用于新興污染物降解的酶納米雜化物.pdf

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1、Enzyme Nanohybrids for Emerging Contaminant DegradationYifan Gao,Meng Wang,Kshitija Shah,Shaily MahendraUCLA Civil and Environmental EngineeringAcknowledgmentsUCLA Faculty:Leonard Rome,Bruce Dunn,Eric Hoek,Patrick Allard,Chong Liu,JunyoungPark,Valerie KickhoeferUCLA Students and postdocs:Benjamin Cr

2、oze,Yichang Chen,Esther Lan,David Zgonc,Minhao Xiao,Haiyuan Zou,Glenn Nurwono,Yu Chen,Randy Chen,Jingyu Wang,Yongchao XieUSEPA:Quinn Birch,Mallikarjuna NadagoudaUniversity of Georgia:Qingguo HuangMicrobial Enzymes Biotransform Contaminants3Created by BioRNarayanan et al.,JEM,2023Enzyme Immobilizatio

3、n4Surface BindingCrosslinkingEntrapment+-GlassPolymersActivated CarbonNanoparticlesImmobilizationEnzymes immobilized into/ontonanomaterials for stability and longevityYifan Gao et al.,Biotech.Adv.,2022Nanoscale:Size Matters Nanoscale=1-100 nm in at least 1 dimension Increased surface area=increase r

4、eactivity Wave/Particle duality=unique nanoscale propertiesKlaine et al.2011The ratio of a nanomaterial to a tennis ball is about the same as the ratio of tennis ball to the moon!Human hair grows6 inches/year=5 nm/sec!Vault NanoparticlesFrascotti et al.,Cancers,2021;Kickhoefer et al.,PNAS,20056vPARP

5、INT serves as a“Zip code”to help deliver proteinsinto vaultsC-terminalN-terminalMajor vaultprotein(MVP)INTProtein of interestEngineered emptyvaults7Cooper sitesLaccaseCiprofloxacinProteins of Interest:Fungal Laccase and MnPLigninMnPMot and Silaghi-Dumitrescu,2012;Gecco BiotechLets make recombinant p

6、roteinsYifan Gao et al.,Bioresour.Technol.,20228Transfection of Insect CellsLaccaseLigationLaccase-INT-pFastBacPlasmidBacmidE.coliTransformationIsolationRecombinant Laccase-INT-BacmidLaccase-INTBaculovirusLaccaseINTRecombinantLaccase-INTInfected Sf9 CellsProduce recombinant laccase-INT in Sf9 cellsS

7、equence derived fromTrametes versicolor1Encapsulate laccases into vaultsYifan Gao et al.,Bioresour.Technol.,20229Produce empty vaults inyeast cells2Lyse20000gS20100000gDiscardResuspendAmmonium Acetate20000gResuspendPichia pastoris SMD1168Laccase-INT100000gEncapsulationby mixingrecombinantlaccase wit

8、hempty vaults3Laccase activity improved after packaging in vaultsYifan Gao et al.,Bioresour.Technol.,202210nLaccase:natural laccasevLaccase:vault-packaged laccasevLaccase had higher activityat neutral pH rangevLaccase had enhancedthermal stability11 Weak estrogenic activity reproductive toxicity Cau

9、ses chromosomal abnormalities and DNA damage Induces germline apoptosis Reduces brood size Increases embryonic lethality Used in production of plastics,epoxy resins,copy paper,PVC,dental sealants,flame retardants,food packaging containersBisphenol A and its SubstitutesCHOO HC H3C H3Vault-packaged en

10、zymes showed better thermal stability,wider pH adaptation,and biodegraded bisphenolic compounds at higher rates than unpackaged enzymes or live microbes.Wang et al.,ACS Sus Chem Eng,2019Improved Biodegradation RatesVault MnP Lowers BPA Product ToxicityWang et al.,ACS Sus Chem Eng,2019 MnP maintained

11、 100%activity after 12 recycles,90%activity after 25 recycles No significant decrease in BPA removal in four repeated uses of the same MnP-INT/vault/silicaReusability of MnP-INT/vault/silicaWang et al.,Chem Eng J,2021Degradation of Nitro-Amino-Aromatics16(Lothe et al.,Chemosphere,2020)Vault-packaged

12、 enzymes were required in lower amounts than free enzymes to achieve the same degradation rates of dinitrotoluenes(DNT),aminonitrotoluenes(ANT),and diaminotoluene(DAT).Synthetic Dyes as Contaminants of Concern 17Ecological impactHurt animals!Toxic to humansDecolorization of Synthetic DyesGao et al.,

13、Bioresour.Technol.,202218Reactive blue 19Detoxification of DyesGao et al.,Bioresour.Technol.,202219Insect cellsvlaccasenlaccasecontrolE.coliS.epidermidisC.vulgarisHigh chlorophyll=Low toxicityModel organismsChlorophyllUnexposedRemoval of PFOA by vLaccase20290 ppb260 ppbNon-detectableF-ionsRemoval of

14、 6:2 FtS by vLaccase21F-tentative39 ppb312 ppb600 ppbAtrazine Why do we care?Among the most widely used herbicides in agricultureto control annual weeds and grasses Endocrine disruption,carcinogenesis,andbioaccumulation in aquatic animals and plants.In soil,half-life is more than 100 days;in surface

15、 water,half-life is approx.85 days.22Texas Corn ProducersPereira de Albuquerque et al.,STOTEN,2020;Perez et al.,Chemosphere,2022Atrazine Degradation TechnologiesElectrochemical advancedoxidative processAdsorptionKomtchou et al.,Water Research,2017Cao et al.,J.Environ.Chem.Eng.,2021Atrazine Biodegrad

16、ation PhytoextractionBacteriaJiang et al.,Chemosphere,2020Qu et al.,Chemosphere,2018Laccase-mediated Atrazine Degradation Free plant laccase only achieved 30%of atrazine degradation after 3 days.Fungi,Bjerkandera adusta,showed atrazine removal(not degradation).We used laccase from Pycnoporus sp.SYBC

17、-L3,gifted from Huang Lab at University ofGeorgia.25Dhiman et al.,Chemosphere,2020;Huang et al.,JAFC,2016Boron Nitride as Enzyme Supporting Material Bulk hexagonal boron nitride(hBN):Insoluble in water Lower access to enzymes and contaminants Boron nitride nanosheet(BNNS):Soluble High surface area:2

18、600m2g1;bulk hBN:10m2g1(Rasul et al.,2021)High biocompatibility(Li et al.,2014)Inert(Li et al.,Chem.Commun.,2014)Boron nitride nanosheets need to be functionalizedfor enzyme immobilizationLin and Connell,Nanoscale,2012;Dibaji et al.,Materials Today,2020BNNS for Laccase ImmobilizationGao et al.,ES&T

19、202427EDC/NHSBoron nitride nanosheets“White Graphene”ArginineUltrasonication“Peptide bond”InertBiocompatibleWidely used in proteindrug deliveryBut need proper functionalization(Lei et al.,Nat.Commun,2015)Stability and Reusability of BNNS-ArgLacBNNS-ArgLac retained higheractivity at near neutral pH e

20、xhibited higher activity around 30 C can be used for up to6 consecutive cycles28Higher Atrazine Degradation by BNNS-ArgLac29Atrazine degradation rateProduct yieldAtrazine Degradation in Agricultural Wastewater30Different mediatorsSynthetic agricultural wastewaterBiodegradation Products31Different en

21、d product profileMolecular dockingDegradation leading to Detoxification32Less toxic small moleculesSummary and Significance Innovative enzyme immobilization platforms Vault nanoparticles Functionalized BNNS Both demonstrated enhanced stability,catalytic activity,and reusability.Can be multiplexed for contaminant mixtures or intermediates in biodegradation pathways Generally energy-and cost-efficient for production and scaling up Combined with abiotic technologies for industrial processes and water treatment33