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Improved Thermal and Reusability Properties of Xylanase by Genipin Cross-Linking to Magnetic Chitosan Particles

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Abstract

Enzymes are gradually increasingly preferred over chemical processes, but commercial enzyme applications remain limited due to their low stability and low product recovery, so the application of an immobilization technique is required for repeated use. The aims of this work were to produce stable enzyme complexes of cross-linked xylanase on magnetic chitosan, to describe some characteristics of these complexes, and to evaluate the thermal stability of the immobilized enzyme and its reusability. A xylanase was cross-linked to magnetite particles prepared by in situ co-precipitation of iron salts in a chitosan template. The effect of temperature, pH, kinetic parameters, and reusability on free and immobilized xylanase was evaluated. Magnetization, morphology, size, structural change, and thermal behavior of immobilized enzyme were described. 1.0 ± 0.1 μg of xylanase was immobilized per milligram of superparamagnetic chitosan nanoparticles via covalent bonds formed with genipin. Immobilized xylanase showed thermal, pH, and catalytic velocity improvement compared to the free enzyme and can be reused three times. Heterogeneous aggregates of 254 nm were obtained after enzyme immobilization. The immobilization protocol used in this work was successful in retaining enzyme thermal stability and could be important in using natural compounds such as Fe3O4@Chitosan@Xylanase in the harsh temperature condition of relevant industries.

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References

  1. Sorek, N., Yeats, T. H., Szemenyei, H., Youngs, H., & Somerville, C. R. (2014). The implications of lignocellulosic biomass chemical composition for the production of advanced biofuels. BioScience, 64(3), 192–201. https://doi.org/10.1093/biosci/bit037.

    Article  Google Scholar 

  2. Saka, S., & Bae, H. J. (2016). Secondary xylem for bioconversion. In K. Yoon soo, F. Ryo, & P. S. Adya (Eds.), Secondary xylem biology: origins, functions, and applications (pp. 213–231). Academic Press. https://doi.org/10.1016/B978-0-12-802185-9.00011-5.

  3. Dutta, S. K., & Chakraborty, S. (2015). Kinetic analysis of two-phase enzymatic hydrolysis of hemicellulose of xylan type. Bioresource Technology, 198, 642–650. https://doi.org/10.1016/j.biortech.2015.09.066.

    Article  CAS  PubMed  Google Scholar 

  4. Sukri, S. S. M., & Munaim, M. S. A. (2017). Combination of entrapment and covalent binding techniques for xylanase immobilisation on alginate beads: screening process parameters. Chemical Engineering Transactions, 56, 169–174. https://doi.org/10.3303/CET1756029.

    Article  Google Scholar 

  5. Bibi, Z., Ansari, A., Zohra, R. R., Aman, A., & Ul Qader, S. A. (2014). Production of xylan degrading endo-1, 4-β-xylanase from thermophilic Geobacillus stearothermophilus KIBGE-IB29. Journal of Radiation Research and Applied Sciences, 7(4), 478–485. https://doi.org/10.1016/j.jrras.2014.08.001.

    Article  Google Scholar 

  6. Bhushan, B., Pal, A., & Jain, V. (2015). Improved enzyme catalytic characteristics upon glutaraldehyde cross-linking of alginate entrapped xylanase isolated from Aspergillus flavus MTCC 9390. Enzyme Research, 2015, 1–9. https://doi.org/10.1155/2015/210784.

    Article  CAS  Google Scholar 

  7. Lyu, F., Zhang, Y., Zare, R. N., Ge, J., & Liu, Z. (2014). One-pot synthesis of protein-embedded metal-organic frameworks with enhanced biological activities. Nano Letters, 14(10), 5761–5765. https://doi.org/10.1021/nl5026419.

    Article  CAS  PubMed  Google Scholar 

  8. Li, Z., Xia, H., Li, S., Pang, J., Zhu, W., & Jiang, Y. (2017). In situ hybridization of enzymes and their metal-organic framework analogues with enhanced activity and stability by biomimetic mineralisation. Nanoscale, 9(40), 15298–15302. https://doi.org/10.1039/c7nr06315f.

    Article  CAS  PubMed  Google Scholar 

  9. Xia, H., Zhong, X., Li, Z., & Jiang, Y. (2019). Palladium-mediated hybrid biocatalysts with enhanced enzymatic catalytic performance via allosteric effects. Journal of Colloid and Interface Science, 533, 1–8. https://doi.org/10.1016/j.jcis.2018.08.052.

    Article  CAS  PubMed  Google Scholar 

  10. Romo Sánchez, S., Gil Sánchez, I., Arévalo-Villena, M., & Briones Pérez, A. (2015). Production and immobilization of enzymes by solid-state fermentation of agroindustrial waste. Bioprocess and Biosystems Engineering, 38(3), 587–593. https://doi.org/10.1007/s00449-014-1298-y.

    Article  CAS  PubMed  Google Scholar 

  11. Kaur, S., & Dhillon, G. S. (2014). The versatile biopolymer chitosan: potential sources, evaluation of extraction methods and applications. Critical Reviews in Microbiology, 40(2), 155–175. https://doi.org/10.3109/1040841X.2013.770385.

    Article  CAS  PubMed  Google Scholar 

  12. Manickam, B., Sreedharan, R., & Elumalai, M. (2014). “Genipin”—the natural water soluble cross-linking agent and its importance in the modified drug delivery systems: an overview. Current Drug Delivery, 11(1), 139–145. https://doi.org/10.2174/15672018113106660059.

    Article  CAS  PubMed  Google Scholar 

  13. Klein, M. P., Hackenhaar, C. R., Lorenzoni, A. S. G., Rodrigues, R. C., Costa, T. M. H., Ninow, J. L., & Hertz, P. F. (2016). Chitosan crosslinked with genipin as support matrix for application in food process: support characterization and β-d-galactosidase immobilization. Carbohydrate Polymers, 137, 184–190. https://doi.org/10.1016/j.carbpol.2015.10.069.

    Article  CAS  PubMed  Google Scholar 

  14. Lau, Y. T., Kwok, L. F., Tam, K. W., Chan, Y. S., Shum, D. K. Y., & Shea, G. K. H. (2018). Genipin-treated chitosan nanofibers as a novel scaffold for nerve guidance channel design. Colloids and Surfaces B: Biointerfaces, 162, 126–134. https://doi.org/10.1016/j.colsurfb.2017.11.061.

    Article  CAS  PubMed  Google Scholar 

  15. Pozzo L., d. Y., da Conceição, T. F., Spinelli, A., Scharnagl, N., & Pires, A. T. N. (2018). Chitosan coatings crosslinked with genipin for corrosion protection of AZ31 magnesium alloy sheets. Carbohydrate Polymers, 181(September 2017), 71–77. https://doi.org/10.1016/j.carbpol.2017.10.055.

    Article  CAS  Google Scholar 

  16. Oryan, A., Kamali, A., Moshiri, A., Baharvand, H., & Daemi, H. (2018). Chemical crosslinking of biopolymeric scaffolds: current knowledge and future directions of crosslinked engineered bone scaffolds. International Journal of Biological Macromolecules, 107(PartA), 678–688. https://doi.org/10.1016/j.ijbiomac.2017.08.184.

    Article  CAS  PubMed  Google Scholar 

  17. Feng, J., Yu, S., Li, J., Mo, T., & Li, P. (2016). Enhancement of the catalytic activity and stability of immobilized aminoacylase using modified magnetic Fe3O4 nanoparticles. Chemical Engineering Journal, 286, 216–222. https://doi.org/10.1016/j.cej.2015.10.083.

    Article  CAS  Google Scholar 

  18. Liu, M. Q., Dai, X. J., Guan, R. F., & Xu, X. (2014). Immobilization of Aspergillus niger xylanase A on Fe3O4-coated chitosan magnetic nanoparticles for xylooligosaccharide preparation. Catalysis Communications, 55, 6–10. https://doi.org/10.1016/j.catcom.2014.06.002.

    Article  CAS  Google Scholar 

  19. Morales, M. A., De Souza Rodrigues, E. C., De Amorim, A. S. C. M., Soares, J. M., & Galembeck, F. (2013). Size selected synthesis of magnetite nanoparticles in chitosan matrix. Applied Surface Science, 275, 71–74. https://doi.org/10.1016/j.apsusc.2013.01.123.

    Article  CAS  Google Scholar 

  20. Sheldon, R. A., & Van Pelt, S. (2013). Enzyme immobilisation in biocatalysis: why, what and how. Chemical Society Reviews, 42(42), 6223–6235. https://doi.org/10.1039/c3cs60075k.

    Article  CAS  PubMed  Google Scholar 

  21. Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31(3), 426–428. https://doi.org/10.1021/ac60147a030.

    Article  CAS  Google Scholar 

  22. Length, F. (2011). Homologue expression of a fungal endo-1 , 4- -D- xylanase using submerged and solid substrate fermentations. Journal of Biotechnology, 10(10), 1760–1767. https://doi.org/10.5897/AJB10.1952.

    Article  Google Scholar 

  23. Goluguri, B. R., Thulluri, C., Addepally, U., & Shetty, P. R. (2016). Novel alkali-thermostable xylanase from Thielaviopsis basicola (MTCC 1467): purification and kinetic characterization. International Journal of Biological Macromolecules, 82, 823–829. https://doi.org/10.1016/j.ijbiomac.2015.10.055.

    Article  CAS  PubMed  Google Scholar 

  24. Lineweaver, H., & Burk, D. (1934). The determination of enzyme dissociation constants. Journal of the American Chemical Society, 56(3), 658–666. https://doi.org/10.1021/ja01318a036.

    Article  CAS  Google Scholar 

  25. Tokareva, M. I., Ivantsova, M. N., & Mironov, M. A. (2017). Heterocycles of natural origin as non-toxic reagents for cross-linking of proteins and polysaccharides. Chemistry of Heterocyclic Compounds, 53(1), 21–35. https://doi.org/10.1007/s10593-017-2016-x.

    Article  CAS  Google Scholar 

  26. Mehnati-Najafabadi, V., Taheri-Kafrani, A., & Bordbar, A.-K. (2017). Xylanase immobilization on modified superparamagnetic graphene oxide nanocomposite: effect of PEGylation on activity and stability. International Journal of Biological Macromolecules., 107(Pt A), 418–425. https://doi.org/10.1016/j.ijbiomac.2017.09.013.

    Article  CAS  PubMed  Google Scholar 

  27. Shahrestani, H., Taheri-Kafrani, A., Soozanipour, A., & Tavakoli, O. (2016). Enzymatic clarification of fruit juices using xylanase immobilized on 1,3,5-triazine-functionalized silica-encapsulated magnetic nanoparticles. Biochemical Engineering Journal, 109, 51–58. https://doi.org/10.1016/j.bej.2015.12.013.

    Article  CAS  Google Scholar 

  28. Soozanipour, A., Taheri-Kafrani, A., & Landarani Isfahani, A. (2015). Covalent attachment of xylanase on functionalized magnetic nanoparticles and determination of its activity and stability. Chemical Engineering Journal, 270, 235–243. https://doi.org/10.1016/j.cej.2015.02.032.

    Article  CAS  Google Scholar 

  29. Selvarajan, E., & Veena, R. (2017). Recent advances and future perspectives of thermostable xylanase. Biomedical & Pharmacology Journal, 10(1), 261–279. https://doi.org/10.13005/bpj/1106.

    Article  Google Scholar 

  30. Vaz, R. P., de Souza Moreira, L. R., & Ferreira Filho, E. X. (2016). An overview of holocellulose-degrading enzyme immobilization for use in bioethanol production. Journal of Molecular Catalysis B: Enzymatic, 133, 127–135. https://doi.org/10.1016/j.molcatb.2016.08.006.

    Article  CAS  Google Scholar 

  31. Mohamad, N. R., Marzuki, N. H. C., Buang, N. A., Huyop, F., & Wahab, R. A. (2015). An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes. Biotechnology and Biotechnological Equipment, 29(2), 205–220. https://doi.org/10.1080/13102818.2015.1008192.

    Article  CAS  PubMed  Google Scholar 

  32. Kumar, S., Haq, I., Prakash, J., & Raj, A. (2017). Improved enzyme properties upon glutaraldehyde cross-linking of alginate entrapped xylanase from Bacillus licheniformis. International Journal of Biological Macromolecules, 98, 24–33. https://doi.org/10.1016/j.ijbiomac.2017.01.104.

    Article  CAS  PubMed  Google Scholar 

  33. Liu, M. Q., Huo, W. K., Xu, X., & Jin, D. F. (2015). An immobilized bifunctional xylanase on carbon-coated chitosan nanoparticles with a potential application in xylan-rich biomass bioconversion. Journal of Molecular Catalysis B: Enzymatic, 120, 119–126. https://doi.org/10.1016/j.molcatb.2015.07.002.

    Article  CAS  Google Scholar 

  34. Hou, L., Sun, X., Sui, J., & Ding, C. (2015). Immobilization of a 22kDa xylanase on Eudragit L-100 for xylo-oligosaccharide production. Advance Journal of Food Science and Technology, 7(6), 401–407. https://doi.org/10.19026/ajfst.7.1332.

    Article  CAS  Google Scholar 

  35. Bagewadi, Z. K., Mulla, S. I., Shouche, Y., & Ninnekar, H. Z. (2016). Xylanase production from Penicillium citrinum isolate HZN13 using response surface methodology and characterization of immobilized xylanase on glutaraldehyde-activated calcium-alginate beads. 3 Biotech, 6(2), 1–18. https://doi.org/10.1007/s13205-016-0484-9.

    Article  Google Scholar 

  36. Chen, J., Leng, J., Yang, X., Liao, L., Liu, L., & Xiao, A. (2017). Enhanced performance of magnetic graphene oxide-immobilized laccase and its application for the decolorization of dyes. Molecules, 22(2), 221. https://doi.org/10.3390/molecules22020221.

    Article  CAS  PubMed Central  Google Scholar 

  37. Al-Qodah, Z., Al-Shannag, M., Al-Busoul, M., Penchev, I., & Orfali, W. (2017). Immobilized enzymes bioreactors utilizing a magnetic field: a review. Biochemical Engineering Journal, 121, 94–106. https://doi.org/10.1016/j.bej.2017.02.003.

    Article  CAS  Google Scholar 

  38. Pati, S. S., Singh, L. H., Guimaraes, E. M., Mantilla, J., Coaquira, J. A. H., Oliveira, A. C., et al. (2016). Magnetic chitosan-functionalized Fe3O4@Au nanoparticles: synthesis and characterization. Journal of Alloys and Compounds, 684, 68–74. https://doi.org/10.1016/j.jallcom.2016.05.160.

    Article  CAS  Google Scholar 

  39. Prabha, G., & Raj, V. (2016). Preparation and characterization of chitosan-polyethylene glycol-polyvinylpyrrolidone-coated superparamagnetic iron oxide nanoparticles as carrier system: drug loading and in vitro drug release study. Journal of Biomedical Materials Research - Part B Applied Biomaterials, 104(4), 808–816. https://doi.org/10.1002/jbm.b.33637.

    Article  CAS  Google Scholar 

  40. Saikia, C., Das, M. K., Ramteke, A., & Maji, T. K. (2016). Effect of crosslinker on drug delivery properties of curcumin loaded starch coated iron oxide nanoparticles. International Journal of Biological Macromolecules, 93(Pt A), 1121–1132. https://doi.org/10.1016/j.ijbiomac.2016.09.043.

    Article  CAS  PubMed  Google Scholar 

  41. Bhattacharya, A., & Pletschke, B. I. (2014). Magnetic cross-linked enzyme aggregates (CLEAs): a novel concept towards carrier free immobilization of lignocellulolytic enzymes. Enzyme and Microbial Technology, 61–62, 17–27. https://doi.org/10.1016/j.enzmictec.2014.04.009.

    Article  CAS  PubMed  Google Scholar 

  42. Jia, L., Budinova, G. A. L. G., Takasugi, Y., Noda, S., Tanaka, T., Ichinose, H., Goto, M., & Kamiya, N. (2016). Synergistic degradation of arabinoxylan by free and immobilized xylanases and arabinofuranosidase. Biochemical Engineering Journal, 114, 268–275. https://doi.org/10.1016/j.bej.2016.07.013.

    Article  CAS  Google Scholar 

  43. Waifalkar, P. P., Parit, S. B., Chougale, A. D., Sahoo, S. C., Patil, P. S., & Patil, P. B. (2016). Immobilization of invertase on chitosan coated γ-Fe2O3 magnetic nanoparticles to facilitate magnetic separation. Journal of Colloid and Interface Science, 482, 159–164. https://doi.org/10.1016/j.jcis.2016.07.082.

    Article  CAS  PubMed  Google Scholar 

  44. Li, L., Li, G., Cao, L. C., Ren, G. H., Kong, W., Di Wang, S., et al. (2015). Characterization of the cross-linked enzyme aggregates of a novel β-galactosidase, a potential catalyst for the synthesis of galacto-oligosaccharides. Journal of Agricultural and Food Chemistry, 63(3), 894–901. https://doi.org/10.1021/jf504473k.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by the Consejo Nacional de Ciencia y Tecnología, Mexico [CB-2014-241208].

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Correspondence to Aldo Amaro-Reyes.

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Gracida, J., Arredondo-Ochoa, T., García-Almendárez, B.E. et al. Improved Thermal and Reusability Properties of Xylanase by Genipin Cross-Linking to Magnetic Chitosan Particles. Appl Biochem Biotechnol 188, 395–409 (2019). https://doi.org/10.1007/s12010-018-2928-7

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