Immobilization of β-glucuronidase in lysozyme-induced biosilica particles to improve its stability

Abstract

Mesoporous silica particles were prepared for efficient immobilization of the β-glucuronidase (GUS) through a biomimetic mineralization process, in which the solution containing lysozyme and GUS were added into the prehydrolyzed tetraethoxysilane (TEOS) solution. The silica particles were formed in a way of biomineralization under the catalysis of lysozyme and GUS was immobilized into the silica particles simultaneously during the precipitation process. The average diameter of the silica particles is about 200 nm with a pore size of about 4 nm. All the enzyme molecules are tightly entrapped inside the biosilica nanoparticles without any leaching even under a high ionic strength condition. The immobilized GUS exhibits significantly higher thermal and pH stability as well as the storage and recycling stability compared with GUS in free form. No loss in the enzyme activity of the immobilized GUS was found after 30-day’s storage, and the initial activity could be well retained after 12 repeated cycles.

Keywords

silica nanoparticles / biocatalysis / biomimetic synthesis / β-glucuronidase encapsulation / storage and recycling stability

References

[1] Lee C H, Lin T S, Mou C Y. Mesoporous materials for encapsulating enzymes. Nano Today, 2009, 4(2): 165–179

[2] Schmid A, Dordick J S, Hauer B, Kiener A, Wubbolts M, Witholt B. Industrial biocatalysis today and tomorrow. Nature, 2001, 409(6817): 258–268

[3] Bornscheuer U T. Immobilizing enzymes: How to create more suitable biocatalysts. Angewandte Chemie International Edition, 2003, 42(29): 3336–3337

[4] Eggers D K, Valentine J S. Molecular confinement influences protein structure and enhances thermal protein stability. Protein Science, 2008, 10(2): 250–261

[5] Kim J, Grate J W, Wang P. Nanostructures for enzyme stabilization. Chemical Engineering Science, 2006, 61(3): 1017–1026

[6] Pioselli B, Bettati S, Mozzarelli A. Confinement and crowding effects on tryptophan synthase α₂β₂ complex. FEBS Letters, 2005, 579(10): 2197–2202

[7] Reátegui E, Aksan A.Structural changes in confined lysozyme. Journal of biomechanical engineering, 2009, 131(7): 074520.1–074520.4

[8] Zhou H X. Protein folding in confined and crowded environments. Archives of Biochemistry and Biophysics, 2008, 469(1): 76–82

[9] Zhou H X. Protein folding and binding in confined spaces and in crowded solutions. Journal of Molecular Recognition, 2004, 17(5): 368–375

[10] Zhou H X, Dill K A. Stabilization of proteins in confined spaces. Biochemistry, 2001, 40(38): 11289–11293

[11] Avnir D, Coradin T, Lev O, Livage J. Recent bio-applications of sol-gel materials. Journal of Materials Chemistry, 2006, 16(11): 1013–1030

[12] Kim Y H, Lee I, Choi S H, Lee O K, Shim J, Lee J, Kim J, Lee E Y. Enhanced stability and reusability of marine epoxide hydrolase using ship-in-a-bottle approach with magnetically-separable mesoporous silica. Journal of Molecular Catalysis. B, Enzymatic, 2013, 89: 48–51

[13] Pastor I, Ferrer M L, Lillo M P, Gómez J, Mateo C R. Structure and dynamics of lysozyme encapsulated in a silica sol-gel matrix. Journal of Physical Chemistry B, 2007, 111(39): 11603–11610

[14] Khanna S, Goyal A, Moholkar V S. Mechanistic investigation of ultrasonic enhancement of glycerol bioconversion by immobilized clostridium pasteurianum on silica support. Biotechnology and Bioengineering, 2013, 110(6): 1637–1645

[15] Luckarift H R, Spain J C, Naik R R, Stone M O. Enzyme immobilization in a biomimetic silica support. Nature Biotechnology, 2004, 22(2): 211–213

[16] Pouget E, Dujardin E, Cavalier A, Moreac A, Valéry C, Marchi-Artzner V, Weiss T, Renault A, Paternostre M, Artzner F. Hierarchical architectures by synergy between dynamical template self-assembly and biomineralization. Nature Materials, 2007, 6(6): 434–439

[17] Rusu V M, Ng C H, Wilke M, Tiersch B, Fratzl P, Peter M G. Size-controlled hydroxyapatite nanoparticles as self-organized organic–inorganic composite materials. Biomaterials, 2005, 26(26): 5414–5426

[18] Zhang Y F, Wu H, Li L, Li J, Jiang Z Y, Jiang Y J, Chen Y. Enzymatic conversion of baicalin into baicalein by β-glucuronidase encapsulated in biomimetic core-shell structured hybrid capsules. Journal of Molecular Catalysis. B, Enzymatic, 2009, 57(1-4): 130–135

[19] Naik R R, Tomczak M M, Luckarift H R, Spain J C, Stone M O. Entrapment of enzymes and nanoparticles using biomimetically synthesized silica. Chemical Communications, 2004, (15): 1684–1685

[20] Miller S A, Hong E D, Wright D. Rapid and efficient enzyme encapsulation in a dendrimer silica nanocomposite. Macromolecular Bioscience, 2006, 6(10): 839–845

[21] Zhang Y F, Wu H, Li J, Li L, Jiang Y J, Jiang Z Y, Jiang Z. Protamine-templated biomimetic hybrid capsules: Efficient and stable carrier for enzyme encapsulation. Chemistry of Materials, 2008, 20(3): 1041–1048

[22] Naik R R, Brott L L, Clarson S J, Stone M O. Silica-precipitating peptides isolated from a combinatorial phage display peptide library. Journal of Nanoscience and Nanotechnology, 2002, 2(1): 95–100

[23] Kroger N, Deutzmann R, Sumper M. Silica-precipitating peptides from diatoms. Journal of Biological Chemistry, 2001, 276(28): 26066–26070

[24] Luckarift H R, Dickerson M B, Sandhage K H, Spain J C. Rapid, room-temperature synthesis of antibacterial bionanocomposites of lysozyme with amorphous silica or titania. Small, 2006, 2(5): 640–643

[25] Coradin T, Coupé A, Livage J. Interactions of bovine serum albumin and lysozyme with sodium sil<?Pub Caret?>icate solutions. Colloids and Surfaces. B, Biointerfaces, 2003, 29(2-3): 189–196

[26] Shiomi T, Tsunoda T, Kawai A, Mizukami F, Sakaguchi K. Synthesis of a cagelike hollow aluminosilicate with vermiculate micro-through-holes and its application to ship-in-bottle encapsulation of protein. Small, 2009, 5(1): 67–71

[27] Ramanathan M, Luckarift H R, Sarsenova A, Wild J R, Ramanculov E K, Olsen E V, Simonian A L. Lysozyme-mediated formation of protein-silica nano-composites for biosensing applications. Colloids and Surfaces. B, Biointerfaces, 2009, 73(1): 58–64

[28] Garakani T M, Wang H H, Krappitz T, Liebeck B M, Vanrijn P, Boker A. Lysozyme-silica hybrid materials: From nanoparticles to capsules and double emulsion mineral capsules. Chemical Communications, 2012, 48(82): 10210–10212

[29] Ivnitski D, Artyushkova K, Rincon R A, Atanassov P, Luckarift H R, Johnson G R. Entrapment of enzymes and carbon nanotubes in biologically synthesized silica: Glucose oxidase-catalyzed direct electron transfer. Small, 2008, 4(3): 357–364

[30] Luckarift H R, Balasubramanian S, Paliwal S, Johnson G R, Simonian A L. Enzyme-encapsulated silica monolayers for rapid functionalization of a gold surface. Colloids and Surfaces. B, Biointerfaces, 2007, 58(1): 28–33

[31] Cao X D, Yu J C, Zhang Z Q, Liu S Q. Bioactivity of horseradish peroxidase entrapped in silica nanospheres. Biosensors & Bioelectronics, 2012, 35(1): 101–107

[32] Cushnie T, Lamb A J. Antimicrobial activity of flavonoids. International Journal of Antimicrobial Agents, 2005, 26(5): 343–356

[33] Ma Z, Otsuyama K i, Liu S, Abroun S, Ishikawa H, Tsuyama N, Obata M, Li F J, Zheng X, Maki Y. Baicalein, a component of scutellaria radix from Huang-Lian-Jie-Du-Tang (HLJDT), leads to suppression of proliferation and induction of apoptosis in human myeloma cells. Blood, 2005, 105(8): 3312–3318

[34] Zhu J T, Choi R C, Chu G K, Cheung A W, Gao Q T, Li J, Jiang Z Y, Dong T T, Tsim K W. Flavonoids possess neuroprotective effects on cultured pheochromocytoma PC12 cells: A comparison of different flavonoids in activating estrogenic effect and in preventing β-amyloid-induced cell death. Journal of Agricultural and Food Chemistry, 2007, 55(6): 2438–2445

[35] Matte C R, Nunes M R, Benvenutti E V, Schöffer J N, Ayub M A Z, Hertz P F. Schöffer J d N, Ayub M A Z, Hertz P F. Characterization of cyclodextrin glycosyltransferase immobilized on silica microspheres via aminopropyltrimethoxysilane as a “spacer arm”. Journal of Molecular Catalysis. B, Enzymatic, 2012, 78: 51–56

[36] Martín M T, Plou F J, Alcalde M, Ballesteros A. Immobilization on Eupergit C of cyclodextrin glucosyltransferase (CGTase) and properties of the immobilized biocatalyst. Journal of Molecular Catalysis. B, Enzymatic, 2003, 21(4-6): 299–308

[37] Miller S A, Hong E D, Wright D. Rapid and efficient enzyme encapsulation in a dendrimer silica nanocomposite. Macromolecular Bioscience, 2006, 6(10): 839–845

Acknowledgments

The authors thank the financial support from the Specialized Research Fund for the Doctoral Program of Higher Education (20130032110023), National Science Foundation of China (21076145), the Program for New Century Excellent Talents in University (NCET-10-0623), the National Basic Research Program of China (2009CB724705), National Science Fund for Distinguished Young Scholars (21125627).

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2014 Higher Education Press and Springer-Verlag Berlin Heidelberg