Adsorption of microbial esterases on Bacillus subtilis-templated cobalt oxide nanoparticlesInternational Journal of Biological Macromolecules


Eunjin Jang, Bum Han Ryu, Hyun-Woo Shim, Hansol Ju, Dong-Wan Kim, T. Doohun Kim
Molecular Biology / Structural Biology / Biochemistry


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Alfonso Ciccone, Cristina Motto, Elisabetta Aritzu, Alessandra Piana, Livia Candelise, on behalf of the Group

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Krisztián Kordás, Géza Tóth, Juhani Levoska, Mika Huuhtanen, Riitta Keiski, Matti Härkönen, Thomas F George, Jouko Vähäkangas


International Journal of Biological Macromolecules 65 (2014) 188–192

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules j ourna l ho me pa g e: www.elsev ier .com

Adsorp ubt oxide n

Eunjin Ja a, D

T. Doohu a Department o b Department o a r t i c l

Article history:

Received 10 O

Received in re

Accepted 13 Ja

Available onlin


Cobalt-oxide n

Bacillus subtili

Hydrolase surfa lizati ate an featu catte ylum tures e im pres used candidates for the preparation of effective nanobiocatalysts. © 2014 Elsevier B.V. All rights reserved. 1. Introdu

The stab resents a h to its abilit formation,

Therefore, m of immobil large numb

Recently, n for the deli volume rati excellent m als includin have been a bio-inspir [7,8]. In th particles, D nanomateri tion of Baci their applic ∗ Correspon


E-mail add 0141-8130/$ – http://dx.doi.oction le immobilization of enzymes on solid supports repighly important and useful industrial technique due y to promote ease of separation, controlled product and adaptability to various engineering designs [1]. any studies have been conducted on the preparation ized enzymes, and these studies have investigated a er of solid supports and immobilization methods [2,3]. anomaterials have received great interest as platforms very of immobilized enzymes due to large surface-toos, strong adsorption ability, low cost of production, and echanical resistance [4,5]. To date, several nanomaterig carbon nanotubes, mesoporous silica, and iron oxide fabricated for enzyme immobilization [6]. In addition, ed method of nanomaterials synthesis was developed is process, biological macromolecules such as virus

NAs, or proteins were used as templates to develop als as green methods of synthesis. Here, the preparallus subtilis-templated cobalt oxide nanoparticles and ation as a support for the immobilization of microbial ding author at: Prof. Thomas Doohun Kim, Ajou University, San 5, g, Youngtong-gu, Suwon, 443749, Korea. Tel.: +1 617 916 5736. ress: (T.D. Kim). hydrolases is described. This method of synthesis produces rodshaped nanostructures, and is achieved on the bacterial surface, which allows the large-scale synthesis of nanostructures at room temperature [9]. Microbial carboxylesterases were chosen for immobilization because they are among the most widely used enzymes in the biotechnological industries involved in food, fine chemicals, and biomedical device production [10–12]. The morphological properties and size distributions of the nanostructures were analyzed by electron microscopy (EM) and dynamic light scattering (DLS). Efficient immobilization, operational stability, and economical reusability were explored for the use of Bacillus subtilis-templated cobalt oxide nanoparticles in industrial processes. 2. Materials and methods 2.1. Bacterial strains, cultures, and reagents

Bacillus subtilis cells were obtained from the Korean Collection for Type Culture (KCTC, Daejon, Korea). The cells were grown in a liquid medium of Luria-Bertani broth at 37 ◦C and 200 rpm in a shaking incubator. After 12 h, the bacterial cells were harvested at 5000 rpm for 20 min, and resuspended in distilled water. The final optical density at 600 nm (OD600) in distilled water was ∼1.6. All other chemicals were of reagent grade and used without further purification. see front matter © 2014 Elsevier B.V. All rights reserved. rg/10.1016/j.ijbiomac.2014.01.027tion of microbial esterases on Bacillus s anoparticles nga, Bum Han Ryua, Hyun-Woo Shimb, Hansol Ju n Kima,∗ f Applied Chemistry and Biological Engineering, Korea f Energy Systems Research and Department of Material Science and Engineering, Korea e i n f o ctober 2013 vised form 9 January 2014 nuary 2014 e 18 January 2014 anoparticles s a b s t r a c t

Due to low diffusion rates and large porting materials for enzyme immobi

Bacillus subtilis as a biological templ lization is described. Morphological microscopy (EM) and dynamic light s tures were investigated using 4-meth substrates. Enzyme-coated nanostruc successive reaction cycles, and enzym a loss of immobilization potential. The nanoparticles have the potential to be/ locate / i jb iomac ilis-templated cobalt ong-Wan Kimb, ce areas, nanomaterials have received great interest as supon. Here, the preparation of a cobalt oxide nanoparticle using d use of the nanostructure for microbial esterase immobires and size distributions were investigated using electron ring (DLS). Catalytic properties of enzyme-coated nanostrucbelliferyl acetate and p-nitrophenyl (PNP) acetate as model were observed to retain ∼85% of the initial activity after 15 mobilization processes could be repeated four times without ent work demonstrates that B. subtilis-templated cobalt oxide as biocompatible immobilization materials, and are promising

E. Jang et al. / International Journal of Biological Macromolecules 65 (2014) 188–192 189 2.2. Synthesis and preparation of enzyme-cobalt oxide nanoparticles

The cobalt oxide nanoparticles were synthesized as described previously,

Briefly, 50 m lus subtilis, solution wa vested at 5 washed sev removal of t 300 ◦C for 1 and 20 mM centrifuged (EfEstA, Est prepared ac (20 g) was room temp coated nano 20 mM Tris isoelectric p (http://www rithm. 2.3. Charac

All elect emission s

Zeiss, Germ high-resolu model Tec scattering sizer Nano


Fluoresc hydrolysis

UV-incubat bated for 2 50 mM sodi (250 M), a was determ standard a (p-nitrophe 8.0) and 0. ture was in released d a VersaMa

U.S.A.). 2.4. Multip

To inves ticles, nano reaction and (20 mM Tris for another cobalt oxid coated nano solution con step (∼10 mi from nanop ity. Then, fr enzyme act above. 3. Results and discussion 3.1. Syntheses and preparation of cobalt oxide nanoparticles ostr hrou ) oxid tem emp that asse of th prod nano ep sh dim . sorp en m ng N orpti stud eir ble 1 ions rans urfac