β-sheet Engineering of IsPETase for PET Depolymerization

Songfeng Gao, Lixia Shi, Hongli Wei, Pi Liu, Wei Zhao, Lanyu Gong, Zijian Tan, Huanhuan Zhai, Weidong Liu, Haifeng Liu, Leilei Zhu

Engineering ›› 2025, Vol. 47 ›› Issue (4) : 180-193.

PDF(4004 KB)
PDF(4004 KB)
Engineering ›› 2025, Vol. 47 ›› Issue (4) : 180-193. DOI: 10.1016/j.eng.2024.10.015
Research
Article

β-sheet Engineering of IsPETase for PET Depolymerization

Author information +
History +

Abstract

The enzymatic depolymerization of polyethylene terephthalate (PET) offers a sustainable approach for the recycling of PET waste. Great efforts have been devoted to engineering PET depolymerases on the substrate binding cleft and the surrounding loops/α-helices on the surface. Here, we report the systematic engineering of whole β-sheet regions in the core of IsPETase (a PETase from Ideonella sakaiensis) via a fluorescent high-throughput screening assay. Twenty-one beneficial substitutions were obtained and iteratively recombined. The best variant, DepoPETase β, with an increase in the melting temperatures (Tm) of 22.9 °C, exhibited superior depolymerization performance and enabled complete depolymerization of 100.5 g of untreated post-consumer PET (pc-PET; 0.26% Wenzyme/WPET enzyme loading) in liter-scale bioreactor at 50 °C within 4 d. Crystallization and molecular dynamics simulations revealed that the improved activity and thermostability of DepoPETase β were due to enhanced hydrogen bonds and salt bridges in the β-sheet region, a more tightly packed structure of the core sheets and the surrounding helix, and improved binding of PET to the active sites. This study not only demonstrates the importance of engineering strategy in the β-sheet region of PET hydrolases but also provides a potential PET depolymerase for large-scale PET recycling.

Graphical abstract

Keywords

Directed evolution / High-throughput screening / PETase / PET depolymerization / Thermostability / β-sheet engineering

Cite this article

Download citation ▾
Songfeng Gao, Lixia Shi, Hongli Wei, Pi Liu, Wei Zhao, Lanyu Gong, Zijian Tan, Huanhuan Zhai, Weidong Liu, Haifeng Liu, Leilei Zhu. β-sheet Engineering of IsPETase for PET Depolymerization. Engineering, 2025, 47(4): 180‒193 https://doi.org/10.1016/j.eng.2024.10.015

References

[1]
Welle F.Twenty years of PET bottle to bottle recycling—an overview.Resour Conserv Recycling 2011; 55(11):865-875.
[2]
Nicholson SR, Rorrer NA, Carpenter AC, Beckham GT.Manufacturing energy and greenhouse gas emissions associated with plastics consumption.Joule 2021; 5(3):673-686.
[3]
Bornscheuer UT.Feeding on plastic.Science 2016; 351(6278):1154-1155.
[4]
Webb HK, Arnott J, Crawford RJ, Ivanova EP.Plastic degradation and its environmental implications with special reference to poly(ethylene terephthalate).Polymers 2013; 5(1):1-18.
[5]
Li Q, Hai R, Lin A.Human health risk assessment on contaminants in recycled plastic bags packaged foods.Environ Sci Technol 2010; 33(11):181-185.
[6]
Tournier V, Duquesne S, Guillamot F, Cramail H, Taton D, Marty A, et al.Enzymes’ power for plastics degradation.Chem Rev 2023; 123(9):5612-5701.
[7]
Chae Y, Kim D, Kim SW, An YJ.Trophic transfer and individual impact of nano-sized polystyrene in a four-species freshwater food chain.Sci Rep 2018; 8(1):274.
[8]
Hahladakis JN, Velis CA, Weber R, Iacovidou E, Purnell P.An overview of chemical additives present in plastics: migration, release, fate and environmental impact during their use, disposal and recycling.J Hazard Mater 2018; 344:179-199.
[9]
Pabortsava K, Lampitt RS.High concentrations of plastic hidden beneath the surface of the Atlantic ocean.Nat Commun 2020; 11(1):4073.
[10]
Jambeck JR, Geyer R, Wilcox C, Siegler TR, Perryman M, Andrady A, et al.Plastic waste inputs from land into the ocean.Science 2015; 347(6223):768-771.
[11]
Ellis LD, Rorrer NA, Sullivan KP, Otto M, McGeehan JE, Roman-Leshkov Y, et al.Chemical and biological catalysis for plastics recycling and upcycling.Nat Catal 2021; 4(7):539-556.
[12]
Wei R, Tiso T, Bertling J, O K’Connor, Blank LM, Bornscheuer UT.Possibilities and limitations of biotechnological plastic degradation and recycling.Nat Catal 2020; 3(11):867-871.
[13]
Coviello CG, Lassandro P, Sabb MFà, Foti D.Mechanical and thermal effects of using fine recycled PET aggregates in common screeds.Sustainability 2023; 15(24):16692.
[14]
Lerna M, Foti D, Petrella A, Sabb MFà, Mansour S.Effect of the chemical and mechanical recycling of PET on the thermal and mechanical response of mortars and premixed screeds.Materials 2023; 16(8):3155.
[15]
Brzozowski AM, Savage H, Verma CS, Turkenburg JP, Lawson DM, Svendsen A, et al.Structural origins of the interfacial activation in Thermomyces (humicola) lanuginosa lipase.Biochemistry 2000; 39(49):15071-15082.
[16]
Parisi D, Riley C, Srivastava AS, McCue HV, Johnson JR, Carnell AJ.PET hydrolysing enzymes catalyse bioplastics precursor synthesis under aqueous conditions.Green Chem 2019; 21(14):3827-3833.
[17]
Ronkvist AM, Xie WC, Lu WH, Gross RA.Cutinase-catalyzed hydrolysis of poly(ethylene terephthalate).Macromolecules 2009; 42(14):5128-5138.
[18]
Muller RJ, Schrader H, Profe J, Dresler K, Deckwer WD.Enzymatic degradation of poly(ethylene terephthalate): rapid hydrolyse using a hydrolase from T. fusca.Macromol Rapid Commun 2005; 26(17):1400-1405.
[19]
Then J, Wei R, Oeser T, Barth M, Belisario-Ferrari MR, Schmidt J, et al.Ca2+ and Mg2+ binding site engineering increases the degradation of polyethylene terephthalate films by polyester hydrolases from Thermobifida fusca.Biotechnol J 2015; 10(4):592-598.
[20]
Kawabata T, Oda M, Kawai F.Mutational analysis of cutinase-like enzyme, Cut190, based on the 3D docking structure with model compounds of polyethylene terephthalate.J Biosci Bioeng 2017; 124(1):28-35.
[21]
Oda M, Yamagami Y, Inaba S, Oida T, Yamamoto M, Kitajima S, et al.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.
[22]
Sulaiman S, Yamato S, Kanaya E, Kim JJ, Koga Y, Takano K, et al.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.
[23]
Tournier V, Topham CM, Gilles A, David B, Folgoas C, Moya-Leclair E, et al.An engineered PET depolymerase to break down and recycle plastic bottles.Nature 2020; 580(7802):216-219.
[24]
Ding ZD, Xu GS, Miao RJ, Wu NF, Zhang W, Yao B, et al.Rational redesign of thermophilic PET hydrolase LCCICCG to enhance hydrolysis of high crystallinity polyethylene terephthalates.J Hazard Mater 2023; 453:131386.
[25]
Li QB, Zheng Y, Su TY, Wang Q, Liang QF, Zhang ZD, et al.Computational design of a cutinase for plastic biodegradation by mining molecular dynamics simulations trajectories.Comput Struct Biotechnol J 2022; 20:459-470.
[26]
Yoshida S, Hiraga K, Takehana T, Taniguchi I, Yamaji H, Maeda Y, et al.A bacterium that degrades and assimilates poly(ethylene terephthalate).Science 2016; 351(6278):1196-1199.
[27]
Chen ZZ, Wang YY, Cheng YY, Wang X, Tong SW, Yang HT, et al.Efficient biodegradation of highly crystallized polyethylene terephthalate through cell surface display of bacterial PETase.Sci Total Environ 2020; 709:136138.
[28]
Chen K, Hu Y, Dong XY, Sun Y.Molecular insights into the enhanced performance of ekylated PETase toward PET degradation.ACS Catal 2021; 11(12):7358-7370.
[29]
Liu K, Xu ZP, Zhao ZY, Chen YX, Chai YT, Ma L, et al.A dual fluorescence assay enables high-throughput screening for poly(ethylene terephthalate) hydrolases.ChemSusChem 2023; 16(5):e202202019.
[30]
Feng SS, Yue Y, Zheng MN, Li YW, Zhang QZ, Wang WX.IsPETase and IsMHETase-catalyzed cascade degradation mechanism toward polyethylene terephthalate.ACS Sustain Chem Eng 2021; 9(29):9823-9832.
[31]
Zheng MN, Li YW, Dong WL, Zhang WX, Feng SS, Zhang QZ, et al.Depolymerase-catalyzed polyethylene terephthalate hydrolysis: a unified mechanism revealed by quantum mechanics/molecular mechanics analysis.ACS Sustain Chem Eng 2022; 10(22):7341-7348.
[32]
Ma Y, Yao MD, Li BZ, Ding MZ, He B, Chen S, et al.Enhanced poly(ethylene terephthalate) hydrolase activity by protein engineering.Engineering 2018; 4(6):888-893.
[33]
Pfaff L, Gao J, Li ZS, Jaeckering A, Weber G, Mican J, et al.Multiple substrate binding mode-guided engineering of a thermophilic pet hydrolase.ACS Catal 2023; 12(15):9790-9800.
[34]
Erickson E, Gado JE, Avilán L, Bratti F, Brizendine RK, Cox PA, et al.Sourcing thermotolerant poly(ethylene terephthalate) hydrolase scaffolds from natural diversity.Nat Commun 2022; 13(1):7850.
[35]
Sagong HY, Son HF, Seo H, Hong H, Lee D, Kim KJ.Implications for the PET decomposition mechanism through similarity and dissimilarity between PETases from Rhizobacter gummiphilus and Ideonella sakaiensis.J Hazard Mater 2021; 416:126075.
[36]
Nakamura A, Kobayashi N, Koga N, Iino R.Positive charge introduction on the surface of thermostabilized PET hydrolase facilitates PET binding and degradation.ACS Catal 2021; 11(14):8550-8564.
[37]
Eiamthong B, Meesawat P, Wongsatit T, Jitdee J, Sangsri R, Patchsung M, et al.Discovery and genetic code expansion of a polyethylene terephthalate (PET) hydrolase from the human saliva metagenome for the degradation and bio-functionalization of PET.Angew Chem Int Ed 2022; 61(37):e202203061.
[38]
White MFM, Wallace S.A new PETase from the human saliva metagenome and its functional modification via genetic code expansion in bacteria.Angew Chem Int Ed 2023; 135(12):e202216963.
[39]
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.
[40]
Li A, Sheng Y, Cui H, Wang M, Wu L, Song Y, et al.Discovery and mechanism-guided engineering of BHET hydrolases for improved PET recycling and upcycling.Nat Commun 2023; 14(1):4169.
[41]
Zhang S, Hu Q, Zhang YX, Guo H, Wu Y, Sun M, et al.Depolymerization of polyesters by a binuclear catalyst for plastic recycling.Nat Sustain 2023; 6(8):965-973.
[42]
Velasco-Lozano S, MÀGalm és, Olazabal I, Sardon H, López-Gallego F, et al.Mechanistic studies of a lipase unveil effect of pH on hydrolysis products of small PET modules.Nat Commun 2023; 14(1):3556.
[43]
Hayes HC, Luk LYP.Investigating the effects of cyclic topology on the performance of a plastic degrading enzyme for polyethylene terephthalate degradation.Sci Rep 2023; 13(1):1267.
[44]
Kalathil S, Miller M, Reisner E.Microbial fermentation of polyethylene terephthalate (PET) plastic waste for the production of chemicals or electricity.Angew Chem Int Ed 2022; 61(45):e202211057.
[45]
Ye M, Li Y, Yang Z, Yao C, Sun W, Zhang X, et al.Ruthenium/TiO2-catalyzed hydrogenolysis of polyethylene terephthalate: reaction pathways dominated by coordination environment.Angew Chem Int Ed 2023; 62(19):e202301024.
[46]
Liu Y, Zhang C, Feng J, Wang X, Ding Z, He L, et al.Integrated photochromic–photothermal processes for catalytic plastic upcycling.Angew Chem Int Ed 2023; 62(38):e202308930.
[47]
Gopal MR, Dickey RM, Butler ND, Talley MR, Nakamura DT, Mohapatra A, et al.Reductive enzyme cascades for valorization of polyethylene terephthalate deconstruction products.ACS Catal 2023; 13(7):4778-4789.
[48]
Gong X, Ma F, Zhang Y, Li Y, Wang Z, Liu Y, et al.In situ construction of an intramolecular donor–acceptor conjugated copolymer via terephthalic acid derived from plastic waste for photocatalysis of plastic to hydrogen peroxide.ACS Catal 2023; 13(18):12338-12349.
[49]
Arnal G, Anglade J, Gavalda S, Tournier V, Chabot N, Bornscheuer UT, et al.Assessment of four engineered pet degrading enzymes considering large-scale industrial applications.ACS Catal 2023; 13(20):13156-13166.
[50]
von G Haugwitz, Han X, Pfaff L, Li Q, Wei HL, Gao J, et al.Structural insights into (tere)phthalate-ester hydrolysis by a carboxylesterase and its role in promoting PET depolymerization.ACS Catal 2022; 12(24):15259-15270.
[51]
Palm GJ, Reisky L, Böttcher D, Müller H, Michels EAP, Walczak MC, et al.Structure of the plastic-degrading Ideonella sakaiensis MHETase bound to a substrate.Nat Commun 2019; 10(1):1717.
[52]
Tarazona NA, Wei R, Brott S, Pfaff L, Bornscheuer UT, Lendlein A, et al.Rapid depolymerization of poly(ethylene terephthalate) thin films by a dual-enzyme system and its impact on material properties.Chem Catal 2022; 2(12):3573-3589.
[53]
Shi LX, Liu HF, Gao SF, Weng YX, Zhu LL.Enhanced extracellular production of IsPETase in Escherichia coli via engineering of the pelB signal peptide.J Agric Food Chem 2021; 69(7):2245-2252.
[54]
Falkenstein P, Zhao Z, Di A Pede-Mattatelli, Künze G, Sommer M, Sonnendecker C, et al.On the binding mode and molecular mechanism of enzymatic polyethylene terephthalate degradation.ACS Catal 2023; 13(10):6919-6933.
[55]
Zheng MN, Li YW, Xue R, Dong WL, Zhang QZ, Wang WX.Hydrolases catalyzed nanosized polyethylene terephthalate depolymerization: new insights from QM/MM analysis.J Clean Prod 2022; 377:134429.
[56]
Chen CC, Han X, Ko TP, Liu W, Guo RT.Structural studies reveal the molecular mechanism of PETase.FEBS J 2018; 285(20):3717-3723.
[57]
Han X, Liu W, Huang JW, Ma J, Zheng Y, Ko TP, et al.Structural insight into catalytic mechanism of PET hydrolase.Nat Commun 2017; 8(1):2106.
[58]
Wang N, Guan F, Lv X, Han D, Zhang Y, Wu N, et al.Enhancing secretion of polyethylene terephthalate hydrolase PETase in Bacillus subtilis WB600 mediated by the SPamysignal peptide.Lett Appl Microbiol 2020; 71(3):235-241.
[59]
Liu YD, Liu ZZ, Guo ZY, Yan TT, Jin CX, Wu J.Enhancement of the degradation capacity of IsPETase for PET plastic degradation by protein engineering.Sci Total Environ 2022; 834:154947.
[60]
Son HF, Cho IJ, Joo S, Seo H, Sagong HY, Choi SY, et al.Rational protein engineering of thermo-stable PETase from Ideonella sakaiensis for highly efficient PET degradation.ACS Catal 2019; 9(4):3519-3526.
[61]
Cui YL, Chen YC, Liu XY, Dong SJ, Tian YE, Qiao YX, et al.Computational redesign of a PETase for plastic biodegradation under ambient condition by the grape strategy.ACS Catal 2021; 11(3):1340-1350.
[62]
Lu HY, Diaz DJ, Czarnecki NJ, Zhu CZ, Kim WT, Shroff R, et al.Machine learning-aided engineering of hydrolases for PET depolymerization.Nature 2022; 604(7907):662.
[63]
Shi LX, Liu P, Tan ZJ, Zhao W, Gao JF, Gu Q, et al.Complete depolymerization of PET wastes by an evolved PET hydrolase from directed evolution.Angew Chem Int Ed 2023; 62(14):e202218390.
[64]
Erickson E, Shakespeare TJ, Bratti F, Buss BL, Graham R, Hawkins MA, et al.Comparative performance of PETase as a function of reaction conditions, substrate properties, and product accumulation.ChemSusChem 2022; 15(1):e202101932.
[65]
Meng X, Yang L, Liu H, Li Q, Xu G, Zhang Y, et al.Protein engineering of stable IsPETase for PET plastic degradation by premuse.Int J Biol Macromol 2021; 180:667-676.
[66]
Chen ZZ, Duan RD, Xiao YJ, Wei Y, Zhang HX, Sun XZ, et al.Biodegradation of highly crystallized poly(ethylene terephthalate) through cell surface codisplay of bacterial PETase and hydrophobin.Nat Commun 2022; 13(1):7138.
[67]
Kosiorowska KE, Moreno AD, Iglesias R, Leluk K, Mironczuk AM.Production of PETase by engineered Yarrowia lipolytica for efficient poly(ethylene terephthalate) biodegradation.Sci Total Environ 2022; 846:157358.
[68]
Guo BY, Vanga SR, Lopez-Lorenzo X, Saenz-Mendez P, Ericsson SR, Fang Y, et al.Conformational selection in biocatalytic plastic degradation by PETase.ACS Catal 2022; 12(6):3397-3409.
[69]
Yin QD, You SP, Zhang JX, Qi W, Su RX.Enhancement of the polyethylene terephthalate and mono-(2-hydroxyethyl) terephthalate degradation activity of Ideonella sakaiensis PETase by an electrostatic interaction-based strategy.Bioresour Technol 2022; 364:128026.
[70]
Achatz S, Jarasch A, Skerra A.Structural plasticity in the loop region of engineered lipocalins with novel ligand specificities, so-called anticalins.J Struct Biol X 2022; 6:100054.
[71]
Bell EL, Smithson R, Kilbride S, Foster J, Hardy FJ, Ramachandran S, et al.Directed evolution of an efficient and thermostable PET depolymerase.Nat Catal 2022; 5(8):673-681.
[72]
Brott S, Pfaff L, Schuricht J, Schwarz JN, Bottcher D, Badenhorst CPS, et al.Engineering and evaluation of thermostable IsPETase variants for PET degradation.Eng Life Sci 2022; 22(3–4):192-203.
[73]
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.
[74]
Austin HP, Allen MD, Donohoe BS, Rorrer NA, Kearns FL, Silveira RL, et al.Characterization and engineering of a plastic-degrading aromatic polyesterase.Proc Natl Acad Sci USA 2018; 115(19):E4350-E4357.
[75]
Fecker T, Galaz-Davison P, Engelberger F, Narui Y, Sotomayor M, Parra LP, et al.Active site flexibility as a hallmark for efficient PET degradation by I. sakaiensis PETase.Biophys J 2018; 114(6):1302-1312.
[76]
Wang CH, Lu LH, Huang C, He BF, Huang RB.Simultaneously improved thermostability and hydrolytic pattern of alpha-amylase by engineering central beta strands of TIM barrel.Appl Biochem Biotechnol 2020; 192(1):57-70.
[77]
Lou D, Tan J, Zhu L, Ji S, Wang B.The β-sheet core is the favored candidate of engineering SDR for enhancing thermostability but not for activity.Protein Pept Lett 2017; 24(6):511-516.
[78]
Lou D, Tan J, Zhu L, Ji S, Tang S, Yao K, et al.Engineering Clostridium absonum 7α-hydroxysteroid dehydrogenase for enhancing thermostability based on flexible site and ΔΔG prediction.Protein Pept Lett 2018; 25(3):230-235.
[79]
Crooks GE, Hon G, Chandonia JM, Brenner SE.Weblogo: a sequence logo generator.Genome Res 2004; 14(6):1188-1190.
[80]
Joo S, Cho IJ, Seo H, Son HF, Sagong HY, Shin TJ, et al.Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation.Nat Commun 2018; 9(1):382.
[81]
Ashkenazy H, Abadi S, Martz E, Chay O, Mayrose I, Pupko T, et al.Consurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules.Nucleic Acids Res 2016; 44(W1):W344-W350.
[82]
Landau M, Mayrose I, Rosenberg Y, Glaser F, Martz E, Pupko T, et al.Consurf 2005: the projection of evolutionary conservation scores of residues on protein structures.Nucleic Acids Res 2005; 33(Suppl 2):S299-S302.
[83]
Ashkenazy H, Erez E, Martz E, Pupko T, Ben-Tal N.Consurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids.Nucleic Acids Res 2010; 38(Suppl 2):W529-W533.
[84]
Delano WL.The pymol molecular graphics system.Proteins Struct Funct Bioinf 2002; 30:442-454.
[85]
Guerois R, Nielsen JE, Serrano L.Predicting changes in the stability of proteins and protein complexes: a study of more than 1000 mutations.J Mol Biol 2002; 320(2):369-387.
[86]
Otwinowski Z, Minor W.Processing of X-ray diffraction data collected in oscillation mode.Methods Enzymol 1997; 276:307-326.
[87]
McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ.Phaser crystallographic software.J Appl Cryst 2007; 40(Pt 4):658-674.
[88]
Adams PD, Afonine PV, Bunkóczi G, Chen VB, Davis IW, Echols N, et al.Phenix: a comprehensive python-based system for macromolecular structure solution.Acta Crystallogr D 2010; 66(Pt 2):213-221.
[89]
Terwilliger TC, Grosse-Kunstleve RW, Afonine PV, Moriarty NW, Zwart PH, Hung LW, et al.Iterative model building, structure refinement and density modification with the phenix autobuild wizard.Acta Crystallogr D 2008; 64(Pt 1):61-69.
[90]
Terwilliger TC, Grosse-Kunstleve RW, Afonine PV, Moriarty NW, Adams PD, Read RJ, et al.Iterative-build omit maps: map improvement by iterative model building and refinement without model bias.Acta Crystallogr D 2008; 64(Pt 5):515-524.
[91]
Brünger AT.Assessment of phase accuracy by cross validation: the free R value. Methods and applications.Acta Crystallogr D 1993; 49(Pt 1):24-36.
[92]
Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML.Comparison of simple potential functions for simulating liquid water.J Chem Phys 1998; 79(2):926-935.
[93]
Pearlman DA, Connelly PR.Determination of the differential effects of hydrogen bonding and water release on the binding of FK506 to native and Tyr82→Phe82 FKBP-12 proteins using free energy simulations.J Mol Biol 1995; 248(3):696-717.
[94]
Andersen HC.Rattle: a “velocity” version of the shake algorithm for molecular dynamics calculations.J Comput Phys 1983; 52(1):24-34.
[95]
Chow KH, Ferguson DM.Isothermal–isobaric molecular dynamics simulations with monte carlo volume sampling.Comput Phys Commun 1995; 91(1–3):283-289.
[96]
Grand SL, GoTz AW, Walker RC.SPFP: speed without compromise—a mixed precision model for GPU accelerated molecular dynamics simulations.Comput Phys Commun 2013; 184(2):374-380.
AI Summary AI Mindmap
PDF(4004 KB)

Accesses

Citations

Detail

Sections
Recommended

/