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Enhanced Catalytic Performance of Trichoderma reesei Cellulase Immobilized on Magnetic Hierarchical Porous Carbon Nanoparticles

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Abstract

Cellulase from Trichoderma reesei was immobilized by covalent or non-covalent binding onto magnetic hierarchical porous carbon (MHPC) nanomaterials. The immobilization yield and the enzyme activity were higher when covalent immobilization approach was followed. The covalent immobilization approach leads to higher immobilization yield (up to 96%) and enzyme activity (up to 1.35 U mg−1) compared to the non-covalent cellulase binding. The overall results showed that the thermal, storage and operational stability of the immobilized cellulase was considerably improved compared to the free enzyme. The immobilized cellulose catalyzed the hydrolysis of microcrystalline cellulose up to 6 consecutive successive reaction cycles, with a total operation time of 144 h at 50 °C. The half-life time of the immobilized enzyme in deep eutectic solvents-based media was up to threefold higher compared to the soluble enzyme. The increased pH and temperature tolerance of the immobilized cellulase, as well as the increased operational stability in aqueous and deep eutectic solvents-based media indicate that the use of MHPCs as immobilization nanosupport could expand the catalytic performance of cellulolytic enzymes in various reaction conditions.

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Abbreviations

BET:

Brunauer–Emmett–Teller

CMC:

Carboxymethyl cellulose sodium salt

ChCl:

Choline chloride

DES:

Deep eutectic solvents

DNSA:

3,5-Dinitrosalicylic acid

EDC:

1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride

EG:

Ethylene glycol

FT-IR:

Fourier transform infrared

Gly:

Glycerol

HPCs:

Hierarchical porous carbons

MHPC:

Magnetic hierarchical porous carbon

NHS:

N-Hydroxysuccinimide

SEM:

Scanning electron microscopy

U:

Urea

References

  1. Zhou CH, Xia X, Lin CX et al (2011) Catalytic conversion of lignocellulosic biomass to fine chemicals and fuels. Chem Soc Rev 40:5588–5617

    Article  CAS  PubMed  Google Scholar 

  2. Ikeda Y, Parashar A (2015) Reusability of immobilized cellulases with highly retained enzyme activity and their application for the hydrolysis of model substrates and lignocellulosic biomass. J Thermodyn Catal. https://doi.org/10.4172/2157-7544.1000149

    Article  Google Scholar 

  3. Ebner G, Vejdovszky P, Wahlström R et al (2014) The effect of 1-ethyl-3-methylimidazolium acetate on the enzymatic degradation of cellulose. J Mol Catal B Enzym 99:121–129. https://doi.org/10.1016/j.molcatb.2013.11.001

    Article  CAS  Google Scholar 

  4. Woolridge E (2014) Mixed enzyme systems for delignification of lignocellulosic biomass. Catalysts 4:1–35. https://doi.org/10.3390/catal4010001

    Article  CAS  Google Scholar 

  5. Alfenore S, Molina-jouve C (2016) Current status and future prospects of conversion of lignocellulosic resources to biofuels using yeasts and bacteria. Process Biochem 51:1747–1756. https://doi.org/10.1016/j.procbio.2016.07.028

    Article  CAS  Google Scholar 

  6. Guerriero G, Hausman J, Strauss J et al (2015) Destructuring plant biomass: focus on fungal and extremophilic cell wall hydrolases. Plant Sci 234:180–193. https://doi.org/10.1016/j.plantsci.2015.02.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Bornscheuer U, Buchholz K, Seibel J (2014) Enzymatic degradation of (ligno)cellulose. Angew Chemie Int Ed 53:10876–10893

    Article  CAS  Google Scholar 

  8. Vaz RP, de Souza Moreira LR, Ferreira Filho EX (2016) An overview of holocellulose-degrading enzyme immobilization for use in bioethanol production. J Mol Catal B Enzym 133:127–135

    Article  CAS  Google Scholar 

  9. Khoshnevisan K, Vakhshiteh F, Barkhi M et al (2017) Immobilization of cellulase enzyme onto magnetic nanoparticles: applications and recent advances. Mol Catal 442:66–73

    Article  CAS  Google Scholar 

  10. Gokhale AA, Lee I (2012) Cellulase immobilized nanostructured supports for efficient saccharification of cellulosic substrates. Topics Catal 55:1231–1246

    Article  CAS  Google Scholar 

  11. Rai M, Ingle AP, Pandit R et al (2019) Emerging role of nanobiocatalysts in hydrolysis of lignocellulosic biomass leading to sustainable bioethanol production. Catal Rev Sci Eng 61:1–26. https://doi.org/10.1080/01614940.2018.1479503

    Article  CAS  Google Scholar 

  12. Quoc Viet T, Phuoc Minh N, Thi Anh Dao D (2013) Immobilization of cellulase enzyme in calcium alginate gel and its immobilized stability. Am J Res Commun 1:254–267

    Google Scholar 

  13. Tu M, Chandra RP, Saddler JN (2007) Evaluating the distribution of cellulases and the recycling of free cellulases during the hydrolysis of lignocellulosic substrates. Biotechnol Prog 23:398–406. https://doi.org/10.1021/bp060354f

    Article  CAS  PubMed  Google Scholar 

  14. Asgher M, Shahid M, Kamal S et al (2014) Recent trends and valorization of immobilization strategies and ligninolytic enzymes by industrial biotechnology. J Mol Catal B Enzym 101:56–66. https://doi.org/10.1016/j.molcatb.2013.12.016

    Article  CAS  Google Scholar 

  15. Pavlidis IV, Patila M, Bornscheuer UT et al (2014) Graphene-based nanobiocatalytic systems: recent advances and future prospects. Trends Biotechnol 32:312–320. https://doi.org/10.1016/j.tibtech.2014.04.004

    Article  CAS  PubMed  Google Scholar 

  16. Ahmad R, Khare SK (2018) Immobilization of Aspergillus niger cellulase on multiwall carbon nanotubes for cellulose hydrolysis. Bioresour Technol 252:72–75. https://doi.org/10.1016/j.biortech.2017.12.082

    Article  CAS  PubMed  Google Scholar 

  17. Mubarak NM, Wong JR, Tan KW et al (2014) Immobilization of cellulase enzyme on functionalized multiwall carbon nanotubes. J Mol Catal B Enzym 107:124–131. https://doi.org/10.1016/j.molcatb.2014.06.002

    Article  CAS  Google Scholar 

  18. Khoshnevisan K, Bordbar AK, Zare D et al (2011) Immobilization of cellulase enzyme on superparamagnetic nanoparticles and determination of its activity and stability. Chem Eng J 171:669–673. https://doi.org/10.1016/j.cej.2011.04.039

    Article  CAS  Google Scholar 

  19. Poorakbar E, Shafiee A, Saboury AA et al (2018) Synthesis of magnetic gold mesoporous silica nanoparticles core shell for cellulase enzyme immobilization: improvement of enzymatic activity and thermal stability. Process Biochem 71:92–100. https://doi.org/10.1016/j.procbio.2018.05.012

    Article  CAS  Google Scholar 

  20. Khoshnevisan K, Poorakbar E, Baharifar H, Barkhi M (2019) Recent advances of cellulase immobilization onto magnetic nanoparticles: an update review. Magnetochemistry 5:36. https://doi.org/10.3390/magnetochemistry5020036

    Article  Google Scholar 

  21. Khoshnevisan K, Barkhi M, Ghasemzadeh A et al (2016) Fabrication of coated/uncoated magnetic nanoparticles to determine their surface properties. Mater Manuf Process 31:1206–1215. https://doi.org/10.1080/10426914.2015.1048362

    Article  CAS  Google Scholar 

  22. Wu L, Yuan X, Sheng J (2005) Immobilization of cellulase in nanofibrous PVA membranes by electrospinning. J Memb Sci 250:167–173. https://doi.org/10.1016/j.memsci.2004.10.024

    Article  CAS  Google Scholar 

  23. Ho KM, Mao X, Gu L, Li P (2008) Facile route to enzyme immobilization: core-shell nanoenzyme particles consisting of well-defined poly(methyl methacrylate) cores and cellulase shells. Langmuir 24:11036–11042. https://doi.org/10.1021/la8016529

    Article  CAS  PubMed  Google Scholar 

  24. Romo-Sánchez S, Camacho C, Ramirez HL, Arévalo-Villena M (2014) Immobilization of commercial cellulase and xylanase by different methods using two polymeric supports. Adv Biosci Biotechnol 05:517–526. https://doi.org/10.4236/abb.2014.56062

    Article  CAS  Google Scholar 

  25. Wang Y, Chen D, Wang G et al (2018) Immobilization of cellulase on styrene/maleic anhydride copolymer nanoparticles with improved stability against pH changes. Chem Eng J 336:152–159. https://doi.org/10.1016/j.cej.2017.11.030

    Article  CAS  Google Scholar 

  26. Tsai CT, Meyer AS (2014) Enzymatic cellulose hydrolysis: enzyme reusability and visualization of β-glucosidase immobilized in calcium alginate. Molecules 19:19390–19406. https://doi.org/10.3390/molecules191219390

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Tébéka IRM, Silva AGL, Petti DFS (2009) Hydrolytic activity of free and immobilized cellulase. Langmuir 25:1582–1587. https://doi.org/10.1021/la802882s

    Article  CAS  PubMed  Google Scholar 

  28. Cheng C, Chang KC (2013) Development of immobilized cellulase through functionalized gold nano-particles for glucose production by continuous hydrolysis of waste bamboo chopsticks. Enzyme Microb Technol 53:444–451. https://doi.org/10.1016/j.enzmictec.2013.09.010

    Article  CAS  PubMed  Google Scholar 

  29. Abraham RE, Verma ML, Barrow CJ, Puri M (2014) Suitability of magnetic nanoparticle immobilised cellulases in enhancing enzymatic saccharification of pretreated hemp biomass. Biotechnol Biofuels 7:1–12. https://doi.org/10.1186/1754-6834-7-90

    Article  CAS  Google Scholar 

  30. Das R, Mishra H, Srivastava A, Kayastha AM (2017) Covalent immobilization of Β-amylase onto functionalized molybdenum sulfide nanosheets, its kinetics and stability studies: a gateway to boost enzyme application. Chem Eng J 328:215–227. https://doi.org/10.1016/j.cej.2017.07.019

    Article  CAS  Google Scholar 

  31. Orfanakis G, Patila M, Catzikonstantinou AV et al (2018) Hybrid nanomaterials of magnetic iron nanoparticles and graphene oxide as matrices for the immobilization of β-glucosidase: synthesis, characterization, and biocatalytic properties. Front Mater 5:1–11. https://doi.org/10.3389/fmats.2018.00025

    Article  Google Scholar 

  32. Vinu A, Hossian KZ, Srinivasu P et al (2007) Carboxy-mesoporous carbon and its excellent adsorption capability for proteins. J Mater Chem 17:1819–1825. https://doi.org/10.1039/b613899c

    Article  CAS  Google Scholar 

  33. Estevez L, Dua R, Bhandari N et al (2013) A facile approach for the synthesis of monolithic hierarchical porous carbons-high performance materials for amine based CO2 capture and supercapacitor electrode. Energy Environ Sci 6:1785–1790. https://doi.org/10.1039/c3ee40549d

    Article  CAS  Google Scholar 

  34. Fujita S, Yamanoi S, Murata K et al (2014) A repeatedly refuelable mediated biofuel cell based on a hierarchical porous carbon electrode. Sci Rep 4:1–8. https://doi.org/10.1038/srep04937

    Article  CAS  Google Scholar 

  35. Baruah J, Nath BK, Sharma R et al (2018) Recent trends in the pretreatment of lignocellulosic biomass for value-added products. Front Energy Res 6:1–19. https://doi.org/10.3389/fenrg.2018.00141

    Article  Google Scholar 

  36. Kucharska K, Rybarczyk P, Hołowacz I et al (2018) Pretreatment of lignocellulosic materials as substrates for fermentation processes. Molecules 23:1–32. https://doi.org/10.3390/molecules23112937

    Article  CAS  Google Scholar 

  37. Kumar AK, Sharma S (2017) Recent updates on different methods of pretreatment of lignocellulosic feedstocks: a review. Bioresour Bioprocess. https://doi.org/10.1186/s40643-017-0137-9

    Article  PubMed  PubMed Central  Google Scholar 

  38. Xu GC, Ding JC, Han RZ et al (2016) Enhancing cellulose accessibility of corn stover by deep eutectic solvent pretreatment for butanol fermentation. Bioresour Technol 203:364–369. https://doi.org/10.1016/j.biortech.2015.11.002

    Article  CAS  PubMed  Google Scholar 

  39. Kumar AK, Parikh BS, Pravakar M (2016) Natural deep eutectic solvent mediated pretreatment of rice straw: bioanalytical characterization of lignin extract and enzymatic hydrolysis of pretreated biomass residue. Environ Sci Pollut Res 23:9265–9275. https://doi.org/10.1007/s11356-015-4780-4

    Article  CAS  Google Scholar 

  40. Zhang CW, Xia SQ, Ma PS (2016) Facile pretreatment of lignocellulosic biomass using deep eutectic solvents. Bioresour Technol 219:1–5. https://doi.org/10.1016/j.biortech.2016.07.026

    Article  CAS  PubMed  Google Scholar 

  41. Chen Y, Zhang L, Yu J et al (2019) High-purity lignin isolated from poplar wood meal through dissolving treatment with deep eutectic solvents. R Soc Open Sci 6:181757. https://doi.org/10.1098/rsos.181757

    Article  PubMed  PubMed Central  Google Scholar 

  42. Nagoor Gunny AA, Arbain D, Nashef EM, Jamal P (2015) Applicability evaluation of deep eutectic solvents-cellulase system for lignocellulose hydrolysis. Bioresour Technol 181:297–302. https://doi.org/10.1016/j.biortech.2015.01.057

    Article  CAS  Google Scholar 

  43. Gupta R, Sadaf A, Grewal J, Khare K (2017) Deep eutectic solvents compatible Aspergillus niger cellulase and its utility for in situ pre-treatment and saccharification of wheat straw. J Energy Environ Sustain 4:52–57

    Google Scholar 

  44. Papadopoulou AA, Tzani A, Polydera AC et al (2018) Green biotransformations catalysed by enzyme-inorganic hybrid nanoflowers in environmentally friendly ionic solvents. Environ Sci Pollut Res 25:26707–26714. https://doi.org/10.1007/s11356-017-9271-3

    Article  CAS  Google Scholar 

  45. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. https://doi.org/10.1016/0003-2697(76)90527-3

    Article  CAS  PubMed  Google Scholar 

  46. Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem 59:257–268. https://doi.org/10.1111/j.1468-2389.1995.tb00038.x

    Article  CAS  Google Scholar 

  47. Han J, Rong J, Wang Y et al (2018) Immobilization of cellulase on thermo-sensitive magnetic microspheres: improved stability and reproducibility. Bioprocess Biosyst Eng 41:1051–1060. https://doi.org/10.1007/s00449-018-1934-z

    Article  CAS  PubMed  Google Scholar 

  48. Patila M, Diamanti EK, Bergouni D et al (2018) Preparation and biochemical characterisation of nanoconjugates of functionalized carbon nanotubes and cytochrome. Nanomed Res J 3:10–18. https://doi.org/10.22034/nmrj.2018.01.002

    Article  CAS  Google Scholar 

  49. Dutta N, Biswas S, Saha MK (2016) Biophysical characterization and activity analysis of nano-magnesium supplemented cellulase obtained from a psychrobacterium following graphene oxide immobilization. Enzyme Microb Technol 95:248–258. https://doi.org/10.1016/j.enzmictec.2016.04.012

    Article  CAS  PubMed  Google Scholar 

  50. Kudina O, Zakharchenko A, Trotsenko O et al (2014) Highly efficient phase boundary biocatalysis with enzymogel nanoparticles. Angew Chemie Int Ed 53:483–487. https://doi.org/10.1002/anie.201306831

    Article  CAS  Google Scholar 

  51. Jordan J, Kumar CSSR, Theegala C (2011) Preparation and characterization of cellulase-bound magnetite nanoparticles. J Mol Catal B Enzym 68:139–146. https://doi.org/10.1016/j.molcatb.2010.09.010

    Article  CAS  Google Scholar 

  52. Gao J, Lu C-L, Wang Y et al (2018) Rapid immobilization of cellulase onto graphene oxide with a hydrophobic spacer. Catalysts 8:180. https://doi.org/10.3390/catal8050180

    Article  CAS  Google Scholar 

  53. Manasa P, Saroj P, Korrapati N (2017) Immobilization of Cellulase enzyme on zinc ferrite nanoparticles in increasing enzymatic hydrolysis on ultrasound-assisted alkaline pretreated Crotalaria juncea biomass. Indian J Sci Technol 10:1–7. https://doi.org/10.17485/ijst/2017/v10i24/112798

    Article  CAS  Google Scholar 

  54. Zhou J (2010) Immobilization of cellulase on a reversibly soluble-insoluble support: properties and application. J Agric Food Chem 58:6741–6746. https://doi.org/10.1021/jf100759c

    Article  CAS  PubMed  Google Scholar 

  55. Zang L, Qiu J, Wu X et al (2014) Preparation of magnetic chitosan nanoparticles as support for cellulase immobilization. Ind Eng Chem Res 53:3448–3454. https://doi.org/10.1021/ie404072s

    Article  CAS  Google Scholar 

  56. Hartono SB, Qiao SZ, Liu J et al (2010) Functionalized mesoporous silica with very large pores for cellulase immobilization. J Phys Chem C 114:8353–8362. https://doi.org/10.1021/jp102368s

    Article  CAS  Google Scholar 

  57. Hosseini SH, Hosseini SA, Zohreh N et al (2018) Covalent immobilization of cellulase using magnetic poly(ionic liquid) support: improvement of the enzyme activity and stability. J Agric Food Chem 66:789–798. https://doi.org/10.1021/acs.jafc.7b03922

    Article  CAS  PubMed  Google Scholar 

  58. Ungurean M, Paul C, Peter F (2013) Cellulase immobilized by sol–gel entrapment for efficient hydrolysis of cellulose. Bioprocess Biosyst Eng 36:1327–1338. https://doi.org/10.1007/s00449-012-0835-9

    Article  CAS  PubMed  Google Scholar 

  59. Wang S, Su P, Ding F, Yang Y (2013) Immobilization of cellulase on polyamidoamine dendrimer-grafted silica. J Mol Catal B Enzym 89:35–40. https://doi.org/10.1016/j.molcatb.2012.12.011

    Article  CAS  Google Scholar 

  60. Simon P, Lima JS, Valério A et al (2018) Cellulase immobilization on poly(methyl methacrylate) nanoparticles by miniemulsion polymerization. Brazilian J Chem Eng 35:649–658. https://doi.org/10.1590/0104-6632.20180352s20160094

    Article  CAS  Google Scholar 

  61. Nagoor Gunny AA, Arbain D, Javed M et al (2019) Deep eutectic solvents-halophilic cellulase system: an efficient route for in situ saccharification of lignocellulose. Process Biochem 81:99–103. https://doi.org/10.1016/j.procbio.2019.03.003

    Article  CAS  Google Scholar 

  62. Papadopoulou AA, Efstathiadou E, Patila M et al (2016) Deep eutectic solvents as media for peroxidation reactions catalyzed by heme-dependent biocatalysts. Ind Eng Chem Res 55:5145–5151. https://doi.org/10.1021/acs.iecr.5b04867

    Article  CAS  Google Scholar 

  63. Hammond OS, Bowron DT, Edler KJ (2017) The effect of water upon deep eutectic solvent nanostructure: an unusual transition from ionic mixture to aqueous solution. Angew Chem Int Ed 56:9782–9785. https://doi.org/10.1002/anie.201702486

    Article  CAS  Google Scholar 

  64. Guajardo N, Domínguez de María HP, Ahumada K et al (2017) Water as cosolvent: nonviscous deep eutectic solvents for efficient lipase-catalyzed esterifications. ChemCatChem 9:1393–1396. https://doi.org/10.1002/cctc.201601575

    Article  CAS  Google Scholar 

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Acknowledgments

We acknowledge support of this work by the project MIS 5005434 which has been co-financed by the Operational Program “Human Resources Development, Education and Lifelong Learning” and is co-financed by the European Union (European Social Fund) and Greek national funds. We are grateful to Dr. Michaela Patila and Dr. Konstantinos Spyrou for the synthesis and characterization of MHPC nanoparticles.

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Authors and Affiliations

  1. Laboratory of Biotechnology, Department of Biological Applications and Technologies, University of Ioannina, 45110, Ioannina, Greece

    Athena Papadopoulou & Haralambos Stamatis

  2. Institute of Chemical Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, 11635, Athens, Greece

    Dimitra Zarafeta

  3. Faculty of Biology, University of Athens, Panepistimioupolis, 15784, Athens, Greece

    Anastasia P. Galanopoulou

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  1. Athena Papadopoulou
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Papadopoulou, A., Zarafeta, D., Galanopoulou, A.P. et al. Enhanced Catalytic Performance of Trichoderma reesei Cellulase Immobilized on Magnetic Hierarchical Porous Carbon Nanoparticles. Protein J 38, 640–648 (2019). https://doi.org/10.1007/s10930-019-09869-w

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