Engineering a robust IsPETase for energy-efficient PET depolymerization in natural seawater at ambient temperatures

Abstract

Enzymatic depolymerization of polyethylene terephthalate (PET), the world’s most widely used polyester, in seawater at ambient temperature offers a promising and energy-efficient route for freshwater-free plastic recycling. While a number of PET hydrolases have been reported in recent years, their potential under saline conditions remains largely unexplored. Here, we screened eight enzymes in artificial seawater at 30 °C and engineered the most active one, IsPETase, using a semi-rational strategy focused on rigidifying flexible sites. The resulting variant M8 showed simultaneous enhancementsin thermostability (ΔT_m = + 27.3 °C), activity (1.14-fold increase) and soluble expression yield (14.3-fold increase). The overall depolymerization efficiency of M8 surpassed that of the thermostable benchmark enzymes DuraPETase and LCC-ICCG by 32.2- and 10.4-fold, respectively. Notably, M8 achieved continuous and efficient depolymerization of 15% (w/v) PET powder in natural seawater at 37 °C, yielding monomers at a rate of 15.4 mM/day, a concentration sufficient to support downstream bacterial assimilation. This work provides an efficient enzymatic platform and paves the way for fully integrated, seawater-based plastic bioconversion processes.

Keywords

PET hydrolase / Bio-recycling / Enzymatic depolymerization / Enzyme engineering / Seawater bio-catalysis / Simultaneous enzymatic depolymerization and fermentation (SEDF)

References

[1] Abraham MJ, Murtola T, Schulz R, Páll S, Smith JC, Hess B, Lindahl E. GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX, 2015, 1–2: 19-25

[2] Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, et al. . PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Struct Biol, 2010, 66: 213-221

[3] American society for testing and materials. Standard practice for the preparation of substitute ocean water. ASTM International. 2021. https://store.astm.org/d1141-98r21.html.

[4] Austin HP, Allen MD, Donohoe BS, Rorrer NA, Kearns FL, Silveira RL, Pollard BC, Dominick G, Duman R, El Omari K, et al. . Characterization and engineering of a plastic-degrading aromatic polyesterase. P Natl Acad Sci u s a, 2018, 115(19): E4350-E4357

[5] Bell EL, Smithson R, Kilbride S, Foster J, Hardy FJ, Ramachandran S, Tedstone AA, Haigh SJ, Garforth AA, Day PJR, et al. . Directed evolution of an efficient and thermostable PET depolymerase. Nat Catal, 2022, 5(8): 673-681

[6] Bigman LS, Levy Y. Proteins: molecules defined by their trade-offs. Curr Opin Struct Biol, 2020, 60: 50-56

[7] Bollinger A, Thies S, Knieps-Gruenhagen E, Gertzen C, Kobus S, Hoeppner A, Ferrer M, Gohlke H, Smits SHJ, Jaeger KE. A novel polyester hydrolase from the marine bacterium Pseudomonas aestusnigri-structural and functional insights. Front Microbiol, 2020, 11: 114

[8] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976, 72: 248-254

[9] Brandenberg OF, Schubert OT, Kruglyak L. Towards synthetic PETtrophy: engineering Pseudomonas putida for concurrent polyethylene terephthalate (PET) monomer metabolism and PET hydrolase expression. Microb Cell Fact, 2022, 21(1): 119

[10] Buss O, Rudat J, Ochsenreither K. FoldX as protein engineering tool: better than random based approaches?. Comput Struct Biotechnol J, 2018, 16: 25-33

[11] Bussi G, Donadio D, Parrinello M. Canonical sampling through velocity rescaling. J Comput Chem., 2007, 126(1): 014101

[12] Chen GQ, Jiang XR. Next generation industrial biotechnology based on extremophilic bacteria. Curr Opin Biotechnol, 2018, 50: 94-100

[13] Chen VB, Arendall WBIIIHeadd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS, Richardson DC. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Struct Biol, 2010, 66: 12-21

[14] Chen J, Jia Y, Sun Y, Liu K, Zhou C, Liu C, Li D, Liu G, Zhang C, Yang T, et al. . Global marine microbial diversity and its potential in bioprospecting. Nature, 2024, 633(8029): 371-379

[15] Cottom JW, Cook E, Velis CA. A local-to-global emissions inventory of macroplastic pollution. Nature, 2024, 633(8028): 101-108

[16] Cowger W, Willis KA, Bullock S, Conlon K, Emmanuel J, Erdle LM, Eriksen M, Farrelly TA, Hardesty BD, Kerge K, et al. Global producer responsibility for plastic pollution. Sci Adv. 2024;10(17):eadj8275.

[17] Cui Y, Chen Y, Sun J, Zhu T, Pang H, Li C, Geng WC, Wu B. Computational redesign of a hydrolase for nearly complete PET depolymerization at industrially relevant high-solids loading. Nat Commun, 2024, 15(1): 1417

[18] Cui Y, Chen Y, Liu X, Dong S, Tian Ye, Qiao Y, et al. Computational redesign of a PETase for plastic biodegradation under ambient condition by the GRAPE strategy. ACS Catal. 2021;11(3):1340–1350.

[19] Darden T, York D, Pedersen L. Particle mesh Ewald: anN.log(N) method for Ewald sums in large systems. J Chem Phys, 1993, 98(12): 10089-10092

[20] Deng H, Wei R, Luo W, Hu L, Li B, Di Yn, et al. Microplastic pollution in water and sediment in a textile industrial area. Environ Pollut. 2020;258:113658.

[21] Dieckhaus H, Brocidiacono M, Randolph NZ, Kuhlman B. Transfer learning to leverage larger datasets for improved prediction of protein stability changes. P Natl Acad Sci U S A. 2024;121(6):e2314853121.

[22] Duan L, Liu X, Zhang JZH. Interaction entropy: a new paradigm for highly efficient and reliable computation of protein-ligand binding free energy. J Am Chem Soc, 2016, 138(17): 5722-5728

[23] Dvorak P, Nikel PI, Damborsky J, de Lorenzo V. Bioremediation 3.0: engineering pollutant-removing bacteria in the times of systemic biology. Biotechnol Adv, 2017, 35(7): 845-866

[24] Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr D Struct Biol, 2010, 66: 486-501

[25] Erickson E, Gado JE, Avilán L, Bratti F, Brizendine RK, Cox PA, Gill R, Graham R, Kim DJ, König G, et al. . Sourcing thermotolerant poly(ethylene terephthalate) hydrolase scaffolds from natural diversity. Nat Commun., 2022, 13(1): 7850

[26] Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, et al. Gaussian 09, revision D.01. Gaussian, Inc., Wallingford CT. 2013.

[27] Gao S, Shi L, Wei H, Liu P, Zhao W, Gong L, Tan Z, Zhai H, Liu W, Liu H, et al. . β-sheet engineering of IsPETase for PET depolymerization. Engineering, 2025, 47: 180-193

[28] Gordon JC, Myers JB, Folta T, Shoja V, Heath LS, Onufriev A. H++: a server for estimating pK_as and adding missing hydrogens to macromolecules. Nucleic Acids Res, 2005, 33: W368-W371

[29] Han X, Liu W, Huang JW, Ma J, Zheng Y, Ko TP, Xu L, Cheng YS, Chen CC, Guo R-T. Structural insight into catalytic mechanism of PET hydrolase. Nat Commun, 2017, 8: 2106

[30] Hanwell MD, Curtis DE, Lonie DC, Vandermeersch T, Zurek E, Hutchison GR. Avogadro: an advanced semantic chemical editor, visualization, and analysis platform. J Cheminform, 2012, 4: 17

[31] Hess B, Bekker H, Berendsen HJC, Fraaije JGEM. LINCS: a linear constraint solver for molecular simulations. J Comput Chem, 1997, 18(12): 1463-1472

[32] Hong H, Ki D, Seo H, Park J, Jang J, Kim KJ. Discovery and rational engineering of PET hydrolase with both mesophilic and thermophilic PET hydrolase properties. Nat Commun, 2023, 14(1): 4556

[33] Huang R, Chen H, Zhong C, Kim JE, Zhang YH. High-throughput screening of coenzyme preference change of thermophilic 6-phosphogluconate dehydrogenase from NADP(+) to NAD(.). Sci Rep, 2016, 6: 32644

[34] Huang X, Cao L, Qin Z, Li S, Kong W, Liu Y. Tat-independent secretion of polyethylene terephthalate hydrolase PETase in Bacillus subtilis 168 mediated by its native signal peptide. J Agric Food Chem, 2018, 66(50): 13217-13227

[35] Joo S, Cho IJ, Seo H, Son HF, Sagong HY, Shin TJ, Choi SY, Lee SY, Kim KJ. Structural insight into molecular mechanism of poly (ethylene terephthalate) degradation. Nat Commun., 2018, 9: 382

[36] Kawai F, Oda M, Tamashiro T, Waku T, Tanaka N, Yamamoto M, Mizushima H, Miyakawa T, Tanokura M. A novel Ca²⁺-activated, thermostabilized polyesterase capable of hydrolyzing polyethylene terephthalate from Saccharomonospora viridis AHK190. Appl Microbiol Biotechnol, 2014, 98(24): 10053-10064

[37] Kille S, Acevedo-Rocha CG, Parra LP, Zhang ZG, Opperman DJ, Reetz MT, Pablo Acevedo J. Reducing codon redundancy and screening effort of combinatorial protein libraries created by saturation mutagenesis. ACS Synth Biol, 2013, 2(2): 83-92

[38] Li G, Zhang H, Sun Z, Liu X, Reetz MT. Multiparameter optimization in directed evolution: engineering thermostability, enantioselectivity, and activity of an epoxide hydrolase. ACS Catal, 2016, 6(6): 3679-3687

[39] Liu CG, Xiao Y, Xia XX, Zhao XQ, Peng L, Srinophakun P, Bai FW. Cellulosic ethanol production: progress, challenges and strategies for solutions. Biotechnol Adv, 2019, 37(3): 491-504

[40] Liu X, Park H, Ackermann YS, Averous L, Ballerstedt H, Besenmatter W, et al. Exploring biotechnology for plastic recycling, degradation and upcycling for a sustainable future. Biotechnol Adv. 2025a;81:108544.

[41] Liu YJ, Zhou J, Li Y, Yan X, Xu A, Zhou X, et al. State-of-the-art advances in biotechnology for polyethylene terephthalate bio-depolymerization. Green Carbon. 2025b;3(3):303-19.

[42] Lu T. A comprehensive electron wavefunction analysis toolbox for chemists, Multiwfn. J Chem Phys, 2024, 161(8): 082503

[43] Lu T, Chen F. Multiwfn: a multifunctional wavefunction analyzer. J Comput Chem, 2012, 33(5): 580-592

[44] Lu H, Diaz DJ, Czarnecki NJ, Zhu C, Kim W, Shroff R, Acosta DJ, Alexander BR, Cole HO, Zhang Y, et al. . Machine learning-aided engineering of hydrolases for PET depolymerization. Nature, 2022, 604(7907): 662-667

[45] Maier JA, Martinez C, Kasavajhala K, Wickstrom L, Hauser KE, Simmerling C. ff14SB: improving the accuracy of protein side chain and backbone parameters from ff99SB. J Chem Theory Comput, 2015, 11(8): 3696-3713

[46] McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ. Phaser crystallographic software. J Appl Crystallogr, 2007, 40: 658-674

[47] Mehta A, Gaur U, Wunderlich B. Equilibrium melting parameters of poly(ethylene terephthalate). J Polym Sci Polym Phys Ed (USA), 1978, 16(2): 289-296

[48] Mican J, Jaradat DSMM, Liu W, Weber G, Mazurenko S, Bornscheuer UT, et al. Exploring new galaxies: perspectives on the discovery of novel PET-degrading enzymes. Appl Catal B: Environ. 2024;342:123404.

[49] Narancic T, Salvador M, Hughes GM, Beagan N, Abdulmutalib U, Kenny ST, Wu H, Saccomanno M, Um J, O'Connor KE, et al. . Genome analysis of the metabolically versatile Pseudomonas umsongensis GO16: the genetic basis for PET monomer upcycling into polyhydroxyalkanoates. Microb Biotechnol, 2021, 14(6): 2463-2480

[50] Oda M, Yamagami Y, Inaba S, Oida T, Yamamoto M, Kitajima S, Kawai F. Enzymatic hydrolysis of PET: functional roles of three Ca²⁺ ions bound to a cutinase-like enzyme, Cut190*, and its engineering for improved activity. Appl Microbiol Biotechnol, 2018, 102(23): 10067-10077

[51] Onufriev A, Bashford D, Case DA. Exploring protein native states and large-scale conformational changes with a modified generalized born model. Proteins Struct Funct Bioinform, 2004, 55(2): 383-394

[52] Otwinowski Z, Minor W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol, 1997, 276: 307-326

[53] Park H, He H, Yan X, Liu X, Scrutton NS, Chen GQ. PHA is not just a bioplastic!. Biotechnol Adv., 2024, 71: 108320

[54] Parrinello M, Rahman A. Polymorphic transitions in single crystals: a new molecular dynamics method. J Appl Phys, 1981, 52(12): 7182-7190

[55] Pottinger AS, Geyer R, Biyani N, Martinez CC, Nathan N, Morse MR, Liu C, Hu S, de Bruyn M, Boettiger C, et al. . Pathways to reduce global plastic waste mismanagement and greenhouse gas emissions by 2050. Science, 2024, 386(6726): 1168-1173

[56] Ragaert K, Delva L, Van Geem K. Mechanical and chemical recycling of solid plastic waste. Waste Manag, 2017, 69: 24-58

[57] Reetz MT, Carballeira JD. Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat Protoc, 2007, 2(4): 891-903

[58] Reetz MT, Kahakeaw D, Lohmer R. Addressing the numbers problem in directed evolution. ChemBioChem, 2008, 9(11): 1797-1804

[59] Reetz MT, Qu G, Sun Z. Engineered enzymes for the synthesis of pharmaceuticals and other high-value products. Nat Synth, 2024, 3(3): 347-356

[60] Ribitsch D, Heumann S, Trotscha E, Acero EH, Greimel K, Leber R, Birner-Gruenberger R, Deller S, Eiteljoerg I, Remler P, et al. . Hydrolysis of polyethyleneterephthalate by p-Nitrobenzylesterase from Bacillus subtilis. Biotechnol Prog, 2011, 27(4): 951-960

[61] Romero PA, Tran TM, Abate AR. Dissecting enzyme function with microfluidic-based deep mutational scanning. Proc Natl Acad Sci U S A, 2015, 112(23): 7159-7164

[62] Schauperl M, Nerenberg PS, Jang H, Wang LP, Bayly CI, Mobley DL, Gilson MK. Non-bonded force field model with advanced restrained electrostatic potential charges (RESP2). Commun Chem., 2020, 3(1): 44

[63] Seo H, Hong H, Park J, Lee SH, Ki D, Ryu A, Sagong HY, Kim KJ. Landscape profiling of PET depolymerases using a natural sequence cluster framework. Science, 2025, 387(6729): eadp5637

[64] Son HF, Cho IJ, Joo S, Seo H, Sagong H-Y, Choi SY, Lee SY, Kim KJ. Rational protein engineering of thermo-stable PETase from Ideonella sakaiensis for highly efficient PET degradation. ACS Catal, 2019, 9(4): 3519-3526

[65] Sulaiman S, Yamato S, Kanaya E, Kim J-J, Koga Y, Takano K, Kanaya S. Isolation of a novel cutinase homolog with polyethylene terephthalate-degrading activity from leaf-branch compost by using a metagenomic approach. Appl Environ Microbiol, 2012, 78(5): 1556-1562

[66] Sun JY, Zhu T, Cui YL, Wu B. Structure-based self-supervised learning enables ultrafast protein stability prediction upon mutation. Innovation., 2025, 6(1): 100750

[67] Tan D, Xue YS, Aibaidula G, Chen GQ. Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD01. Bioresour Technol, 2011, 102(17): 8130-8136

[68] Tan D, Wang Y, Tong Y, Chen GQ. Grand challenges for industrializing polyhydroxyalkanoates (PHAs). Trends Biotechnol. 2021;39(9):953-963.

[69] Taniguchi I, Yoshida S, Hiraga K, Miyamoto K, Kimura Y, Oda K. Biodegradation of PET: current status and application aspects. ACS Catal, 2019, 9(5): 4089-4105

[70] Then J, Wei R, Oeser T, Barth M, Belisario-Ferrari MR, Schmidt J, Zimmermann W. Ca²⁺ and Mg²⁺ binding site engineering increases the degradation of polyethylene terephthalate films by polyester hydrolases from Thermobifida fusca. Biotechnol J, 2015, 10(4): 592-U302

[71] Then J, Wei R, Oeser T, Gerdts A, Schmidt J, Barth M, Zimmermann W. A disulfide bridge in the calcium binding site of a polyester hydrolase increases its thermal stability and activity against polyethylene terephthalate. FEBS Open Bio, 2016, 6(5): 425-432

[72] Thompson RC, Courtene-Jones W, Boucher J, Pahl S, Raubenheimer K, Koelmans AA. Twenty years of microplastic pollution research-what have we learned? Science. 2024;386(6720):eadl2746.

[73] Tiso T, Narancic T, Wei R, Pollet E, Beagan N, Schroder K, Honak A, Jiang M, Kenny ST, Wierckx N, et al. . Towards bio-upcycling of polyethylene terephthalate. Metab Eng, 2021, 66: 167-178

[74] Tournier V, Topham CM, Gilles A, David B, Folgoas C, Moya-Leclair E, Kamionka E, Desrousseaux ML, Texier H, Gavalda S, et al. . An engineered PET depolymerase to break down and recycle plastic bottles. Nature, 2020, 580(7802): 216-219

[75] Tournier V, Duquesne S, Guillamot F, Cramail H, Andre I, Taton D, Marty A. Enzymes? Power for plastics degradation. Chem Rev, 2023, 123(9): 5612-5701

[76] Trott O, Olson AJ. Software news and update AutoDock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem, 2010, 31(2): 455-461

[77] Valdes-Tresanco MS, Valdes-Tresanco ME, Valiente PA, Moreno E. Gmx_MMPBSA: a new tool to perform end-state free energy calculations with GROMACS. J Chem Theory Comput, 2021, 17(10): 6281-6291

[78] Vertommen M, Nierstrasz VA, van der Veer M, Warmoeskerken M. Enzymatic surface modification of poly(ethylene terephthalate). J Biotechnol, 2005, 120(4): 376-386

[79] Wang JM, Wolf RM, Caldwell JW, Kollman PA, Case DA. Development and testing of a general amber force field. J Comput Chem, 2004, 25(9): 1157-1174

[80] Wang J, Wang W, Kollman PA, Case DA. Automatic atom type and bond type perception in molecular mechanical calculations. J Mol Graph Model, 2006, 25(2): 247-260

[81] Wang C, Zhang J, Zhou Z, Jiang L. Genetically engineered Escherichia coli: the new recyclers of PET plastic waste. Biotechnol Adv, 2025, 85: 108713-108713

[82] Wang Z, Xie D, Wu D, Luo X, Wang S, Li Y, et al. Robust enzyme discovery and engineering with deep learning using CataPro. Nat Commun. 2025b;16(1):2736.

[83] Wei R, von Haugwitz G, Pfaff L, Mican J, Badenhorst CPS, Liu WD, Weber G, Austin HP, Bednar D, Damborsky J, et al. . Mechanism-based design of efficient PET hydrolases. ACS Catal, 2022, 12(6): 3382-3396

[84] Wei R, Westh P, Weber G, Blank LM. Bornscheuer UT (2025) Standardization guidelines and future trends for PET hydrolase research. Nat Commun, 2025, 16(1): 4684

[85] Werner AZ, Clare R, Mand TD, Pardo I, Ramirez KJ, Haugen SJ, Bratti F, Dexter GN, Elmore JR, Huenemann JD, et al. . Tandem chemical deconstruction and biological upcycling of poly(ethylene terephthalate) to β-ketoadipic acid by Pseudomonas putida KT2440. Metab Eng, 2021, 67: 250-261

[86] Ye JW, Lin YN, Yi XQ, Yu ZX, Liu X, Chen GQ. Synthetic biology of extremophiles: a new wave of biomanufacturing. Trends Biotechnol, 2023, 41(3): 342-357

[87] Yoshida S, Hiraga K, Takehana T, Taniguchi I, Yamaji H, Maeda Y, Toyohara K, Miyamoto K, Kimura Y, Oda K. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science, 2016, 351(6278): 1196-1199

[88] Yu H, Huang H. Engineering proteins for thermostability through rigidifying flexible sites. Biotechnol Adv, 2014, 32(2): 308-315

[89] Zhang WZ, Tang JC, Wang SS, Wang ZJ, Qin WM, He JH. The protein complex crystallography beamline (BL19U1) at the Shanghai Synchrotron Radiation Facility. Nucl Sci Tech. 2019;30(11):170.

[90] Zheng Y, Li Q, Liu P, Yuan Y, Dian L, Wang Q, Liang Q, Su T, Qi Q. Dynamic docking-assisted engineering of hydrolases for efficient PET depolymerization. ACS Catal, 2024, 14(5): 3627-3639

[91] Zhong-Johnson EZL, Voigt CA, Sinskey AJ. An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films. Sci Rep, 2021, 11(1): 928

[92] Zhong-Johnson EZL, Dong Z, Canova CT, Destro F, Canellas M, Hoffman MC, Marechal J, Johnson TM, Zheng M, Schlau-Cohen GS, et al. . Analysis of poly(ethylene terephthalate) degradation kinetics of evolved IsPETase variants using a surface crowding model. J Biol Chem, 2024, 300(3): 105783

[93] Zhou J, Cui Z, Wei R, Dong W, Jiang M. Interfacial catalysis in enzymatic PET plastic depolymerization. Trends Chem. 2025a;7(4):175–85.

[94] Zhou Y, Zhang J, You S, Zhang C, Qi W. Bio-upcycling PET waste: advances in enzymatic hydrolysis and biosynthesis of value-added products. Biotechnol Adv. 2025b;84:108685.

Funding

National Key Research and Development Program of China(2024YFC3407000)

Guangdong S&T Program(2024B1111130003)

Key Research Program Project of Guangzhou Science and Technology Bureau(2024B03J1276)

National Natural Science Foundation of China(32000031)

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