Molecular rearrangement of glucans from natural starch to form size

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Biotechnology and Biological Transformations

Molecular rearrangement of glucans from natural starch to form size-controlled functional magnetic polymer beads Ke Luo, Ki-Baek Jeong, Sang-Mook You, Da-Hee Lee, and Young-Rok Kim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01590 • Publication Date (Web): 14 Jun 2018 Downloaded from http://pubs.acs.org on June 16, 2018

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Journal of Agricultural and Food Chemistry

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Molecular rearrangement of glucans from natural starch to form size-controlled functional magnetic polymer beads

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Ke Luo,† Ki-Baek Jeong,† Sang-Mook You, Da-Hee Lee, and Young-Rok Kim*

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Institute of Life Sciences and Resources & Department of Food Science and

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Biotechnology, College of Life Sciences, Kyung Hee University, Yongin, 17104

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South Korea

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* Corresponding author.

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Tel: +82-31-201-3830

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Fax: +82-31-204-8116

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E-mail address: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT: Herein, we report a fairly simple and environmentally friendly

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approach for the fabrication of starch-based magnetic polymer beads (SMPBs) with

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uniform shape and size through spontaneous rearrangement of short chain glucan

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(SCG) produced by enzymatic debranching of waxy maize starch. The paramagnetic

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materials, dextran-coated iron oxide nanoparticles (Dex@IONPs), were readily

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incorporated into the starch microstructure and rendered a superparamagnetic

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property to the SMPBs. The morphology and size of resulting SMPBs turned out to be

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modulated by Dex@IONPs in concentration dependent manner, of which

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Dex@IONPs was assumed to be acting as a seed inducing the epitaxial crystallization

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of SCG and further transforming it into homogeneous microparticles. The surface of

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SMPBs was readily functionalized with antibody through one step reaction using a

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linker protein. The immuno-SMPBs showed great capture efficiency (>90%) for

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target bacteria. The colloidal stability and favorable surface environment for

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biomolecules are believed to be responsible for the high capture efficiency and

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specificity of the SMPBs. Furthermore, the captured bacteria along with antibody and

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linker protein were effectively eluted from the surface of SMPBs by adding free

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maltose, making this new material suitable for various chromatographic applications.

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KEYWORDS: polymeric magnetic beads, waxy maize starch, debranching, self-

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assembly, epitaxial growth.

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Polymeric magnetic beads (PMBs) are spherical microstructure of polymeric

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materials containing magnetic particles in dispersed or core-shell form. Due to the

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paramagnetic nature, PMBs have mainly been utilized to separate target components

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from heterogeneous matrices by external magnetic force upon functionalizing the

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surface of PMBs with a specific ligand that binds to the target. The colloidal stability

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of PMBs in aqueous environment along with versatile surface functionalization

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techniques have extended its applications to many areas, such as targeted drug

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delivery, magnetic resonance imaging (MRI), magnetic hyperthermia, bio-separation,

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and biosensing.1-4 The size and surface functionality are among the most critical

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factors that determine the application of PMBs. In particular, micrometer-sized

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spherical PMBs receive considerable attention in analytical field, including

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immunomagnetic separation, column-based chromatography, and flow cytometry,

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where its shape, size, porosity, surface functionality, and monodispersity should be

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strictly controlled.5-6 A range of natural and synthetic polymers, such as dextran,

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alginate, chitosan, polyaspartate, polystyrene, and polyacrylamide, are currently used

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as a base materials for the synthesis of PMBs.7 The synthesis of PMBs using those

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materials is typically carried out by emulsion polymerization and sol-gel process,8-9

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microwave-assisted hydrothermal,10 sonochemical methods,11 which often require

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complicated procedures and large energy consumption. They could also lead to

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negative environmental impacts as well as causing a limited applications on large-

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scale production. Achieving a high colloidal stability as well as controlling the size of

INTRODUCTION

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PMBs for desired applications is another challenging tasks that need to be resolved.

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Starch, as one of the most abundant polysaccharide in nature, is consisting of a

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large number of glucose molecules joined by glycosidic bonds and serves as an

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energy reserve in plants. Amylose is one of the major components in starch and is

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mostly linear homopolymer of glucose linked with α(1,4) glycosidic bonds.

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Amylopectin, another major component of starch, is a branched macromolecule

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composed of α(1,4)-D-glucan chains linked with 5-6% α(1,6) bonds. The ratio of

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amylose and amylopectin in starch varies among different types of plants. A short-

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chain amylose or short-chain glucan (SCG) have been reported to recrystallize in

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aqueous solution to form spherical microstructures and its mechanisms of self-

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assembly have been intensively studied to understand the structural changes in starch

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granules and to produce amylose-based microstructures.12 Due to their structural

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stability, renewable and biocompatible nature, starch microparticles have emerged as

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an effective carrier or encapsulation agent for various guest molecules, such as carbon

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nanotubes,13 iron oxide nanoparticles,14 fatty acids,15 and β-carotene.16 SCG can be

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produced through polymerization of glucose molecules into a linear glucan chain

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using specific enzymes, such as phosphorylase or amylosucrase.13, 17 However, these

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enzymes require a highly selective glycosylation reaction between a donor and an

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acceptor molecule to form α(1,4)-linked glucan. For example, phosphorylase needs

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expensive

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oligosacchraides as a glycosyl acceptor.12 On the other hand, amylosucrase provides a

glucose-1-phosphate

(G-1-P)

as

a

glucosyl

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donor

and

malto-

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better means of producing linear glucan since it requires sucrose as a sole substrate for

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the synthesis of linear glucan with a DP of 40~50.13 However, the conversion rate of

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the substrate into a linear glucan in the amylosucrase-mediated catalytic process was

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shown to be ~20%,18 which limits its applications in mass production.

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Another approach to produce SCG is debranching amylopectins that contain a

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large number of short-chain glucans linked by α(1,6) bonds. Debranching enzymes,

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such as pullulanase and isoamylase, provide simple, effective and environmentally

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friendly means of producing SCG by cleaving α(1,6)-linkages bonds in pullulan,

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amylopectin, or related polysaccharides.19 Waxy starches, such as waxy maize, waxy

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potato, and waxy rice starch, are good candidate to produce SCG by debranching

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reaction since their main component is amylopectin with only trace amount of

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amylose present. The catalytic action of these enzymes has been reported to produce

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SCG, which can be crystallized directly into spherical microstructure in high yield

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(85%) in aqueous environment without the need of any organic solvent and energy

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consumption.20-21 However, their morphologies and sizes were highly heterogeneous,

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limiting their applications in biomedical and analytical fields. Herein, we present a

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fairly simple and eco-friendly approach for fabrication of monodisperse starch-based

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magnetic polymer beads (SMPBs) with controlled particle size by modulating the

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reaction with epitaxial seeding effect using Dex@IONPs, which could regulate the

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nucleation and crystal growth during self-assembly process. The factors affecting the

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size and polydispersity of SMPBs were also investigated. Furthermore, its potential as

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a highly efficient immunomagnetic separation material is presented.

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EXPERIMENTAL SECTION

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Materials. Pullulanase, ferrous chloride tetrahydrate (FeCl2·4H2O), dextran (Mw

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9000-11000), γ-Fe2O3, Tris–HCl, lysozyme, and isopropyl-β-D-thiogalactopyranoside

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(IPTG), 5(6)-carboxyfluoroscein diacetate (CFDA) were purchased from Sigma-

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Aldrich (St. Louis, MO, USA). Ferric chloride hexahydrate (FeCl3·6H2O), ammonium

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hydroxide, and acetone were purchased from Daejung (Siheung, Korea). Anti-

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Escherichia coli O157 monoclonal antibody (FITC conjugate) was purchased from

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Thermo Fisher Scientific Inc. (Cambridge, MA, USA). Sodium acetate trihydrate and

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waxy maize starch were obtained from Yakuri Pure Chemicals (Kyoto, Japan) and

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Samyang Co (Seoul, Korea), respectively. Maize Starch was provided from Daesang

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Co (Seoul, Korea). Ampicillin was supplied by Biosesang (Seongnam, Korea). All

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restriction enzymes were acquired from New England Biolabs (Ipswich, MA, USA).

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Ni-NTA Superflow resin was obtained from Qiagen (Valencia, CA, USA).

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Preparation of dextran-coated iron oxide nanoparticles (Dex@IONPs).

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Dex@IONPs were synthesized by the coprecipitation process using dextran and iron

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chloride as reported by Ahmadi with modification.22 Briefly, 80 mM of FeCl3·6H2O,

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40 mM of FeCl2·4H2O, and 150 mg of dextran were dissolved in 20 ml deionized

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water (DW). The mixture was purged with nitrogen gas to remove dissolved oxygen

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in solution, followed by ultrasonication by a Q500 Sonicator (VC 750, Sonics &

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Materials Inc., Newtown, CT, USA) with on/off cycle of 3s/3s in an ice bath for 3 min 6


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at 30% amplitude concurrently through a 6-mm ultrasound probe. During the

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sonication, 60 % ammonium hydroxide solution was added dropwise into the mixture

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using pipette until the mixture turned to dark suspension. The synthesized

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Dex@IONPs was washed several times with absolute ethanol and DW to remove

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residual ammonium hydroxide and dextran, followed by sonication for 10 s. The final

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product was stored at 4 °C until use. The mean particle size of Dex@IONPs was

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estimated by counting at least 100 particles from the field emission scanning electron

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microscopy (FE-SEM) images.

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Preparation of iron oxide nanoparticles (IONPs). Pristine IONPs were

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synthesized by coprecipitation process as described above in the absence of dextran.

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The synthesized IONPs were dissolved in 10 ml DW with a final concentration of 20

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mg/ml and sonicated (Q500 Sonicator, Qsonica, Newtown, CT) with on/off cycle of

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10s/10s in an ice bath for 30 min at 30% amplitude concurrently through a 13-mm

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ultrasound probe. The sonicated sample was centrifuged at 3000xg for 20 min, and

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the supernatant containing well-dispersed IONPs were transferred to a fresh tube. The

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final product was stored at 4 °C until use. The mean particle size of IONPs was

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estimated by counting at least 100 particles from the SEM images.

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Preparation of starch magnetic polymer beads (SMPBs). Three grams of

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waxy maize starch was dissolved in 30 ml of distilled-deionized water (DDW) and

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boiled at 100 °C for 30 min for gelatinization. After cooling to 60 °C, the gelatinized

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starch was treated with pullulanase through two-step reaction for debranching of 7



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amylopectin. In the first reaction, pullulanase (30 ASPU/ml) was added to the reaction,

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which was subsequently incubated at 60 °C for 4 h. The reaction was stirred with a

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glass stick every hour. After the incubation, 20 ml supernatant of the reaction solution

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was transferred to a conical tube and the volume was adjusted to 50 ml with DDW. In

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the second debranching reaction, the sample was treated with a fresh pullulanase to a

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final concentration of 8 ASPU/ml and incubated at 65 °C for overnight. The sample

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was centrifuged at 15000xg for 5 min, and 0.8 ml of the supernatant was transferred

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to a fresh EP tube containing varying concentrations of IONPs or Dex@IONPs

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ranging from 0 to 10 mg/ml. The mixture was then incubated at 4 °C for 24 h to

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induce the self-assembly of SMPBs. The prepared SMPBs was washed 3 times with

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DW and stored at 4 °C until use. The morphology and composition of the synthesized

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SMPBs were analyzed by FE-SEM and TEM equipped with EDS elemental mapping

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of iron, carbon and oxygen. Magnetic properties of SMPBs were measured using

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physical property measurement system (16 T PPMS Dynacool, Quantum Design,

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USA) at room temperature from −12000 to 12000 Oe.

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Preparation of Maltose binding protein-tagged streptococcal protein G

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(MBP-SPG) fusion protein. The recombinant MBP-SPG fusion protein was

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prepared as described elsewhere.14 Briefly, E. coli DH5α harboring the MBP-SPG-His

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expression vector were cultured in 500 mL LB broth containing ampicillin (0.1 mg/ml)

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at 37 °C with shaking at 250 rpm. When reaching an OD600 of 0.7-0.8, 0.1 mM IPTG

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was added to induce overexpression of the fusion protein and incubated further at

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18 °C for 18 h. The cells were harvested by centrifugation (3000xg for 20 min at 4 °C)

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and resuspended in 5ml of a lysis buffer (50 mM NaH2PO4, 300 mM NaCl, and10

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mM imidazole, pH 8.0) for 20 min at 4 °C, followed by sonication (Q500 Sonicator)

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with on/off cycle of 10s/10s in an ice bath for 10 min at 20% amplitude concurrently

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using a 6-mm ultrasound probe. After centrifugation at 3000xg for 20 min, the

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supernatant was passed through a column packed with Ni-NTA resin (Qiagen). The

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Ni-NTA column was washed with a washing buffer (50 mM NaH2PO4, 300 mM NaCl,

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20 mM imidazole, pH 8.0), and the MBP-SPG proteins were eluted with an elution

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buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). The purified

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MBP-SPG was stored at 4 °C until needed.

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Conjugation of antibody to SMPBs using MBP-SPG fusion protein. To

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conjugate antibodies to the surface of SAMBs, the recombinant MBP-SPG fusion

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protein was used as a cross-linker with the specific affinity of MBP and SPG to

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glucan and the Fc region of the antibody, respectively.14 The synthesized SMPBs were

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suspended in an aqueous solution containing 30 µg/ml of MBP-SPG, incubated at

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4 °C for 30 min in a rotary shaker, washed 3 times with 1X PBS (pH 7.4), and then

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resuspended in 1X PBS to a final concentration of 50 mg/ml. The FITC-labelled anti-

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E. coli O157 antibody with a final concentration of 2 µg/ml was added to the solution

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containing MBP-SPG-functionalized SMPBs. After incubating at 4 °C for 60 min in a

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rotary shaker, the antibody-labelled SMPBs were washed 3 times with 1X PBS and

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stored at 4 °C until needed. The conjugation of FITC-labeled antibodies on the surface

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of the SMPBs were confirmed by fluorescence microscopy (Nikon TE2000U, Tokyo,

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Japan).

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Immunomagnetic separation of target bacteria. Freshly cultured E. coli

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O157:H7 was diluted serially to the concentrations ranging from 102 to 106 CFU/ml in

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1X PBS. The antibody-labelled SMPBs were introduced to the serially diluted

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samples to a final concentration of 10 mg/ml. After incubating the sample at room

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temperature for 30 min with gentle rotation, the target bacteria were separated along

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with the immuno-SAMBs to a side of tube by magnet, and 0.1 ml of the solution

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containing unbound bacteria was plated on LB agar plates. All plates were incubated

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at 37 ºC for 24 h, and expressed as log CFU/ml. The capture efficiency of the SMPBs

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was determined by following equation:

%CE=

Ncon − Nunbound ×100 Ncon

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where %CE is relative capture efficiency to the target bacteria, Ncon is the initial

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concentration of target bacteria, Nunbound is the concentration of unbound bacteria. For

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CFDA staining of E. coli O157:H7, the cultured cells were harvested and washed

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twice in 50 mM phosphate buffer (pH 7) by centrifugation at 3000×g for 10 min at 4 °

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C. One ml of the bacterial suspension was mixed with 10 µl of CFDA stock solution

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(10 mM), followed by incubation at 37 °C for 30 min. For recycling, the antibody-

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conjugated SMPBs were treated with an elution buffer containing 10 mM maltose for

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5 min with rotating at 10 rpm, followed by washing three times with 1X PBS. The

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recycled SMPBs were labeled again with anti-E. coli O157:H7 IgG in the presence of

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MBP-SPG as aforementioned. The capture efficiency of the immuno-SMPBs were

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tested through three successive recycling of the same material.

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Statistical analysis. Capture efficiency of the immuno-SMPBs for target bacteria,

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E. coli O157:H7, was compared over a range of bacterial concentration through two-

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way analysis of variance (ANOVA) using the GraphPad Prism 7 software package

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(Graphpad Software, Inc., San Diego, CA; www.graphpad.com). Statistical

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significance was accepted for P-value of

Molecular rearrangement of glucans from natural starch to form size

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