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Harnessing the potential of laccases for the synthesis of biopolymers

Harnessing the potential of laccases for the synthesis of biopolymers. Catherine Hautphenne , Julie Blavier , Frédéric Debaste. 25/08/2014, paper n°G3.2. What is an organic micropollutant ?.

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Harnessing the potential of laccases for the synthesis of biopolymers

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  1. Harnessing the potential of laccases for the synthesis of biopolymers Catherine Hautphenne, Julie Blavier, Frédéric Debaste 25/08/2014, paper n°G3.2

  2. Whatis an organicmicropollutant? Organicchemical compounds present in the environmentmostly due to humanactivities, and inducenegativeeffectson living organisms and theirdescendentsevenpresent at verylow concentrations(fromng/l to µg/l) 2

  3. Whatis an organicmicropollutant? Organicchemical compounds present in the environmentmostly due to humanactivities, and inducenegativeeffectson living organisms and theirdescendentsevenpresent at verylow concentrations(fromng/l to µg/l) Diverse structures and origins Interferencewithmetabolism Chronicexposureto acocktail of molecules Développement d’une nouvelle technologie 2

  4. Whatis an organicmicropollutant? Organicchemical compounds present in the environmentmostly due to humanactivities, and inducenegativeeffectson living organisms and theirdescendentsevenpresent at verylow concentrations(fromng/l to µg/l) Diverse structures and origins Concentrations ng/l to µg/l Insufficientremovalwithconventional technologies Développement d’une nouvelle technologie 2

  5. Whatis an organicmicropollutant? Organicchemical compounds present in the environmentmostly due to humanactivities, and inducenegativeeffectson living organisms and theirdescendentsevenpresent at verylow concentrations(fromng/l to µg/l) Diverse structures and origins Concentrations ng/l to µg/l Interferencewithmetabolism Chronicexposureto acocktail of molecules Insufficientremovalwithconventional technologies Development of a new technology 2

  6. Laccases degrade micropollutants • White Rot Fungi • Broad spectrum of substrates • Non toxiccosubstrates (no need of H2O2) • Relative stability Good candidates for an industrial application 3

  7. Good candidates for industrial applications but… • Need to be immobilized • Biocatalysts formation • Enhancestability over reaction variations (temperature, pH, etc.) • Recovery and recycling • Widerchoice of reactors • Cost effective in most cases Flow direction laccase support • End of pipe fixedbedreactor 4

  8. Degradation performance in fixed bed reactor • Good degradationresultsobserved However • Decrease of reactor performance concerning micropollutants degradation • Mixing of the bed • Recovery of a certain percentage of the biocatalysts activity • Probable presence of polymers deposits on biocatalysts surfaces But… 5

  9. Objective Thorough study of the phenomenon of insoluble productsformation comingfrom the reactionbetweensubstrates and laccases in order to reducebiocatalysts deactivationin a fixed bedreactor 6

  10. Literature review • High complexity to identify polymerization pathways and formed by-products • By-products structures variation depending on the considered enzyme-subtrate system • Laccases -phénol [1][2][3] • Laccases - triclosan [4] • Laccases - nonylphenol [5] • Laccases - 17-βestradiol [6] • Laccases - bisphenol A [7][8][9] • Etc. • ConnexionsC-C ou C-O between the different units of polymers • In majority, production of small oligomers with 2 to 6 units 7

  11.  Initial simplification of the system • Studying and modeling the kinetics formation of insoluble products in a simplified system: • Free enzymes - substrate system • Laccases (Trametes versicolor) - bisphenol A (BPA) system • In a BATCH reactor 8

  12. Strategy 9

  13. Polymerizationprotocoldevelopment • For each BATCH • 200ml phosphate buffer pH 6.0 • 1.1mM BPA • 740U laccases activity • 24°C • Constant agitation • Same number of BATCH reactors than the number of measurements to do during 6h of polymerization reaction • + 2 controls: • Without BPA • Without laccases • Samplig every 30 minutes • Insoluble by-products recovery by filtration • Filters with particles retention < 2.5µm • Filters drying in a dessiccator during 3 days • Analytical mass weighing 10

  14. Polymerization kinetics: first results Polymers production over time 11

  15. Polymerization kinetics: first results Conclusions • Observation of insoluble by-products formation in all tested conditions • The monitoring of polymers formation over time is conclusive • Experiments are reproducible However, • Potential over estimation of RMP • Laccase maybe retained on filters or adsorbed on formed polymers 12

  16. Experimental Design • Use of experimental design • Acquisition of a maximum of imformation in a minimum of experiments • Permit to establish a connexion between one or more experimental variables and the obtained results • Doehlert Design • 2 factors (all other conditions are keeping • constants) • BPA concentration [mM] • Enzyme concentration [U/200ml] • 1 studied response: Relative mass of BPA • polymers (RMP) • 7 experiments 13

  17. Linear regression and two-way ANOVA • Evaluation of potential interaction between one of the factors and the response Two-way ANOVA • H0: There is no contribution of one factor to the response • H1: At least one factor has a contribution to the response Conclusions • Enzyme concentration has an influence on the evolution of RMP, but not BPA concentration 14

  18. Polymerization kinetic model Reactional system M= insoluble product k1= first kinetic constant Introduction of two corrective terms: First order equation for BPA concentration: Mass balance in a batch reactor: Initial conditions: 15

  19. Polymerization kinetic model Model acquisition with Graphic representation of experimental results for two tests from the Doehlert design, and the corresponding model 16

  20. Productscharacterization • Scanning electronmicroscopy (SEM) • Qualitative observation of sphericalparticles • Fourier TransformInfraredSpectrometry (FTIR) • Presence of ester bonds • Polymers in the samples • Gel permeationChromatography (GPC) • Potentialpresence of smalloligomers • Differential Scanning Calorimetry (DSC) • Traces or lack of BPA as a monomer in the samples Conclusion Presence of smallpolymersin the samples, mostlyassociated by ester bonds betweendifferentunits. 17

  21. Conclusions • Polymerizationexperiments • 7 experimentswithdifferent combinaisons of enzyme-BPA concentrations • Doehlert Design • Linearregression and two-way ANOVA • Influence of enzymeconcentration on RMP • Monitoring of the evolutionof RMP over time • Reproducibility • Polymerizationkinetic model • First order for BPA concentration • Good fittingwithexperimental data • Characterization of producedby-products • Small oligomers • Association betweendifferentunitswith ester bonds • Good correspondance withliterature data 18

  22. Outlook • Literaturereview to identify few adequate enzymes supports • Adaptation of the polymerizationprotocol to the case of immobilized enzymes • Follow of the biocatalystsactivity • Oxygen probe? • Thoroughcharacterization of formedreaction by-product • NMR, MS and HPLC • Systematicsamples analyses 19

  23. Thankyou for your attention

  24. Bibliography [1] Dasgupta, S. and Taylor, K.E. and Bewtra, J.K.andBiswas, N. (2014).Inactivation of enzyme laccase and role of cosubstrate oxygen in enzymatic removal of phenol from water. Water EnvironmentResearch79:858-867. [2] Kurniawati, S. and Nicell, J.A. (2009). A comprehensivekinetic model of laccase-catalyzedoxidation of aqueousphenol. Biotechnology Progress25:763-773. [3] Mita, N. and Tawaki, S-I. and Uyama, H. and Kobayashi, S.(2003). Laccase-catalyzedoxidativepolymerization of phenols. MacromolecularBioscience3:253-257. [4] Murugesan, K. and Chang, Y-Y. and Kim, Y-M. and Jeon, J-R. and Kim, E-J. and Chang, Y-S. (2010). Enhancedtransformation of triclosan by laccase in the presence of redox mediators. Water Research44:298-308. [5]Cabana, H. and Habib Jiwan, J.L. and Rozenberg, R. and Elisahvili, V. and Penninckx, M. and AGathos, S.N. and Jones, J.P. (2007). Elimination of endocrine disruptingchemicalsnonylphenol and bisphenol A and personal care productingredienttriclosanusing enzyme preparationfrom the white rot fungusCoriolopsispolyzona. Chemosphere 67:770-778. [6] Alum, A. and Yoon, Y. and Westerhoff, P. and Abbaszadegan, M. (2004). Oxidationof bisphenol A, 17beta-estradiol and 17alpha-ethynyl estradiol and byproductestrogenicity. EnvironmentalToxicology19:257-264. [7]Fukuda, T. and Uchida, H. and Takashima, Y. and Uwajima, T. and Kawabata, T. and Suzuki, M. (2001). Degradation of bisphenol A by purified laccase Trametesvillosa. Biochemical and BiophysicalResearch Communications 284:704-706. [8] Huang, Q. and Weber, W.J. (2005). Transformation and removal of bisphenol A fromaqueous phase via peroxidase-mediatedoxidativecouplingreactions : Efficacy, products and pathways. EnvironmentalScience and Technology39:6029-6036. [9]Tsutsumi, Y. and Haneda, T. and Nishida, T. (2000). Removal of estrogenicactivities of bisphenol A and nonylphenol by oxidative enzymes fromlignin-degradingbasidiomycetes. Chemosphere42:271-276.

  25. Literaturereview 13

  26. Doehlert matrix for two variables

  27. Productscharacterization Scanning electronmicroscopy (SEM) SEM picture of insolubilized by-productsobtainedafter 4h reactionbetween 1.1mMBPA and 740U/200ml laccases

  28. Productscharacterization FTIR (Fourier TransformInfraredSpectrometry) Spectre FTIR spectrum of sampleobtainedafter 6h reactionbetween 1.1mM de BPA et 740U/200ml laccases in 200ml volume reactor

  29. Productscharacterization FTIR (Fourier TransformInfraredSpectrometry) Spectre FTIR spectrum of a commercial poly(BPA)carbonate samplepurchased by SIGMA.

  30. Productscharacterization FTIR (Fourier TransformInfraredSpectrometry) Comparaison of the peaksobserved on FTIR spectrumfromexperimentalsamples and commercial samples (SIGMA)

  31. Graphicrepresentation of experimentalresults for the test 1.5mM and 740U/200ml fromDoehlert, and the corresponding model

  32. Graphicrepresentation of experimentalresults for the test 1.3mM and 948U/200ml fromDoehlert, and the corresponding model

  33. Graphicrepresentation of experimentalresults for the test 0,9mM and 948U/200ml fromDoehlert, and the corresponding model

  34. Graphicrepresentation of experimentalresults for the test 0,7mM and 740U/200ml fromDoehlert, and the corresponding model

  35. Graphicrepresentation of experimentalresults for the test 0,9mM and 532U/200ml fromDoehlert, and the corresponding model

  36. Linearrepresentations of experimentalresults for three tests of the Doehlert and the correspondinglinearregressions

  37. Représentation graphique linéaire des résultats expérimentaux pour deux tests du Doehlert et les régressions linéaires correspondantes

  38. Linearregression of the enzyme. Dottedlines correspond to a confident interval of 95% Linearregression of BPA concentration. Dottedlines correspond to a confident interval of 90%

  39. Response surface analysis • polynomial model Conclusions • No maximum • Enzyme concentration seems to have an impact on R_MP

  40. Polymerizationkinetic model Model acquisition with Lineargraphicrepresentation of experimentalresults for two tests of the Doehlert and correspondinglinearregressions 20

  41. Polymerizationkinetic model Observations • As ratio REBincreases, k1globallyincreases • La vitesse de la réaction augmente • As ratio REBdecreases, E2 tends to decrease • Part of BPA/modified BPA not available for the reactionincreases 25

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