Hittite Journal of Science and Engineering, 2018, 5 (2) 115-118 ISSN NUMBER: 2148-4171 DOI: 10.17350/HJSE19030000081

Enhanced Enzyme Activity with Ferritin Nanocages

Eylem Turan Gazi University, Department of Chemistry, Besevler, Ankara, Turkey.

Article History: ABSTRACT Received: 2017/09/26 Accepted: 2017/11/27 Online: 2017/12/22 n this study, a novel enzyme system based on ferritin nanocages was designed. α I -Amylase was covalently crosslinked on surface of ferritin nanocages by EDC/NHS. Correspondence to: Eylem Turan The activity of immobilized enzyme was monitored by using UV-vis spectrophotometer. Gazi University, Department of Chemistry, Besevler, Ankara, Turkey o o The optimum temperature of immobilized enzyme shifted from 50 C to 70 C due to the Tel: +90 (312) 202-1145 ferritin nanocages. In addition, The Km and Vmax values of immobilized enzymes were E-Mail: [email protected] 5.19 mg mL-1 and 3.3x10-5 U mg-1, respectively. This novel enzyme system displayed higher catalytic activity and enhanced stability.

Keywords: Ferritin nanocages; α-amylase; Enzyme immobilization; EDC/NHS; Covalently crosslinked

INTRODUCTION

n recent years, the combined use of chemistry, biomedical applications have a promising potential biology and nanotechnology has made significant for sensitive and robust bioassays. Based on these Icontributions to the field of medical diagnosis properties, ferritin is not only due to its inherent nano- and treatment. For example, quantum dots or size and nanostructure, but also by its high stability fluorochrome-linked macromolecules have been and specific structures [3-9]. In addition to these developed as cell and tissue imaging materials. Iron superior properties, nanotecnology researchers have oxide nanoparticles have also been developed for use found that nanosized biomaterials such as ferritin, as biomedical applications, such as hyperthermia, apoferritin in biomedical field, such as diagnosis and cancer diagnosis and therapy, and as a contrast agent treatment of diseases because they are biodegradable, in magnetic resonance imaging (MRI). However, biocompatible and non-toxic properties [10-12]. these nanostructures, which have been subjected so much research on them, they have toxic effects. In this study, it is aimed to developed ferritin For this reason, the search for biocompatible nanocage-based platform to construct an enzyme nanomaterials in diagnosis and treatment of diseases system. To prepare this system, α-amylase enzyme was has become intense. Protein lattices such as ferritin covalently crosslinked on the outer surface of the ferritin and virus capsules (protein layer outside the virus) nanocages. The activity of this novel enzyme system was are versatile nanoscale perfect architectures that investigated for different temperatures and substrate allow both genetic and chemical modification. concentrations using UV-vis spectrophotometer. Nanoscale protein architects with multiple functionalities have a good potential, especially EXPERIMENTAL in medical imaging and treatment fields. Protein Materials lattice structures have different subunits that can All chemicals were used for the preparation enz- be used as the interface between the inner and outer yme immobilization ferritin nanocages. Ferritin na- and subunits. The subunits can be easily modified nocages Type I: From Horse Spleen and α-amylase genetically and chemically to demonstrate versatile (E.C.3.2.1.1, 47 kDa) were obtained from Sigma. functionality. Ferritin is a very large iron storage and detoxification protein found in many organisms [1,2]. Enzyme immobilization In nature, ferritins are filled with iron. Ferritin is a To immobilize α-amylase onto the ferritin nanocages, natural nanocage and also ready to be combined with firstly, ferritin nanocages were suspended in biomolecules for preparing nanobiosensors. Thus, phosphate buffer solution ( pH 7.4) by ultrasonic bath E. Turan / Hittite J Sci Eng, 2018, 5 (2) 115-118 Where [enzyme] Where TEM investigation was performed using a JEOL 100CX 100CX aJEOL using performed was investigation TEM of activity The [enzyme]initial) Characterization Immobilization experiments RESULTS AND DISCUSSION AND RESULTS Firstly, Eq.1: determined by a Shimadzu byaShimadzu determined during 45 min. Then, α Then, min. 45 during concentration of the enzyme. enzyme. the of concentration concentration of substrate was determined via UV-vis UV-vis via determined was of substrate concentration Preparation of novel enzyme system of novel enzyme Preparation Figure 1. with constant shaking. The enzyme immobilized ferritin ferritin immobilized enzyme The shaking. constant with (1:1, buffer phosphate EDC:NHS in w:w) and with nanocages were washed with the buffer solution. Finally Finally solution. buffer the with were washed nanocages nanocages were kept at +4 °C until further use. use. further were until kept °C at +4 nanocages redispersed in PBS (pH 7.4) enzyme immobilized ferritin ferritin PBS (pH 7.4) in immobilized enzyme redispersed (pH 7.4). solution buffer phosphate in After re-dispersed at room temperature. room at spectrophotometer at 560 nm. at 560 spectrophotometer suspansion, and stirred at room temperature for 4h temperature at room stirred and suspansion, nanocage ferritin the to added and min, for 30 solution the reduction of starch in different conditions. The The conditions. different in of starch reduction the in the upper phase and [enzyme] and phase upper the in instrument working at 120 kV. at were 120 working absorbances UV-vis instrument Immobilization efficiency = 1-([enzyme]upper = phase/ efficiency Immobilization the byusing determined was efficiency Immobilization TEM images of a) ferritin nanocages b) enzyme immobilized ferritin nanocages ferritin immobilized b) enzyme nanocages a) of ferritin images TEM α -amylase enzyme was covalently crosslinked crosslinked covalently was enzyme -amylase

α -amylase was determined via examing examing via determined was -amylase supernatant supernatant -amylase was also preactivated preactivated also was -amylase is the enzyme concentration concentration enzyme the is UV-2450 spectrophotometer UV-2450 spectrophotometer

initial is the initial initial the is (1 ) 116 α of immobilization was determined to be 97.3%. This result result 97.3%. be to This determined was of immobilization enzyme was preactivated by EDC/NHS in solution before before solution in byEDC/NHS preactivated was enzyme diameter diameter diameters 1a. Fig. were summarized results characterization TEM The EDC/NHS. via nanocages ferritin onto the with a fixed concentration of enzyme solution. α solution. of enzyme concentration afixed with nanocages and carboxyl groups of enzyme. The efficiency efficiency The of enzyme. groups carboxyl and nanocages nanocages. The bare ferritin nanocages were uniform size size were uniform nanocages ferritin bare The nanocages. reacting a constant concentration of ferritin nanocages nanocages of ferritin concentration aconstant reacting activity ferritin nanocages by reacting between the amino groups of of groups amino the between by reacting nanocages ferritin and no exhibited agglomeration in Fig. 1. Their average average 1. Fig. Their in agglomeration no exhibited and the ferritin nanocages well dispersed with an average average an with dispersed well nanocages ferritin the indicated that, binding process was successful. was process binding that, indicated it was linked on the ferritin nanocage surfaces. Finally, Finally, surfaces. nanocage ferritin on the linked it was Figure 2. -amylase enzyme was immobilized on the surface of the of the surface on the immobilized was enzyme -amylase Thermal stability of immobilized α of immobilized stability Thermal Fig. 1a demonstrated the TEM images of bare ferritin ferritin of bare images TEM the 1aFig. demonstrated Enzyme immobilization was accomplished via via accomplished was immobilization Enzyme Effect of temperature on free and immobilized enzyme enzyme immobilized and free on temperature of Effect 17.3 13.7 ± 0.54 nm 13.7 ± 0.54 ± 0. was determined in Fig. 1b. Fig. in determined was nm 82 . With the enzyme immobilized, -amylase was -amylase -amylase Table1. Kinetic parameters of free and immobilized enzymes

Km Vmax mg mL-1 U mg-1 free enzyme 2.63 10.6x10-5 immobilized enzyme 5.19 3.3x10-5 compared with the free enzyme at different temperatures (20-90 oC). The results were showed in Fig 2. Free enzyme very stable and can be incubated at 50 oC. Optimum temperature of immobilized enzyme was changed from 50 oC to 70 oC. As a result, immobilized Figure 4. enzyme more resistant to heat than the free enzyme. It The reusability of immobilized enzyme can be probably ferritin nanocages were enhanced enzyme the results, the immobilization process and the ferritin activity at higher temperatures. Moreover, it can be the nanocages increased the storage stability and durability of hydration layer around the ferritin nanocages prevented the immobilized α-amylase. thermal denaturation of the enzyme. The reusability of immobilized enzyme is very Kinetic parameters of free and immobilized enzymes important feature for their large-scale applications. The were determined by using the Michaelis-Menten equation. reusability of immobilized enzyme was measured for 10 The results were summarized in Table 1. reuse in 1 day. (Fig.4)

The K values of free enzyme and immobilized m In Fig. 4, after 10 reuses immobilized enzyme protected enzymes were 2.63 and 5.19 mg mL-1, respectively. The about 93% of the initial activity. This result showed that V values of free enzyme and immobilized enzymes were max there was no significant loss after the 10 reuses. Therefore found to be 10.6x10-5 and 3.3x10-5 U mg-1, respectively. The the ferritin nanocages can be reused and they could be an Km value of immobilized enzyme higher then the free economic platform for enzyme immobilization. enzyme .It can be due to the changes in the conformation of the enzyme molecules after the immobilization and it may CONCLUSION be increased enzyme activity [13]. A novel enzyme system was developed by using α Generally, enzymes may lose their activities due to natural protein cages. Model enzyme -amylase environmental conditions. So that it is important their was covalently immobilized onto the nanocages and storage stability for industrial usage. In order to determine optimum immobilization conditions were determined. the storage stabilities of free and immobilized α-amylase The immobilized enzyme showed better temperature enzyme, which were kept in phosphate buffer solution (pH resistance than the free enzyme. In addition, storage 7.4) at +4 oC. Their activities were investigated in every 5 stability of immobilized enzyme was excellent when days during 30 days. (Fig. 3) compared the free enzyme. These results can be attributed to the ferritin nanocages. As a result, ferritin Free enzyme was lost its about all initial activity within nanocages are very important platforms for enzyme 15 days but immobilized enzyme preserved about 96 % of immobilization because of their enzyme mimic activity. the initial activity. After 30 days, the immobilized enzyme 8 retained about 84 % of the initial activity. According to ACKNOWLEDGEMENTS I am thankful to Prof. Dr. Zekiye Suludere for TEM analysis. I am also great thankful to Assoc. Prof. Dr. Hayrettin Tümtürk for his all support.

REFERENCES

1. Bou-Abdallah F, Zhao G, Biasiotto G, Poli M, Arosio P, Chasteen ND, Facilitated diffusion of iron(II) and dioxygen substrates into human H-chain ferritin. A flourrescence

and absorbance study employing the ferroxidase center E. Turan / Hittite J Sci Eng, 2018, 5 (2) 115-11 substitution Y34W. JACS, 130 (2008) 17801-17811. 2. Bulvik BE, Berenshtein E, Meyron-Holtz EG, Konijn Figure 3. Storage stability of free and immobilized enzyme activity

117 E. Turan / Hittite J Sci Eng, 2018, 5 (2) 115-118 4. 7. 3. 6. 5. 15 (2013), 1-9. (2013), 15 Tedesco AC, Oliviera DM, Lacava ZGM, Azevedo RB, Lima RB, Azevedo ZGM, Lacava DM, Oliviera AC, Tedesco Activity Y, Y, Lin Z, Li Zhang Tang Enzyme-Mimic H, Wu Z, Zhen ZP, Tang W, Guo CL, Ferritin nanocages to encapsulate to encapsulate nanocages ZP, Ferritin Zhen CL, Tang W, Guo AM, Chevion M, Cardiac protection by preconditioning preconditioning by protection Cardiac M, Chevion AM, AP, Pereira DIA, A nano-disperse ferritin-core mimetic ferritin-core A nano-disperse DIA, AP, Pereira Applications. Anal. Chem. 83 (2011) 83 8611-8616. Chem. Anal. Applications. degradation of iron proteins. PLoS ONE, 7(2012), 1-9. ONE, PLoS proteins. iron of degradation of Ferric Nano-Core Residing in Ferritin and Its Biosensing Its and Ferritin in Residing Nano-Core Ferric of and deliver photosensitizers for efficient photodynamic efficient for photosensitizers deliver and activity. Nanomedicine: Nanotechnology, Biology, and and stoichiometry of biocompatible cobalt ferrite-based sized monodisperse iron oxide nanoparticles synthesized by remineralization of apoferritin molecules. J. Nanopart. Res. J.Nanopart. molecules. apoferritin of remineralization magnetic fluids to serum albümin. Journal of Magnetism and and Magnetism of Journal albümin. to serum fluids magnetic therapy against cancer. ACS Nano, 7 (2013) 6988-6996. 7(2013) Nano, ACS cancer. against therapy that efficiently corrects anemia without luminal iron redox iron luminal without anemia corrects efficiently that Medicine 10 (2014) 1529-1538. Medicine Brown RJ, Simpson GO, TJ, Latunde-Dada Pennycook N, Hondow LK, SFA, Poots Faria N, Bruggraber JJ, Powell Magnetic Materials. 272–276 (2004) 2404–2405. 272–276 (2004) Materials. Magnetic constant binding the of Investigation PC, ECD, Morais is generated via an iron-signal creatd by proteasomal proteasomal by creatd iron-signal an via generated is Ullrich A, Horn S, Structural investigations on differently on investigations Structural S, Horn A, Ullrich 118 13. 12. 11. 10. 9. 8. TM, Nano/micro Technologies for delivering macromolecular Tümtürk H, Yüksekdağ H, Acetylcholinesterase immobilized Acetylcholinesterase H, Yüksekdağ H, Tümtürk Zhao J, Liu ML, Zhang YY, Li HT, Lin YH, Yao SZ, Apoferritin Yao Apoferritin YY, SZ, HT, Li YH, Lin Zhang ML, Liu J, Zhao detection. Anal. Chim. Acta 759 (2013) 53-60. (2013) 759 Acta Chim. Anal. detection. delivering. Pharmacol. Res., 62 (2010) 62 144-149. Res., Pharmacol. delivering. devices. J. Control. Release 70 (2001) 1-20. 70(2001) Release J.Control. devices. derivatives. J. Control. Release 125 (2008) 193-209. (2008) 125 Release J.Control. derivatives. 27 (2011) 9681-9686. onto PEI-coated silica nanoparticles. Artifi cial Cells, cial Artifi nanoparticles. silica PEI-coated onto Chomoucka J, Drbohlavova J, Huska D, Adam V, D, Adam Kizek Huska J, Drbohlavova J, Chomoucka Soppimath KS, Aminabhavi TM, Kulkarni AR, Rudzinski WE, Rudzinski AR, Kulkarni TM, Aminabhavi KS, Soppimath protein nanoparticles dually labeled with aptamer and aptamer with labeled dually nanoparticles protein therapeutics using poly(D,L-lacticide-co-glycolide)therapeutics its and R, Hubalek J, Magnetic nanoparticles and targeted drug targeted and nanoparticles Magnetic J, Hubalek R, Biodegradable polymeric nanoparticles as drug delivery drug as nanoparticles polymeric Biodegradable Mundargi RC, Babu VR, Rangaswamy V, Patel P, Aminabhavi V, P, Patel Aminabhavi Rangaswamy VR, Babu RC, Mundargi holoferritin of gap band Tuning R, T, Mukhopadhyay Rakshit Nanomedicine, and Biotechnology 44 (2016) 443-447. 44 Biotechnology and Nanomedicine, horseradish peroxidase as a sensing probe for thrombin by metal core reconstitution with Cu, Co and Mn. Langmuir Mn. and Co Cu, with reconstitution core metal by