In response to the increasingly serious issue of plastic pollution, this study screened and heterologously expressed a hypothetical polyethylene terephthalate (PET) hydrolase (PETase) from the genome of the thermophilic microorganism Thermoleophilaceae bacterium, aiming to explore its enzymatic properties and application potential.The function of recombinant enzyme was predicted using the “CLEAN” algorithm.The target gene was cloned into an Escherichia coli expression system, and the recombinant enzyme, named TbPETase, was obtained through IPTG-induced expression and purified via nickel affinity chromatography.The presence and identity of the recombinant enzyme were confirmed by SDS-PAGE and matrix-assisted laser desorption-time of flight-mass spectrometry (MALDI-TOF-MS).Substrate specificity and kinetic properties of TbPETase were subsequently evaluated.Further, the fermentation parameters were optimized through orthogonal design, which significantly improved the expression efficiency of the target protein.The enzymatic properties of TbPETase were systematically investigated.The results showed that the melting temperature (Tm), enthalpy change (ΔH) and denaturation entropy (ΔH/Tm) of TbPETase were 64.87 ℃, 737 kJ/mol and 11.361 J/(K·mol), respectively.Enzyme kinetics analysis indicated that TbPETase exhibited a higher affinity for p-NPAc than for p-NPB.The optimal reaction temperature for p-NPAc hydrolysis was determined to be 55 ℃, which is significantly higher than that of the common mesophilic PETase.TbPETase maintained a half-life of approximately 7 hours at this temperature, indicating enhanced thermostability compared with most previously reported PET hydrolases.Under optimal conditions, TbPETase exhibited PET hydrolysis activity, achieving a hydrolysis rate of approximately 10%-13% after 48 hours.HPLC analysis showed that the hydrolysis products were terephthalic acid (TPA) and monohydroxyethyl terephthalate (MHET).This study provides the first characterization of a PETase derived from a thermophilic bacterium, presenting a promising enzymatic source for the development of high-temperature biodegradation technology and offering a theoretical basis for efficient PET waste recycling and the advancement of the circular economy.
WANG Yanan
,
YANG Ting
,
XIN Yu
,
ZHANG Liang
,
GU Zhenghua
. Recombinant expression and functional characterization of polyethylene terephthalate hydrolase from Thermoleophilaceae bacterium[J]. Food and Fermentation Industries, 2026
, 52(6)
: 43
-50
.
DOI: 10.13995/j.cnki.11-1802/ts.043284
[1] SUN X D, YUAN X Z, JIA Y B, et al.Differentially charged nanoplastics demonstrate distinct accumulation in Arabidopsis thaliana [J].Nature Nanotechnology, 2020, 15(9):755-760.
[2] JAMBECK J R, GEYER R, WILCOX C, et al.Plastic waste inputs from land into the ocean[J].Science, 2015, 347(6223):768-771.
[3] ERIKSEN M, LEBRETON L C M, CARSON H S, et al.Plastic pollution in the world’s oceans:More than 5 trillion plastic pieces weighing over 250, 000 tons afloat at sea[J].PLoS One, 2014, 9(12):e111913.
[4] QI Y L, YANG X M, PELAEZ A M, et al.Macro-and micro-plastics in soil-plant system:Effects of plastic mulch film residues on wheat (Triticum aestivum) growth[J].Science of the Total Environment, 2018, 645:1048-1056.
[5] BORRELLE S B, RINGMA J, LAW K L, et al.Predicted growth in plastic waste exceeds efforts to mitigate plastic pollution[J].Science, 2020, 369(6510):1515-1518.
[6] ELLIS L D, RORRER N A, SULLIVAN K P, et al.Chemical and biological catalysis for plastics recycling and upcycling[J].Nature Catalysis, 2021, 4(7):539-556.
[7] HAN X, LIU W D, HUANG J W, et al.Structural insight into catalytic mechanism of PET hydrolase[J].Nature Communications, 2017, 8(1):2106.
[8] ZHENG M N, LI Y W, DONG W L, et al.Depolymerase-catalyzed polyethylene terephthalate hydrolysis:A unified mechanism revealed by quantum mechanics/molecular mechanics analysis[J].ACS Sustainable Chemistry & Engineering, 2022, 10(22):7341-7348.
[9] MÜLLER R J, SCHRADER H, PROFE J, et al.Enzymatic degradation of poly(ethylene terephthalate):Rapid hydrolyse using a hydrolase from T.fusca[J].Macromolecular Rapid Communications, 2005, 26(17):1400-1405.
[10] YOSHIDA S, HIRAGA K, TAKEHANA T, et al.A bacterium that degrades and assimilates poly(ethylene terephthalate)[J].Science, 2016, 351(6278):1196-1199.
[11] CHEN C C, HAN X, LI X, et al.General features to enhance enzymatic activity of poly(ethylene terephthalate) hydrolysis[J].Nature Catalysis, 2021, 4(5):425-430.
[12] SULAIMAN S, YAMATO S, KANAYA E, et al.Isolation of a novel cutinase homolog with polyethylene terephthalate-degrading activity from leaf-branch compost by using a metagenomic approach[J].Applied and Environmental Microbiology, 2012, 78(5):1556-1562.
[13] HAN Z Y, NINA M R H, ZHANG X Y, et al.Discovery and characterization of two novel polyethylene terephthalate hydrolases:One from a bacterium identified in human feces and one from the Streptomyces genus[J].Journal of Hazardous Materials, 2024, 472:134532.
[14] THOMSEN T B, HUNT C J, MEYER A S.Influence of substrate crystallinity and glass transition temperature on enzymatic degradation of polyethylene terephthalate (PET)[J].New Biotechnology, 2022, 69:28-35.
[15] ERICKSON E, GADO J E, AVILÄN L, et al.Sourcing thermotolerant poly(ethylene terephthalate) hydrolase scaffolds from natural diversity[J].Nature Communications, 2022, 13(1):7850.
[16] SHEIK C S, NATWORA K E, ALEXSON E E, et al.Dolichospermum blooms in lake superior:DNA-based approach provides insight to the past, present and future of blooms[J].Journal of Great Lakes Research, 2022, 48(5):1191-1205.
[17] YU T H, CUI H Y, LI J C, et al.Enzyme function prediction using contrastive learning[J].Science, 2023, 379(6639):1358-1363.
[18] FENG S S, YUE Y, ZHENG M N, et al.IsPETase-and IsMHETase-catalyzed cascade degradation mechanism toward polyethylene terephthalate[J].ACS Sustainable Chemistry & Engineering, 2021, 9(29):9823-9832.
[19] JAEGER K E, KOVACIC F.Determination of Lipolytic Enzyme Activities[M].Pseudomonas Methods and Protocols.New York, NY;Springer New York, 2014:111-134.
[20] GROTE A, HILLER K, SCHEER M, et al.JCat:A novel tool to adapt Codon usage of a target gene to its potential expression host[J].Nucleic Acids Research, 2005, 33(WebServerissue):W526-W531.
