi:10.1016/S1872-2067(24)60232-9

催化学报 ›› 2025, Vol. 71: 375-389.DOI: 10.1016/S1872-2067(24)60232-9

提升未预处理聚对苯二甲酸乙二酯的酶法降解效率

曹玉峰^a^,^e^,¹, 向腊^a^,¹, Nikodinovic-Runic Jasmina^c, Maslak Veselin^d, 金建明^b^,^*(), 梁朝宁^a^,^*(), 唐双焱^a^,^*()

^a中国科学院微生物研究所, 微生物多样性与资源创新利用全国重点实验室, 微生物生理与代谢工程研究室, 北京 100101, 中国
^b北京工商大学, 北京市植物资源研究开发重点实验室, 北京 100048, 中国
^c贝尔格莱德大学, 分子遗传学和遗传工程研究所, 贝尔格莱德, 塞尔维亚
^d贝尔格莱德大学, 有机化学系, 贝尔格莱德, 塞尔维亚
^e中国科学院大学, 北京 100049, 中国

收稿日期:2024-11-15 接受日期:2025-01-08 出版日期:2025-04-18 发布日期:2025-04-13
通讯作者: * 电子信箱: tangsy@im.ac.cn (唐双焱), laraineliang@163.com (梁朝宁), jinjianming@btbu.edu.cn (金建明).
作者简介:
¹共同第一作者.
基金资助:
中国科学院战略性先导科技专项(XDB0810000);国家重点研发项目(2021YFC2103901);国家自然科学基金(31961133016);中国科学院科研仪器设备研制项目(YJKYYQ20210032);中国博士后科学基金(2022M713331)

Improving enzymatic degradation of unpretreated poly(ethylene terephthalate)

Yufeng Cao^a^,^e^,¹, La Xiang^a^,¹, Jasmina Nikodinovic-Runic^c, Veselin Maslak^d, Jian-Ming Jin^b^,^*(), Chaoning Liang^a^,^*(), Shuang-Yan Tang^a^,^*()

^aDepartment of Microbial Physiological & Metabolic Engineering, State Key Laboratory of Microbial Diversity and Innovative Utilization, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
^bBeijing Key Laboratory of Plant Resources Research and Development, Beijing Technology and Business University, Beijing 100048, China
^cInstitute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade 11042, Serbia
^dDepartment of Organic Chemistry, University of Belgrade, Belgrade 11158, Serbia
^eUniversity of Chinese Academy of Sciences, Beijing 100049, China

Received:2024-11-15 Accepted:2025-01-08 Online:2025-04-18 Published:2025-04-13
Contact: * E-mail: tangsy@im.ac.cn (S.-Y. Tang), laraineliang@163.com (C. Liang), jinjianming@btbu.edu.cn (J.-M. Jin).
About author:
¹Contributed equally to the work.
Supported by:
Strategic Priority Research Program of Chinese Academy of Sciences(XDB0810000);National Key Research and Development Program of China(2021YFC2103901);National Natural Science Foundation of China(31961133016);Instrument Developing Project of Chinese Academy of Science(YJKYYQ20210032);China Postdoctoral Science Foundation(2022M713331)

摘要/Abstract

摘要：

聚对苯二甲酸乙二醇酯(PET)是工程塑料之一, 由对苯二甲酸(TPA)和乙二醇以酯键连接而成, 广泛应用于容器、包装材料和纺织行业. 然而, 作为一种高耐久性和高抗性的材料, PET很难通过生物或化学过程降解, 从而造成严重的环境和健康问题. PET污染已成为全球性环境问题之一. 尽管近年来在提升解聚酶降解预处理PET(Goodfellow-PET, gf-PET)的效率方面取得了显著进展, 但在降解未预处理、高结晶度的PET材料方面, 相关研究与技术开发依然面临巨大挑战. 这类PET材料由于结晶度高, 结构稳定, 使得生物酶降解尤为困难. 鉴于此, 本文致力于通过迭代饱和突变策略, 定向进化叶枝堆肥角质酶LCC的突变体WCCG, 以期增强其对未预处理、高结晶度PET底物(结晶度高于40%)的降解效率.
为实现上述目标, 首先构建高效、精准的高通量筛选体系. 该体系专门针对PET降解产物设计, 能够快速识别并筛选出具有优异降解性能的酶突变体. 通过分子对接发现, WCCG的W243残基伸入底物结合沟中央, 并与底物的苯环间形成pi-pi堆叠. 这一强烈的相互作用导致底物方向发生扭转, 以“Z”形与WCCG结合. 考虑到底物结合位点间存在叠加或协同效应, 选择距离底物5 Å内的24个氨基酸残基进行迭代饱和突变. 通过七轮筛选和稳定性设计, 成功获得两个性能显著提升的酶突变体: WCCG-sup1和WCCG-sup2. 其中, WCCG-sup1对未预处理PET(结晶度为42.0%)表现出最高活性, 与WCCG相比, TPA产量提高4.2倍. WCCG-sup2对结晶度9.9%的gf-PET粉末表现出最高活性, 比WCCG提高3.2倍. 有趣的是, WCCG-sup1和WCCG-sup2分别对未预处理PET(结晶度为42.0%)和预处理的gf-PET表现出最高活性. 为了探究造成此差异的原因, 通过荧光法确定了底物对酶的吸附率. 在孵育2 h后, WCCG-sup1在未预处理PET上的吸附率为42%, 是WCCG的2.1倍、WCCG-sup2的1.3倍. 相比之下, WCCG-sup2对gf-PET粉末的吸附率最高. 结果表明, S241D突变导致酶易于吸附于未预处理的PET表面, 而无该突变的酶则更易吸附于预处理的PET表面, 说明对于相同结晶度的底物, 酶突变体的吸附率在PET降解中发挥着重要作用. 然而, 底物结晶度的差异仍是影响降解效果的主要因素. 为探索潜在应用, 比较突变酶降解其他合成聚酯(如聚对苯二甲酸丁二醇酯和聚(醚-酯)弹性体)的能力, 其中WCCG-sup1表现出最高活性, 证明其在广泛降解合成塑料具有巨大潜力. 最后, 在72 ºC, WCCG-sup1仅需36 h即可完全降解商用未预处理PET包装盒(结晶度为16.1%), 这一成果在PET生物降解领域具有重要意义.
综上, 本研究从稳定性, 降解活性和酶吸附作用的角度, 为深入理解LCC的结构-功能关系提供了参考, 不仅有助于推动PET生物降解技术的发展, 更为未来开发更高效、更环保的PET降解酶提供了坚实的理论基础与实践参考.

关键词: 迭代饱和突变, 聚对苯二甲酸乙二醇酯降解效率, 底物吸附, 叶枝堆肥角质酶, 未预处理聚对苯二甲酸乙二醇酯

Abstract:

Although the efficiency of poly(ethylene terephthalate) (PET) degradation has been successfully improved by depolymerase engineering, mostly by using Goodfellow-PET (gf-PET) as a substrate, efforts to degrade unpretreated PET materials with high crystallinity remain insufficient. Here, we endeavored to improve the degradation capability of a WCCG mutant of leaf-branch compost cutinase (LCC) on a unpretreated PET substrate (crystallinity > 40%) by employing iterative saturation mutagenesis. Using this method, we developed a high-throughput screening strategy appropriate for unpretreated substrates. Through extensive screening of residues around the substrate-binding groove, two variants, WCCG-sup1 and WCCG-sup2, showed good depolymerization capabilities with both high- (42%) and low-crystallinity (9%) substrates. The WCCG-sup1 variant completely depolymerized a commercial unpretreated PET product in 36 h at 72 °C. In addition to enzyme thermostability and catalytic efficiency, the adsorption of enzymes onto substrates plays an important role in PET degradation. This study provides valuable insights into the structure-function relationship of LCC.

Key words: Iterative saturation mutagenesis, Poly(ethylene terephthalate) depolymerization efficiency, Substrate adsorption, Leaf-branch compost cutinase, Unpretreated poly(ethylene terephthalate)

曹玉峰, 向腊, Nikodinovic-Runic Jasmina, Maslak Veselin, 金建明, 梁朝宁, 唐双焱. 提升未预处理聚对苯二甲酸乙二酯的酶法降解效率[J]. 催化学报, 2025, 71: 375-389.

Yufeng Cao, La Xiang, Jasmina Nikodinovic-Runic, Veselin Maslak, Jian-Ming Jin, Chaoning Liang, Shuang-Yan Tang. Improving enzymatic degradation of unpretreated poly(ethylene terephthalate)[J]. Chinese Journal of Catalysis, 2025, 71: 375-389.

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图/表 8

Fig. 1. Spectrophotometric screening of PET depolymerization activity. (a) Spectrophotometry of TPA. (b) Positive correlation between absorbance at 290 nm (OD290) and TPA concentrations. (c) Absorbance of the LCC reaction mixtures at 290 nm, compared with the control reactions in the absence of LCC enzyme or PET powder substrate (crystallinity 42.0 ± 3.1%). The reactions were carried out at 72 °C for 36 h. (d) Workflow for the directed evolution of WCCG in Escherichia coli.

Fig. 2. Structure-guided directed evolution of WCCG. (a) The interactions between 2-HE(MHET)4 and W243 in WCCG. The 2-HE(MHET)4 is colored in green, W243 is shown in stick representation, the magenta dashed lines indicate π-π stacking interactions. (b) Structural model of 2-HE(MHET)4 docked in ICCG. (c) The identified 24 amino acid residues within 5 ? to the substrate according to the WCCG-2-HE(MHET)4 complex. The 24 amino acid residues were shown as sticks style in magentas. The catalytic residues were shown as sticks style in red. (d) Results of iterative site-saturation mutagenesis of WCCG for improved PET depolymerization. WCCG was shown as black sphere; Mutantions obtained from Round 1, 2, 3, 4, 5, 6 and FireProt-predicting mutagenesis were shown as grey, blue, green, orange, cyan, purple and red spheres, respectively. The order of mutation was indicated by black arrows. The arrows started from WCCG, and pointed to the best mutation in the next round. The mutants with the highest activity obtained from Round 1, 2, 3, 4, 5, 6 and FireProt-predicting mutagenesis were marked as M1, M2, M3, M4, M5, M6 and WCCG-sup1, respectively. And the amino acid substitutions of these mutants were shown in the table. The reaction was carried out at 72 °C for 36 h, with 30 mg PET powder substrate (crystallinity 42.0 ± 3.1%).

Fig. 3. Characterization of PET depolymerization activity of the obtained mutants. Temperature (a) and pH (b) dependence of WCCG and its mutants (enzyme concertration 25 μg mL-1) on PET powder with 42.0 ± 3.1% crystallinity for 5 h. Time courses of total products released from depolymerization of PET powder with 42.0 ± 3.1% crystallinity (c) or gf-PET powder (d) by various mutants (enzyme concertration 0.4 μg mL-1) performed at 72 °C, pH 8.0. (e) Enzymatic degradation of films of commercial PET materials or gf-PET by various depolymerase mutants (enzyme concertration 5.54 μg mL-1) for 36 h, performed at respective optimum conditions. (f) Degradation of different synthetic polymers by various depolymerase mutants (enzyme concertration 5.54 μg mL-1) for 36 h at respective optimum conditions. PET was degraded to TPA and small amounts of MHET. Polybutylene terephthalate (PBT) was degraded to TPA and small amounts of mono(4-hydroxybutyl)-TPA (MHBT). TPEE was degraded to TPA, small amounts of MHET and MHBT. No bis(4-hydroxybutyl)-TPA (BHBT) or BHET was observed. The reactions were performed at 72 °C (for WCCG, ICCG and their mutants), 75 °C (for LCCICCG_I6M) or 50 °C (for FAST-PETase).

Fig. 4. Complete depolymerization of a piece of unpretreated PET container. (a) Time course of degradation of a piece of unpretreated PET container (length 11.7 cm, width 7.8 cm, weight 2.5 g, crystallinity 16.1 ± 0.2%) with 7.5 mg of WCCG-sup1, ICCG, or FAST-PETase, determined with HPLC. (b) Morphological changes and (c) Scanning electron microscope images for depolymerization of a piece of unpretreated PET container following various exposure times in WCCG-sup1 degradation.

Table 1 Kinetic characterization of WCCG and mutants.

Enzyme	Specific activity (mg_TPAeq h^-1 mg_enzyme^-1)		K_A (L μmol^-1)	k_h (mL mg^-1 min^-1)
Enzyme	gf-PET	PET (42.0 ± 3.1% crystallinity)	M(HET)2 M(HET)2
WCCG	99.6 ± 3.6	21.0 ± 2.6	3.9 ± 0.8	0.016 ± 0.006
M3	107.3 ± 2.1	23.7 ± 1.0	n.d.	n.d.
M5	112.0 ± 4.3	31.3 ± 1.6	n.d.	n.d.
M6	136.0 ± 6.4	34.1 ± 1.8	n.d.	n.d.
WCCG-sup2	187.2 ± 6.1	45.8 ± 1.1	10.1 ± 0.4	0.018 ± 0.00005
WCCG-sup1	169.5 ± 6.3	61.5 ± 2.5	14.5 ± 0.6	0.019 ± 0.0002

Fig. 5. Structure analysis of amino acid substitutions. (a,b) Location of substitutions in the overall structure of WCCG-sup1 docked with 2-HE (MHET)4 (cyan). The WCCG-sup1 structure is shown as cartoon (green), while the Cα atoms of the eight substitutions (G88A, T96S, F125T, S136Q, N239L, S241D, N246M and S247R) are shown as magenta atoms. The key residues are shown in stick representations. (c-i) Structural effects of substitutions of N246M (c), N239L (d), T96S (e), F125T (f), G88A (g), S136Q (h) and S241D/S247R (i) of WCCG-sup1 (green) compared to WCCG (orange). The green dashed lines indicate hydrogen bonds, the yellow dashed lines indicate electrostatic interactions, and the pink dashed lines indicate hydrophobic interactions.

Fig. 6. Structural model of 2-HE(MHET)4 docked in WCCG-sup1 (a), WCCG-sup2 (b), and WCCG (c). The proteins were shown in surface, mutation sites were shown as sticks style in oranges, residue 247 was colored in magentas, and the catalytic residues were shown as sticks style in blue. The 2-HE(MHET)4 was showed as stick model in cyans, W243 was shown in stick model in line.

Fig. 7. Enzyme-PET binding assay. The adsorption of WCCG (a,d), WCCG-sup1 (b,e) and WCCG-sup2 (c,f) to unpretreated PET (42.0 ± 3.1% crystallinity) (a-c) and gf-PET (d-f) monitored by Zeiss Axio Image A2. (g) The adsorption rate of WCCG, WCCG-sup1 and WCCG-sup2 to unpretreated PET and gf-PET monitored by fluorescence analysis.

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提升未预处理聚对苯二甲酸乙二酯的酶法降解效率

Improving enzymatic degradation of unpretreated poly(ethylene terephthalate)

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