Positional orientating co-immobilization of bienzyme CPO/GOx on mesoporous TiO2 thin film for efficient cascade reaction

  • Fengqin Gao
  • Mancheng Hu
  • Shuni Li
  • Quanguo Zhai
  • Yucheng JiangEmail author
Research Paper


A multitude of industrial processes are catalyzed by two or more enzymes working together in a cascade way. However, designing efficient enzymatic cascade reactions is still a challenge. In this work, a TiO2 thin film with mesoporous pores was prepared and used as carrier for co-immobilization of chloroperoxidase (CPO) and glucose peroxidase (GOx). By adjusting the dosage of hexadecyltrimethylammonium bromide (CTAB) and the ratio of the two enzymes, CPO and GOx were well distributed and positional orientated to their own appropriate pores to form an ordered “occupation” based on a “feet in right shoes” effect. Moreover, when the pore size was controlled around 12 nm, the enzymes aggregation was inhibited so as to avoid the decrease of activity of enzyme; The catalytic performance of TiO2–GOx and CPO composites was evaluated by the application of decolorization of Orange G dye in a cascaded manner. The oxidant H2O2 needed by CPO is generated in situ through glucose oxidation by GOx. Upon co-immobilization of CPO and GOx on the same carrier, a large increase in the initial catalytic efficiency was detected when compared to an equimolar mixture of the free enzymes, which was four times greater. Moreover, the affinity of the enzyme toward substrate binding was improved according to the kinetic assay. The thermal stability of TiO2–GOx and CPO composites were greatly improved than free enzymes. The TiO2–GOx and CPO composites can be easily separated from the reaction media which facilitate its recycle use.

Graphical abstract


Co-immobilization Chloroperoxidase Glucose oxidase Mesoporous TiO2 thin film Cascaded reaction 



This work is supported by the National Natural Science Foundation of China (21873061), the Fundamental Research Funds for the Chinese Central Universities (GK201701003) to Y. Jiang and Scientific Research Program Funded by Shaanxi Provincial Education Department (16JK1827) to F. Gao.


  1. 1.
    Schoffelen D, van Hest JCM (2012) Multi-enzyme systems: bringing enzymes together in vitro. Soft Matter 8:1736–1746. CrossRefGoogle Scholar
  2. 2.
    Wheeldon I, Minteer SD, Banta S, Barton SC, Atanassov P, Sigman M (2016) Substrate channeling as an approach to cascade reactions. Nat Chem 8:299–309. CrossRefGoogle Scholar
  3. 3.
    Mathesh M, Liu J, Barrow CJ, Yang W (2017) Graphene-oxide-based enzyme nanoarchitectonics for substrate channeling. Chem Eur J 23:304–311. CrossRefGoogle Scholar
  4. 4.
    Logan TC, Clark DS, Stachowiak TB, Svec F, Frechet JM (2007) Photopatterning enzymes on polymer monoliths in microfluidic devices for steady-state kinetic analysis and spatially separated multi-enzyme reactions. Anal Chem 79: 6592–6598. CrossRefGoogle Scholar
  5. 5.
    Hernandez K, Fernandez-Lafuente R (2011) Control of protein immobilization: coupling immobilization and site-directed mutagenesis to improve biocatalyst or biosensor performance. Enzyme Microb Technol 48:107–122. CrossRefGoogle Scholar
  6. 6.
    Pham TA, Berrin JG, Record E, To KA, Sigoillot JC (2010) Hydrolysis of softwood by Aspergillus mannanase: role of a carbohydrate-binding module. J Biotechnol 148:163–170. CrossRefGoogle Scholar
  7. 7.
    Hirakawa H, Nagamune T (2010) Molecular assembly of P450 with ferredoxin and ferredoxin reductase by fusion to PCNA. Chembiochem 11:1517–1520. CrossRefGoogle Scholar
  8. 8.
    Steinmann B, Christmann A, Heiseler T, Fritz J, Kolmar H (2010) In vivo enzyme immobilization by inclusion body display. Appl Environ Microbiol 76:5563–5569. CrossRefGoogle Scholar
  9. 9.
    Mortensen UH, Albertsen L, Chen Y, Bach LS, Rattleff S, Maury J, Brix S, Nielsen J (2011) Diversion of flux toward sesquiterpene production in saccharomyces cerevisiae by fusion of host and heterologous enzymes. Appl Environ Microbiol 77:1033–1040. CrossRefGoogle Scholar
  10. 10.
    Fan ZM, Wagschal K, Chen W, Montross MD, Lee CC, Yuan L (2009) Multimeric hemicellulases facilitate biomass conversion. Appl Environ Microbiol 75:1754–1757. CrossRefGoogle Scholar
  11. 11.
    Ji X, Su Z, Wang P, Ma G, Zhang S (2014) Polyelectrolyte doped hollow nanofibers for positional assembly of bienzyme system for cascade reaction at O/W interface. ACS Catal 4:4548–4559. CrossRefGoogle Scholar
  12. 12.
    Keighron JD, Keating CD (2010) Enzyme: nanoparticle bioconjugates with two sequential enzymes: stoichiometry and activity of malate dehydrogenase and citrate synthase on Au nanoparticles. Langmuir 26:18992–19000. CrossRefGoogle Scholar
  13. 13.
    Fu J, Liu M, Liu Y, Woodbury NW, Yan H (2012) Interenzyme substrate diffusion for an enzyme cascade organized on spatially addressable DNA nanostructures. J Am Chem Soc 134:5516–5519. CrossRefGoogle Scholar
  14. 14.
    El-Nahass MN, El-keiy MM, Ali EM (2018) Immobilization of horseradish peroxidase into cubic mesoporous silicate, SBA-16 with high activity and enhanced stability. Int J Biol Macromol 116:1304–1309. CrossRefGoogle Scholar
  15. 15.
    Qian X, Ren M, Yue D, Zhu Y, Han Y, Bian Z, Zhao Y (2017) Mesoporous TiO2 films coated on carbon foam based on waste polyurethane for enhanced photocatalytic oxidation of VOCs. Appl Catal B Environ 212:1–6. CrossRefGoogle Scholar
  16. 16.
    Muñoz-Guerrero FA, Águila S, Vazquez-Duhalt R, Alderete JB (2015) Enhancement of operational stability of chloroperoxidase from caldariomyces fumago by immobilization onto mesoporous supports and the use of co-solvents. J Mol Catal B Enzym 116:1–8. CrossRefGoogle Scholar
  17. 17.
    Guo Q, Liu L, Zhang M, Hou H, Song Y, Wang H, Zhong B, Wang L (2017) Hierarchically mesostructured porous TiO2 hollow nanofibers for high performance glucose biosensing. Biosens Bioelectron 92:654–660. CrossRefGoogle Scholar
  18. 18.
    Li D, Jia J, Zhang Y, Wang N, Guo X, Yu X (2016) Preparation and characterization of Nano-graphite/TiO2 composite photoelectrode for photoelectrocatalytic degradation of hazardous pollutant. J Hazard Mater 315:1–10. CrossRefGoogle Scholar
  19. 19.
    Son KJ, Ahn SH, Kim JH, Koh WG (2011) Graft copolymer-templated mesoporous TiO2 films micropatterned with poly(ethylene glycol) hydrogel: novel platform for highly sensitive protein microarrays. ACS Appl Mater Inter 3:573–581. CrossRefGoogle Scholar
  20. 20.
    Dong J, Wen Y, Miao Y, Xie Z, Zhang Z, Yang H (2010) A nanoporous zirconium phytate film for immobilization of redox protein and the direct electrochemical biosensor. Sens Actuators B 150:141–147. CrossRefGoogle Scholar
  21. 21.
    Li G, Nandgaonkar AG, Wang Q, Zhang J, Krause WE, Wei Q, Lucia LA (2017) Laccase-immobilized bacterial cellulose/TiO2 functionalized composite membranes: evaluation for photo- and bio-catalytic dye degradation. J Membr Sci 525:89–98. CrossRefGoogle Scholar
  22. 22.
    Hurtado L, Solís-Casados D, Escobar-Alarcón L, Romero R, Natividad R (2016) Multiphase photo-capillary reactors coated with TiO2 films: preparation, characterization and photocatalytic performance. Chem Eng J 304:39–47. CrossRefGoogle Scholar
  23. 23.
    Bricchi BR, Ghidelli M, Mascaretti L, Zapelli A, Russo V, Casari CS, Terraneo G, Alessandri I, Ducati C, Bassi AL (2018) Integration of plasmonic Au nanoparticles in TiO2 hierarchical structures in a single-step pulsed laser co-deposition. Mater Design 156:311–319. CrossRefGoogle Scholar
  24. 24.
    Zhi L, Jiang Y, Wang Y, Hu M, Li S, Ma Y (2007) Effects of additives on the thermostability of chloroperoxidase. Biotechnol Prog 23:729–733. CrossRefGoogle Scholar
  25. 25.
    Hager LP, Morris DR, Brown FS, Eberwein H (1966) Chloroperoxidase II. utilization of halogen anions. J Biol Chem 241:1769–1777.
  26. 26.
    Lu J, Cheng L, Wang Y, Ding Y, Hu M, Li S, Zhai Q, Jiang Y (2017) Enzymatic-photocatalytic synergetic effect on the decolorization of dyes by single chloroperoxidase molecule immobilization on TiO2 mesoporous thin film. Mater Design 129:219–226. CrossRefGoogle Scholar
  27. 27.
    Aburto J, Ayala M, Bustos-Jaimes I, Montiel C, Terrés E, Domínguez JM, Torres E (2005) Stability and catalytic properties of chloroperoxidase immobilized on SBA-16 mesoporous materials. Microporous Mesoporous Mater 83:193–200. CrossRefGoogle Scholar
  28. 28.
    Zhou Z, Hartmann M (2013) Progress in enzyme immobilization in ordered mesoporous materials and related applications. Chem Soc Rev 42:3894–3912. CrossRefGoogle Scholar
  29. 29.
    Zhang J, Feng M, Jiang Y, Hu M, Li S, Zhai Q (2012) Efficient decolorization /degradation of aqueous azo dyes using buffered H2O2 oxidation catalyzed by a dosage below ppm level of chloroperoxidase. Chem Eng J 191:236–242. CrossRefGoogle Scholar
  30. 30.
    Jiao R, Tan Y, Jiang Y, Hu M, Li S, Zhai Q (2014) Ordered mesoporous silica matrix for immobilization of chloroperoxidase with enhanced biocatalytic performance for oxidative decolorization of azo dye. Ind Eng Chem Res 53:12201–12208. CrossRefGoogle Scholar
  31. 31.
    Jung D, Streb C, Hartmann M (2008) Oxidation of indole using chloroperoxidase and glucose oxidase immobilized on SBA-15 as tandem biocatalyst. Micropor Mesopor Mat 113:523–529. CrossRefGoogle Scholar
  32. 32.
    Hecht HJ, Schomburg D, Kalisz H, Schmid RD (1993) The 3D structure of glucose oxidase from aspergillus niger. implications for the use of GOD as a biosensor enzyme. Biosens Bioelectron 8:197–203. CrossRefGoogle Scholar
  33. 33.
    Wheeldon I, Minteer SD, Banta S, Barton SC, Atanassov P, Sigman M (2016) Substrate channelling as an approach to cascade reactions. Nat Chem 8:299–309. CrossRefGoogle Scholar
  34. 34.
    Wu L, Liu Y, Chi B, Xu Z, Feng X, Li S, Xu H (2015) An innovative method for immobilizing sucrose isomerase on ε-poly-l-lysine modified mesoporous TiO2. Food Chem 187:182–188. CrossRefGoogle Scholar
  35. 35.
    Hou J, Dong G, Ye Y, Chen V (2014) Laccase immobilization on titania nanoparticles and titania-functionalized membranes. J Membr Sci 452:229–240. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Fengqin Gao
    • 1
    • 2
  • Mancheng Hu
    • 1
    • 3
  • Shuni Li
    • 1
    • 3
  • Quanguo Zhai
    • 1
    • 3
  • Yucheng Jiang
    • 1
    • 3
    Email author
  1. 1.School of Chemistry and Chemical EngineeringShaanxi Normal UniversityXi’anPeople’s Republic of China
  2. 2.College of Chemistry and Chemical EngineeringXianyang Normal UniversityXianyangPeople’s Republic of China
  3. 3.Key Laboratory of Macromolecular Science of Shaanxi ProvinceShaanxi Normal UniversityXi’anPeople’s Republic of China

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