Abstract

Tripsinin (TRP) amino grubu ile fonksiyonelleştirilmiş silika kaplı TiO2 nanopartiküller (ASTiNP'ler) üzerine farklı çapraz bağlayıcılar (1,4-fenilen diizotiyosiyanat (PDC), 1,3-fenilen diizotiyosiyanat (MDC), glutaraldehit (GA) ile ya da çapraz bağlayıcılar olmadan immobilizasyonu çalışılmıştır. ASTiNP'ler ve çapraz bağlayıcılarla modifiye edilmiş ASTiNP'ler FTIR ve TEM ile karakterize edilmiştir. ASTiNP'ler üzerine immobilize edilen TRP’nin spesifik aktivitesi incelendiğinde, GA ile bağlı TRP’nin daha yüksek spesifik aktivite göstermiştir. Ancak PDC çapraz bağlayıcı olarak kullanıldığında immobilizasyon verimi (%) ve yükleme kapasitesi diğer çapraz bağlayıcılardan daha yüksektir. TRP immobilizasyonu için çapraz bağlayıcıların optimum konsantrasyonu 20,8 µM PDC, 5,2 µM MDC ve %1,5 v/v GA olarak belirlenmiştir. Çağraz bağlayıcı olmadan immobilize edilen TRP, dört tekrar kullanım sonucunda başlangıç aktivitesinin %5'ini gösterirken GA ile immobilize edilen TRP, 7 tekrar kullanım sonucunda ilk aktivitesinin % 7'sini, ve MDC ve PDC bağlı TRP 10 tekrar kullanım sonucunda başlangıç aktivitesinin sırasıyla % 7'sini ve % 11'ini sürdürmüştür. Cyt C'nin immobilize TRP ile parçalanması, LC-MS/MS analizi ile değerlendirilmiştir. Çapraz bağlayıcı ile ASTiNP'ler üzerine immobilize edilen TRP, çapraz bağlayıcı olmadan ASTiNP’ler üzerine immobilize edilen TRP ile karşılaştırıldığında daha yüksek Cyt C parçalama verimliliği göstermiştir. Sonuç olarak, PDC ile ASTiNP'ler üzerine immobilize edilen TRP; Cyt C parçalama verimliliği, immobilizasyon verimi, yükleme kapasitesi, katalitik aktivite ve tekrar kullanılabilirlik açısından daha iyi sonuçlar vermiştir.

Keywords

• tripsin
• immobilizasyon
• silika kaplama
• çapraz bağlayıcı
• amin fonksiyonelleştirmesi

Immobilization and Characterization of Trypsin on TiO2 Nanoparticles Activated with Crosslinkers

Abstract

The immobilization of trypsin (TRP) on the amine-functionalized silica coated TiO2 nanoparticles (ASTiNPs) with/without different crosslinkers (1,4-phenylene diisothiocyanate (PDC), 1,3-phenylene diisothiocyanate (MDC), glutaraldehyde (GA) has been studied. The ASTiNPs and modified ASTiNPs with the crosslinkers were characterized by FTIR and TEM. When considered the specific activity of immobilized TRP on ASTiNPs, GA-bound TRP showed the higher specific activity. Loading capacity was higher when PDC used as crosslinker. Optimum concentration of the crosslinkers for the TRP immobilization was determined as 20.8 µM of PDC, 5.2 µM of MDC and 1.5 v/v of GA. The direct-bound TRP showed 5% of its initial activity after the four cycles while the GA-bound TRP sustained 7% of its initial activity after the seven cycles and MDC and PDC-bound TRP sustained 7% and 11% of its initial activity after the ten cycles, respectively. The digestion of the Cyt C with immobilized TRP was evaluated by LC-MS/MS analysis. The immobilized TRP on ASTiNPs with crosslinkers showed the higher digestion efficiency of Cyt C when compared to the immobilized TRP on ASTiNPs without crosslinker. Consequently, the PDC-bound TRP on the ASTiNPs gave the better result of digestion efficiency, loading capacity, catalytic activity and reusability than the others.

Keywords

• Trypsin
• immobilization
• crosslinker
• silica coating
• amine functionalization
• TiO2

References

1. L. Li, H. Li, B. Yan, S. Yu, "Preparation of a reversible soluble-insoluble beta-d-Glucosidase with perfect stability and activity." J Biotechnol, 291, pp. 46-51, 2019.
2. C. geor malar, M. Seenuvasan, K. S. Kumar, A. Kumar, R. Parthiban, "Review on surface modification of nanocarriers to overcome diffusion limitations: An enzyme immobilization aspect." Biochemical Engineering Journal, 158, pp. 107574, 2020.
3. J. Andre, D. Saleh, C. Syldatk, R. Hausmann, "Effect of spacer modification on enzymatic synthetic and hydrolytic activities of immobilized trypsin." Journal of Molecular Catalysis B: Enzymatic, 125, pp. 88-96, 2016.
4. M. G. Miljkovic, V. Lazic, K. Banjanac, S. Z. Davidovic, D. I. Bezbradica, A. D. Marinkovic, D. Sredojevic, J. M. Nedeljkovic, S. I. Dimitrijevic Brankovic, "Immobilization of dextransucrase on functionalized TiO2 supports." Int J Biol Macromol, 114, pp. 1216-1223, 2018.
5. L. Wu, S. Wu, Z. Xu, Y. Qiu, S. Li, H. Xu, "Modified nanoporous titanium dioxide as a novel carrier for enzyme immobilization." Biosensors and Bioelectronic, 80, pp. 59-66, 2016.
6. S. Şahin, "Optimization of the Immobilization Conditions of Horseradish Peroxidase on TiO2COOH nanoparticles by Box-Behnken Design." Süleyman Demirel Üniversitesi Fen Bilimleri Enstitüsü Dergisi, 10.19113/sdufenbed.557021, pp. 904-916, 2019.
7. J. N.-M. Calvo, M. Elices, G. V. Guinea, J. Pérez-Rigueiro, M. Arroyo-Hernández, "Stability and activity of lactate dehydrogenase on biofunctional layers deposited by activated vapor silanization (AVS) and immersion silanization (IS)." Applied Surface Science, 416, pp. 965-970, 2017.
8. C. Victor Dos Santos Junior, M. S. Sader, G. C. Goncalves, G. Weissmuller, R. A. Simao, "Effect of pH on the adsorption and interactions of Bovine Serum Albumin with functionalized silicon nitride surface." Colloids Surf B Biointerfaces, 167, pp. 441-447, 2018.

9. H. Q. Wang, Z. Yao, Y. Sun, Z. Zhou, Q. Xiong, Z. X. Zhong, "Immobilization of γ-glutamyltranspeptidase on silylated mesoporous TiO2 whiskers." Biotechnology and Bioprocess Engineering, 19, pp. 304-310, 2014.
10. A. H. A. Al-Dhrub, S. Sahin, I. Ozmen, E. Tunca, M. Bulbul, "Immobilization and characterization of human carbonic anhydrase I on amine functionalized magnetic nanoparticles." Process Biochemistry, 57, pp. 95-104, 2017.
11. E. Aslani, A. Abri, M. Pazhang, "Immobilization of trypsin onto Fe3O4@SiO2 -NH2 and study of its activity and stability." Colloids Surf B Biointerfaces, 170, pp. 553-562, 2018.
12. N. Aissaoui, L. Bergaoui, S. Boujday, J. F. Lambert, C. Methivier, J. Landoulsi, "Enzyme immobilization on silane-modified surface through short linkers: fate of interfacial phases and impact on catalytic activity." Langmuir, 30, pp. 4066-4077, 2014.
13. O. Makrygenni, D. Brouri, A. Proust, F. Launay, R. Villanneau, "Immobilization of polyoxometalate hybrid catalysts onto mesoporous silica supports using phenylene diisothiocyanate as a cross-linking agent." Microporous and Mesoporous Materials, 278, pp. 314-321, 2019.
14. N. Aissaoui, J. Landoulsi, L. Bergaoui, S. Boujday, J. F. Lambert, "Catalytic activity and thermostability of enzymes immobilized on silanized surface: influence of the crosslinking agent." Enzyme Microb Technol, 52, pp. 336-343, 2013.
15. Bergmeyer H.U., Gawehn K., Grassi M. (1974) Methods of enzymatic analysis., 2nd ed., New York.
16. Y. Cao, L. Wen, F. Svec, T. Tan, Y. Lv, "Magnetic AuNP@Fe 3 O 4 nanoparticles as reusable carriers for reversible enzyme immobilization." Chemical Engineering Journal, 286, pp. 272-281, 2016.
17. C. Xia, H. Wang, F. Jiao, F. Gao, Q. Wu, Y. Shen, Y. Zhang, X. Qian, "Rational synthesis of MoS2-based immobilized trypsin for rapid and effective protein digestion." Talanta, 179, pp. 393-400, 2018.
18. P. M. Kumar, S. Badrinarayanan, M. Sastry, "Nanocrystalline TiO2 studied by optical, FTIR and X-ray photoelectron spectroscopy: correlation to presence of surface states." Thin Solid Films 358, pp. 122-130, 2000.
19. N. Majoul, S. Aouida, B. Bessaïs, "Progress of porous silicon APTES-functionalization by FTIR investigations." Applied Surface Science, 331, pp. 388-391, 2015.
20. Lex A., Pacher P., Werzer O., Track A., Shen Q., Schennach R., Koller G., Hlawacek G., Zojer E., Resel R., Ramsey M., Teichert C., Kern W., Trimmel G., "Synthesis of a Photosensitive Thiocyanate-Functionalized Trialkoxysilane and Its Application in Patterned Surface Modifications." Chemistry of Materials, 20, pp. 2009-2015, 2008.
21. G. Martinez-Edo, M. C. Llinas, S. Borros, D. Sanchez-Garcia, "Isothiocyanate-Functionalized Mesoporous Silica Nanoparticles as Building Blocks for the Design of Nanovehicles with Optimized Drug Release Profile." Nanomaterials (Basel), 9, pp., 2019.
22. M. Z. Anwar, D. J. Kim, A. Kumar, S. K. S. Patel, S. Otari, P. Mardina, J. H. Jeong, J. H. Sohn, J. H. Kim, J. T. Park, J. K. Lee, "SnO2 hollow nanotubes: a novel and efficient support matrix for enzyme immobilization." Sci Rep, 7, pp. 15333, 2017.
23. P. Saengdee, W. Chaisriratanakul, W. Bunjongpru, W. Sripumkhai, A. Srisuwan, W. Jeamsaksiri, C. Hruanun, A. Poyai, C. Promptmas, "Surface modification of silicon dioxide, silicon nitride and titanium oxynitride for lactate dehydrogenase immobilization." Biosens Bioelectron, 67, pp. 134-138, 2015.
24. D. Liu, A. M. Pourrahimi, L. K. H. Pallon, R. L. Andersson, M. S. Hedenqvist, U. W. Gedde, R. T. Olsson, "Morphology and properties of silica-based coatings with different functionalities for Fe3O4, ZnO and Al2O3 nanoparticles." RSC Advances, 5, pp. 48094-48103, 2015.
25. C. Daglioglu, F. Zihnioglu, "Covalent immobilization of trypsin on glutaraldehyde-activated silica for protein fragmentation." Artif Cells Blood Substit Immobil Biotechnol, 40, pp. 378-384, 2012.
26. D. Yang, X. Wang, Q. Ai, J. Shi, Z. Jiang, "Performance comparison of immobilized enzyme on the titanate nanotube surfaces modified by poly(dopamine) and poly(norepinephrine)." RSC Advances, 5, pp. 42461-42467, 2015.
27. B. Niu, B. Li, H. Wang, R. Guo, H. Liang, M. Qiao, W. Li, "Preparing bioactive surface of polystyrene with hydrophobin for trypsin immobilization." Materials Research Express, 3, pp. 055402, 2016.
28. S. Sahin, I. Ozmen, "Determination of optimum conditions for glucose-6-phosphate dehydrogenase immobilization on chitosan-coated magnetic nanoparticles and its characterization." Journal of Molecular Catalysis B: Enzymatic, 133, pp. S25-S33, 2016.
29. B. Tural, T. Tarhan, S. Tural, "Covalent immobilization of benzoylformate decarboxylase from Pseudomonas putida on magnetic epoxy support and its carboligation reactivity." Journal of Molecular Catalysis B: Enzymatic, 102, pp. 188-194, 2014.
30. C. Ji, L. N. Nguyen, J. Hou, F. I. Hai, V. Chen, "Direct immobilization of laccase on titania nanoparticles from crude enzyme extracts of P. ostreatus culture for micro-pollutant degradation.", 178, pp. 215-223, 2017.
31. Z. Gao, I. Zharov, "Large Pore Mesoporous Silica Nanoparticles by Templating with a Nonsurfactant Molecule, Tannic Acid." Chemistry of Materials, 26, pp. 2030-2037, 2014.
32. Q. Wang, L. Peng, G. Li, P. Zhang, D. Li, F. Huang, Q. Wei, "Activity of laccase immobilized on TiO2-montmorillonite complexes." Int J Mol Sci, 14, pp. 12520-12532, 2013.
33. J. Jia, W. Zhang, Z. Yang, X. Yang, N. Wang, X. Yu, "Novel Magnetic Cross-Linked Cellulase Aggregates with a Potential Application in Lignocellulosic Biomass Bioconversion." Molecules, 22, pp., 2017.
34. D. Yang, X. Wang, J. Shi, X. Wang, S. Zhang, P. Han, Z. Jiang, "In situ synthesized rGO–Fe3O4 nanocomposites as enzyme immobilization support for achieving high activity recovery and easy recycling." Biochemical Engineering Journal, 105, pp. 273-280, 2016.
35. K. Atacan, A. N. Kursunlu, M. Ozmen, "Preparation of pillar[5]arene immobilized trypsin and its application in microwave-assisted digestion of Cytochrome c." Mater Sci Eng C Mater Biol Appl, 94, pp. 886-893, 2019.
36. R. Nicoli, N. Gaud, C. Stella, S. Rudaz, J. L. Veuthey, "Trypsin immobilization on three monolithic disks for on-line protein digestion." J Pharm Biomed Anal, 48, pp. 398-407, 2008.
37. C. Temporini, E. Perani, F. Mancini, M. Bartolini, E. Calleri, D. Lubda, G. Felix, V. Andrisano, G. Massolini, "Optimization of a trypsin-bioreactor coupled with high-performance liquid chromatography-electrospray ionization tandem mass spectrometry for quality control of biotechnological drugs." J Chromatogr A, 1120, pp. 121-131, 2006.
38. C. Rocha, M. P. Gonçalves, J. A. Teixeira, "Immobilization of trypsin on spent grains for whey protein hydrolysis." Process Biochemistry, 46, pp. 505-511, 2011.
39. P. Zucca, E. Sanjust, "Inorganic materials as supports for covalent enzyme immobilization: methods and mechanisms." Molecules, 19, pp. 14139-14194, 2014.
40. A. B. Jarzębski, K. Szymańska, J. Bryjak, J. Mrowiec-Białoń, "Covalent immobilization of trypsin on to siliceous mesostructured cellular foams to obtain effective biocatalysts." Catalysis Today, 124, pp. 2-10, 2007.
41. R. Abdulla, S. A. Sanny, E. Derman, "Stability studies of immobilized lipase on rice husk and eggshell membrane." IOP Conference Series: Materials Science and Engineering, 206, pp. 012032, 2017.
42. S. Zhang, Q. Deng, Y. Li, M. Zheng, C. Wan, C. Zheng, H. Tang, F. Huang, J. Shi, "Novel amphiphilic polyvinylpyrrolidone functionalized silicone particles as carrier for low-cost lipase immobilization." R Soc Open Sci, 5, pp. 172368, 2018.
43. Y. A. Duman, N. Tekin, "Kinetic and thermodynamic properties of purified alkaline protease from Bacillus pumilus Y7 and non‐covalent immobilization to poly(vinylimidazole)/clay hydrogel." Engineering in Life Sciences, 20, pp. 36-49, 2019.
44. J. A. Torres, M. C. Silva, J. H. Lopes, A. E. Nogueira, F. G. E. Nogueira, A. D. Correa, "Development of a reusable and sustainable biocatalyst by immobilization of soybean peroxidase onto magnetic adsorbent." Int J Biol Macromol, 114, pp. 1279-1287, 2018.
45. L. J. Li, W. J. Xia, G. P. Ma, Y. L. Chen, Y. Y. Ma, "A study on the enzymatic properties and reuse of cellulase immobilized with carbon nanotubes and sodium alginate." AMB Express, 9, pp. 112, 2019.
46. C. Bonzom, L. Schild, H. Gustafsson, L. Olsson, "Feruloyl esterase immobilization in mesoporous silica particles and characterization in hydrolysis and transesterification." BMC Biochem, 19, pp. 1, 2018.