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Nanofibrillated Cellulose-Enzyme Assemblies for Enhanced Biotransformations with In Situ Cofactor Regeneration

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Abstract

We report herein the use of nanofibrillated cellulose (NFC) for development of enzyme assemblies in an oriented manner for biotransformation with in situ cofactor regeneration. This is achieved by developing fusion protein enzymes with cellulose-specific binding domains. Specifically, lactate dehydrogenase and NADH oxidase were fused with a cellulose binding domain, which enabled both enzyme recovery and assembling in essentially one single step by using NFC. Results showed that the binding capacity of the enzymes was as high as 0.9 μmol-enzyme/g-NFC. Compared to native parent free enzymes, NFC-enzyme assemblies improved the catalytic efficiency of the coupled reaction system by over 100%. The lifetime of enzymes was also improved by as high as 27 folds. The work demonstrates promising potential of using biocompatible and environmentally benign bio-based nanomaterials for construction of efficient catalysts for intensified bioprocessing and biotransformation applications.

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References

  1. Schoffelen, S., & van Hest, J. C. M. (2012). Multi-enzyme systems: bringing enzymes together in vitro. Soft Matter, 8(6), 1736–1746.

    CAS  Google Scholar 

  2. Ardao, I., Hwang, E. T., & Zeng, A. P. (2013). In vitro multienzymatic reaction systems for biosynthesis. In Fundamentals and application of new bioproduction systems (pp. 153–184). Berlin Heidelberg: Springer.

    Google Scholar 

  3. Kim, J., Grate, J. W., & Wang, P. (2008). Nanobiocatalysis and its potential applications. Trends in Biotechnology, 26(11), 639–646.

    CAS  PubMed  Google Scholar 

  4. Wang, P. (2006). Nanoscale biocatalyst systems. Current Opinion in Biotechnology, 17(6), 574–579.

    CAS  PubMed  Google Scholar 

  5. Mateo, C., Palomo, J. M., Fernandez-Lorente, G., Guisan, J. M., & Fernandez-Lafuente, R. (2007). Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme and Microbial Technology, 40(6), 1451–1463.

    CAS  Google Scholar 

  6. Jia, H., Zhu, G., Vugrinovich, B., Kataphinan, W., Reneker, D. H., & Wang, P. (2002). Enzyme-carrying polymeric nanofibers prepared via electrospinning for use as unique biocatalysts. Biotechnology Progress, 18(5), 1027–1032.

    CAS  PubMed  Google Scholar 

  7. Moon, R. J., Martini, A., Nairn, J., Simonsen, J., & Youngblood, J. (2011). Cellulose nanomaterials review: structure, properties and nanocomposites. Chemical Society Reviews, 40(7), 3941–3994.

    CAS  PubMed  Google Scholar 

  8. Xu, C., Zhu, S., Xing, C., Li, D., Zhu, N., & Zhou, H. (2015). Isolation and properties of cellulose nanofibrils from coconut palm petioles by different mechanical process. PLoS One, 10(4), e0122123.

    PubMed  PubMed Central  Google Scholar 

  9. Zhao, X. Y., Lu, X., Tze, W. T. Y., & Wang, P. (2010). A single carbon fiber microelectrode with branching carbon nanotubes for bioelectrochemical processes. Biosensors and Bioelectronics, 25(10), 2343–2350.

    CAS  PubMed  Google Scholar 

  10. Misson, M., Dai, S., Jin, B., Chen, B. H., & Zhang, H. (2016). Manipulation of nanofiber-based β-galactosidase nanoenvironment for enhancement of galacto-oligosaccharide production. Journal of Biotechnology, 222, 56–64.

    CAS  PubMed  Google Scholar 

  11. Zhao, X. Y., Jia, H., Kim, J. B., & Wang, P. (2009). Kinetic limitations of a bioelectrochemical electrode using carbon nanotube-attached glucose oxidase for biofuel eells. Biotechnology and Bioengineering, 104(6), 1068–1074.

    CAS  PubMed  Google Scholar 

  12. Pavilids, L. V., Vorhaben, T., Tsoufis, T., Rudolf, P., Bornscheuer, U. T., Gournis, D., & Stamatis, H. (2012). Development of effective nanobiocatalytic systems through the immobilization of hydrolases on functionalized carbon-based nanomaterials. Bioresource Technology, 115, 164–171.

    Google Scholar 

  13. Lee, D., Lee, J., Kim, J., Na, H. B., Kim, B., Shin, C. H., Kwak, J. H., Dohnalkova, A., Grate, J. W., Hyeon, T., & Kim, H. S. (2005). Simple fabrication of a highly sensitive and fast glucose biosensor using enzymes immobilized in mesocellular carbon foam. Advanced Materials, 17, 2828–2833.

    CAS  Google Scholar 

  14. Asuri, P., Karajanagi, S. S., Sellitto, E., Kim, D. Y., Kane, R. S., & Dordick, J. S. (2006). Water-soluble carbon nanotube-enzyme conjugates as functional biocatalytic formulations. Biotechnology and Bioengineering, 95(5), 804–811.

    CAS  PubMed  Google Scholar 

  15. Ahmad, R., & Sardar, M. (2015). Enzyme immobilization: an overview on nanoparticles as immobilization matrix. Biochemistry and Analytical Biochemistry, 4(2). https://doi.org/10.4172/2161-1009.1000178.

  16. Ning, C., Su, E., Tian, Y., & Wei, D. (2014). Combined cross-linked enzyme aggregates (combi-CLEAs) for efficient integration of a ketoreductase and a cofactor regeneration system. Journal of Biotechnology, 184, 7–10.

    CAS  PubMed  Google Scholar 

  17. Meeuwissen, S. A., Rioz-Martínez, A., de Gonzalo, G., Fraaije, M. W., Gotor, V., & van Hest, J. C. (2011). Cofactor regeneration in polymersome nanoreactors: enzymatically catalysed Baeyer-Villiger reactions. Journal of Materials Chemistry, 21(47), 18923–18926.

    CAS  Google Scholar 

  18. Campbell, J., & Chang, T. M. S. (1976). The recycling of NAD+ (free and immobilized) within semipermeable aqueous microcapsules containing a multi-enzyme system. Biochemistry Biophysics Research Communications, 69(2), 562–569.

    CAS  Google Scholar 

  19. Siró, I., & Plackett, D. (2010). Microfibrillated cellulose and new nanocomposite materials: a review. Cellulose, 17(3), 459–494.

    Google Scholar 

  20. Song, J., Kahveci, D., Chen, M., Guo, Z., Xie, E., Xu, X., Besenbacher, F., & Dong, M. (2012). Enhanced catalytic activity of lipase encapsulated in PCL nanofibers. Langmuir, 28(14), 6157–6162.

    CAS  PubMed  Google Scholar 

  21. Sulaiman, S., Mokhtar, M. N., Naim, M. N., Baharuddin, A. S., & Sulaiman, A. (2015). A review: potential usage of cellulose nanofibers (CNF) for enzyme immobilization via covalent interactions. Applied Biochemistry and Biotechnology, 175(4), 1817–1842.

    CAS  PubMed  Google Scholar 

  22. Shoseyov, O., Shani, Z., & Levy, I. (2006). Carbohydrate binding modules: biochemical properties and novel applications. Microbiology and Molecular Biology Reviews, 70(2), 283–295.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Dai, G., Hu, J., Zhao, X., & Wang, P. (2017). A colorimetric paper sensor for lactate assay using a cellulose-binding recombinant enzyme. Sensors and Actuators B: Chemical, 238, 138–144.

    CAS  Google Scholar 

  24. Hwang, S., Ahn, J., Lee, S., Lee, T. G., Haam, S., Lee, K., Ahn, I. S., & Jung, J. K. (2004). Evaluation of cellulose-binding domain fused to a lipase for the lipase immobilization. Biotechnology Letters, 26(7), 603–605.

    CAS  PubMed  Google Scholar 

  25. Levy, I., Ward, G., Hadar, Y., Shoseyov, O., & Dosoretz, C. G. (2003). Oxidation of 4-bromophenol by the recombinant fused protein cellulose binding domain horseradish peroxidase immobilized on cellulose. Biotechnology and Bioengineering, 82(2), 223–231.

    CAS  PubMed  Google Scholar 

  26. Wan, W., Wang, D., Gao, X., & Hong, J. (2011). Expression of family 3 cellulose-binding module (CBM3) as an affinity tag for recombinant proteins in yeast. Applied Microbiology and Biotechnology, 91(3), 789–798.

    CAS  PubMed  Google Scholar 

  27. Kumar, A., Zhang, S., Wu, G., Wu, C. C., Chen, J., Baskaran, R., & Liu, Z. (2015). Cellulose binding domain assisted immobilization of lipase (GSlip-CBD) onto cellulosic nanogel: characterization and application in organic medium. Colloids and Surfaces B: Biointerfaces, 136, 1042–1050.

    CAS  PubMed  Google Scholar 

  28. Ren, S. Z., Li, C. H., Jiao, X. B., Jia, S. R., Jiang, Y. J., Bilal, M., & Cui, J. D. (2019). Recent progress in multienzymes co-immobilization and multienzyme system applications. Chemical Engineering Journal, 373, 1254–1278.

    CAS  Google Scholar 

  29. Gur, S. D., Idil, N., & Aksoz, N. (2018). Optimization of enzyme co-immobilization with sodium alginate and glutaraldehyde-activated chitosan beads. Applied Biochemistry and Biotechnology, 184(2), 538–552.

    CAS  PubMed  Google Scholar 

  30. Lu, J. X., Zhang, Y. H., Sun, D. F., Jiang, W., Wang, S. Z., & Fang, B. S. (2016). The development of leucine dehydrogenase and formate dehydrogenase bifunctional enzyme cascade improves the biosynthsis of l-tert-leucine. Applied Biochemistry and Biotechnology, 180(6), 1180–1195.

    CAS  PubMed  Google Scholar 

  31. Iturrate, L., Sánchez-Moreno, I., Oroz-Guinea, I., Pérez-Gil, J., & García-Junceda, E. (2010). Preparation and characterization of a bifunctional aldolase/kinase enzyme: a more efficient biocatalyst for C-C bond formation. Chemistry - A European Journal, 16(13), 4018–4030.

    CAS  Google Scholar 

  32. Zhang, Y. H. P. (2011). Substrate channeling and enzyme complexes for biotechnological applications. Biotechnology Advances, 29(6), 715–725.

    CAS  PubMed  Google Scholar 

  33. Hirakawa, H., Kakitani, A., & Nagamune, T. (2013). Introduction of selective intersubunit disulfide bonds into self-assembly protein scaffold to enhance an artificial multienzyme complex’s activity. Biotechnology and Bioengineering, 110(7), 1858–1864.

    CAS  PubMed  Google Scholar 

  34. Wang, T. D., Ma, F., Ma, X., & Wang, P. (2015). Spatially programmed assembling of oxidoreductases with single-stranded DNA for cofactor-required reactions. Applied Microbiology and Biotechnology, 99(8), 3469–3477.

    CAS  PubMed  Google Scholar 

  35. Ougiya, H., Hioki, N., Watanabe, K., Morinaga, Y., Yoshinaga, F., & Samejima, M. (1998). Relationship between the physical properties and surface area of cellulose derived from adsorbates of various molecular sizes. Bioscience Biotechnology and Biochemistry, 62(10), 1880–1884.

    CAS  Google Scholar 

  36. Segal, L., Creely, J. J., Martin, A. E., & Conrad, C. M. (1959). An empirical method for estimating the degree of crystallinity of native cellulose using the X-ray diffractometer. Textile Research Journal, 29, 786–794.

    CAS  Google Scholar 

  37. Goldstein, M. A., Takagi, M., Hashida, S., Shoseyov, O., Doi, R. H., & Segel, I. H. (1993). Characterization of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A. Journal of Bacteriology, 175(18), 5762–5768.

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Hensel, R., Mayr, U., Stetter, K. O., & Kandler, O. (1977). Comparative studies of lactic acid dehydrogenases in lactic acid bacteria. Archives of Microbiology, 112(1), 81–93.

    CAS  PubMed  Google Scholar 

  39. Contag, P. R., Williams, M. G., & Rogers, P. (1990). Cloning of a lactate dehydrogenase gene from Clostridium acetobutylicum B643 and expression in Escherichia coli. Applied and Environmental Microbiology, 56(12), 3760–3765.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Hallström, Å., Carlsson, A., Hillered, L., & Uncerstedt, U. (1989). Simultaneous determination of lactate, pyruvate, and ascorbate in microdialysis samples from rat brain, blood, fat, and muscle using high-performance liquid chromatography. Journal of Pharmacology and Toxicological Methods, 22(2), 113–124.

    Google Scholar 

  41. Hong, J., Ye, X., & Zhang, Y. H. P. (2007). Quantitative determination of cellulose accessibility to cellulase based on adsorption of a nonhydrolytic fusion protein containing CBM and GFP with its applications. Langmuir, 23(25), 12535–12540.

    CAS  PubMed  Google Scholar 

  42. Lehtiö, J., Sugiyama, J., Gustavsson, M., Fransson, L., Linder, M., & Teeri, T. T. (2003). The binding specificity and affinity determinants of family 1 and family 3 cellulose binding modules. Proceedings of the National Academy of Sciences of USA, 100(2), 484–489.

    Google Scholar 

  43. Tormo, J., Lamed, R., Chirino, A. J., Morag, E., Bayer, E. A., Shoham, Y., & Steitz, T. A. (1996). Crystal structure of a bacterial family-III cellulose-binding domain: a general mechanism for attachment to cellulose. EMBO Journal, 15(21), 5739.

    CAS  PubMed  Google Scholar 

  44. Parikh, D. V., Thibodeaux, D. P., & Condon, B. (2007). X-ray crystallinity of bleached and crosslinked cottons. Textile Research Journal, 77(8), 612–616.

    CAS  Google Scholar 

  45. Wang, L., Zhang, Y., Gao, P., Shi, D., Liu, H., & Gao, H. (2006). Changes in the structural properties and rate of hydrolysis of cotton fibers during extended enzymatic hydrolysis. Biotechnology and Bioengineering, 93(3), 443–456.

    CAS  PubMed  Google Scholar 

  46. Jäger, G., Girfoglio, M., Dollo, F., Rinaldi, R., Bongard, H., Commandeur, U., Fischer, R., Spiess, A. C., & Büchs, J. (2011). How recombinant swollenin from Kluyveromyces lactis affects cellulosic substrates and accelerates their hydrolysis. Biotechnology for Biofuels, 4(1), 1.

    Google Scholar 

  47. Wang, P. (2012). Nanoscale engineering for smart biocatalysts with fine-tuned properties and functionalities. Topics in Catalysis, 55(16–18), 1107–1113.

    CAS  Google Scholar 

  48. Karajanagi, S. S., Vertegel, A. A., Kane, R. S., & Dordick, J. S. (2004). Structure and function of enzymes adsorbed onto single-walled carbon nanotubes. Langmuir, 20(26), 11594–11599.

    CAS  PubMed  Google Scholar 

  49. Wang, L., Wei, L., Chen, Y., & Jiang, R. (2010). Specific and reversible immobilization of NADH oxidase on functionalized carbon nanotubes. Journal of Biotechnology, 150(1), 57–63.

    PubMed  Google Scholar 

  50. Li, J., Nayak, S., & Mrksich, M. (2010). Rate enhancement of an interfacial biochemical reaction through localization of substrate and enzyme by an adaptor domain. Journal of Physical Chemistry B, 114(46), 15113–15118.

    CAS  Google Scholar 

  51. Malcata, F. X., Reyes, H. R., Garcia, H. S., Hill Jr., C. G., & Amundson, C. H. (1990). Immobilized lipase reactors for modification of fats and oils, a review. Journal of the American Chemical Society, 67(12), 890–910.

    CAS  Google Scholar 

  52. Fan, L., Zhang, Z., Yu, X., Xue, Y., & Tan, T. (2012). Self-surface assembly of cellulosomes with two miniscaffoldins on Saccharomyces cerevisiae for cellulosic ethanol production. Proceedings of the National Academy of Sciences of USA, 109(33), 13260–13265.

    CAS  Google Scholar 

  53. Guo, J., & Catchmark, J. M. (2013). Binding specificity and thermodynamics of cellulose-binding modules from Trichoderma reesei Cel7A and Cel6A. Biomacromolecules, 14(5), 1268–1277.

    CAS  PubMed  Google Scholar 

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Funding

Dai thanks Chinese Scholarship Council for the support of an international scholarship for visiting research at UMN. Branciforti is grateful to CAPES (project Nanobiotec number 13; postdoctoral scholarship for process number 6290-13-2) for funding her research at the University of Minnesota. Wang thanks partial support from IPRIME (Industrial Partnership for Research in Interfacial and Materials Engineering) of University of Minnesota.

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Correspondence to Ping Wang.

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Dai, G., Tze, W.T.Y., Frigo-Vaz, B. et al. Nanofibrillated Cellulose-Enzyme Assemblies for Enhanced Biotransformations with In Situ Cofactor Regeneration. Appl Biochem Biotechnol 191, 1369–1383 (2020). https://doi.org/10.1007/s12010-020-03263-4

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