Nano-magnetic cross-linked enzyme aggregates of naringinase an efficient nanobiocatalyst for naringin hydrolysis Homa Torabizadeh ⁎, Mohaddeseh Mikani Iranian Research Organization for Science and Technology (IROST), Department of Chemical Technologies, Food Science and Technology Group, Mojtama Asre Enghelab Building, Shahid Ehsanirad Street, 33535111 Tehran, Iran abstract article info Article history: Received 23 April 2018 Received in revised form 15 May 2018 Accepted 22 May 2018 Available online 23 May 2018 In this research, the preparation and characterization of a novel biocatalyst comprising nano-magnetic cross- linked enzyme aggregates of naringinase (NM-NGase-CLEAs), which was covalently bounded to lysine-assisted magnetic nanoparticles, were studied. The Schiff base formed between ɛ-amino groups of the lysine residues and aldehyde groups of glutaraldehyde was reduced by ascorbic acid. Among the six different precipitants, tert-butanol performed the best for naringin hydrolysis. The optimal conditions for the immobilization process required 10 mM glutaraldehyde, 1:10 ratio of lysine/enzyme, and 3 h crosslinking at 3–4 °C. The morphology of the NM-NGase-CLEAs implied a non-uniform, semi-pyramid and semi-cubic rods. The dynamic light scattering (DLS) results showed that the nanomagnetite particle size was around 81.9–96.5 nm, with a polydispersity index (PDI) of 0.238. After NM-NGase-CLEAS formation, the particle size was reduced to around 13.2–15.3 nm, with PDI of 0.177, respectively. Moreover, the Ȥ-potential of -28 mV also confirms the improvement of CLEAs stability. The NM-NGase-CLEAs kept 73% of its original activity after 10 cycles, which proposes strong operational stability. In conclusion, the NM-NGase-CLEAs are thermo-stable, reusable, and efficient nanobiocatalyst for debittering of citrus juices. © 2018 Elsevier B.V. All rights reserved. Keywords: Naringinase immobilization MN-NGase-CLEAs Naringin hydrolysis 1. Introduction Naringinase is a hydrolytic multienzyme complex, which consists of α-L-rhamnosidase (EC 3.2.1.40) and β-D-glucosidase (EC 3.2.1.21) activities. Alpha-rhamnosidase catalyzes the hydrolysis of naringin (naringenin 7-rhamnoglucoside) into rhamnose and prunin (4,5,7-tri- hydroxy flavanone-7-glucoside); the prunin is altered to naringenin (4,5,7-trihydroxy flavanone) and glucose by the β-D-glucosidase activ- ity at the same time [1,2]. This enzyme activity attracts growing biotech- nological interest, owing to its role in debittering of citrus fruit juices [1]. Naringinase also finds applications in the fabrication of glycopeptide an- tibiotics, deglycosylation of flavonoids, and gellan depolymerisation [2]. In addition, its hydrolysis products (rhamnose, prunin and naringenin) display biological activities and can be employed as starting materials for the synthesis of substances used in pharmaceutics, food technology, and cosmetics [3]. In the food processing industry, the rhamnosidases have been employed primarily for debittering of citrus juices. The presence of bitterness is a major restriction in the commercial accep- tance of juices [4]. Both free and immobilized α-L-rhamnosidases from various microorganisms have been explored for naringin hydrolysis. Immobilized enzymes are preferred over free enzymes in large-scale applications, due to improved enzyme stability and reusability; further- more, loss of enzyme activity during the immobilization and reaction process, large mass transfer resistance between the enzyme and sub- strates, and the inconvenience of selecting the proper immobilization technique are the drawbacks of their use [5]. Recently, several studies have been performed on the progress of immobilization techniques and materials [6]. Jiang et al. immobilized successfully horseradish per- oxidase and α-amylase by cross-linked enzyme aggregates (CLEAs) method onto an inverse opal [7]. Gao et al. immobilized lipase by mono- disperse core-shell magnetic organosilica nanoflowers [8]. Jiang et al. prepared CLEAs of lipase in three-dimensionally ordered macroporous silica materials [9] also, Cruz-Izquierdo et al. immobilized lipase by mag- netic CLEAs method [10]. Moreover, Cui et al. prepared immobilized phenylalanine ammonia lyase via mesoporous CLEAs-silica composite microparticles for enhancing of enzyme activity and stability [11], as well as, cutinase was immobilized on amino-functionalized magnetic supports by CLEAs method for its use in bio-degradation of polycaprolactone by Singh [12]. Samoylova et al. fabricated esterase CLEAs for application in malathion removal from municipal wastewater [13]. Martínez-Moñino et al. prepared nicotinic acid mononucleotide deamidase CLEAs to obtain highly valuable NAD + -boosters [14]. Many strategies have been recently employed to immobilize naringinase. Nanotechnology is at an innovative stage of knowledge and technical development, with possible applications in numerous spheres such as International Journal of Biological Macromolecules 117 (2018) 134–143 ⁎ Corresponding author. E-mail address: htoraby@alumni.ut.ac.ir (H. Torabizadeh). https://doi.org/10.1016/j.ijbiomac.2018.05.162 0141-8130/© 2018 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac