Molecular Rearrangement of Glucans from Natural ... - ACS Publications

Subscriber access provided by TUFTS UNIV

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34

Journal of Agricultural and Food Chemistry

1 2 3

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

4 5 6

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

7 8

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

9

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

10

South Korea

11 12

* Corresponding author.

13

Tel: +82-31-201-3830

14

Fax: +82-31-204-8116

15

E-mail address: [email protected]

16 17 18

1

ACS Paragon Plus Environment


19

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

20

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

21

uniform shape and size through spontaneous rearrangement of short chain glucan

22

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

23

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

24

incorporated into the starch microstructure and rendered a superparamagnetic

25

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

26

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

27

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

28

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

29

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

30

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

31

target bacteria. The colloidal stability and favorable surface environment for

32

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

33

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

34

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

35

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

36 37 38

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

39

assembly, epitaxial growth.

2


Page 2 of 34

Page 3 of 34


40

41

Polymeric magnetic beads (PMBs) are spherical microstructure of polymeric

42

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

43

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

44

from heterogeneous matrices by external magnetic force upon functionalizing the

45

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

46

of PMBs in aqueous environment along with versatile surface functionalization

47

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

48

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

49

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

50

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

51

spherical PMBs receive considerable attention in analytical field, including

52

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

53

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

54

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

55

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

56

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

57

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

58

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

59

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

60

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

61

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

INTRODUCTION

3



62

Page 4 of 34

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

63

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

64

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

65

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

66

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

67

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

68

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

69

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

70

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

71

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

72

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

73

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

74

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

75

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

76

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

77

produced through polymerization of glucose molecules into a linear glucan chain

78

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

79

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

80

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

81

expensive

82

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

glucose-1-phosphate

(G-1-P)

as

a

glucosyl

4


donor

and

malto-

Page 5 of 34


83

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

84

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

85

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

86

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

87

Another approach to produce SCG is debranching amylopectins that contain a

88

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

89

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

90

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

91

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

92

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

93

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

94

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

95

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

96

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

97

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

98

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

99

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

100

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

101

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

102

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

103

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

5



104

a highly efficient immunomagnetic separation material is presented.

105

EXPERIMENTAL SECTION

106

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

107

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

108

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

109

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

110

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

111

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

112

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

113

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

114

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

115

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

116

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

117

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

118

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

119

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

120

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

121

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

122

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

123

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

124

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


Page 6 of 34

Page 7 of 34


125

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

126

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

127

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

128

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

129

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

130

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

131

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

132

microscopy (FE-SEM) images.

133

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

134

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

135

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

136

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

137

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

138

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

139

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

140

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

141

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

142

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

143

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

144

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

145

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



146

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

147

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

148

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

149

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

150

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

151

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

152

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

153

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

154

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

155

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

156

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

157

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

158

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

159

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

160

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

161

Preparation of Maltose binding protein-tagged streptococcal protein G

162

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

163

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

164

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

165

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

166

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

8


Page 8 of 34

Page 9 of 34


167

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

168

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

169

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

170

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

171

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

172

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

173

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

174

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

175

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

176

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

177

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

178

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

179

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

180

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

181

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

182

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

183

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

184

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

185

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

186

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

187

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

9



188

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

189

Japan).

190

Immunomagnetic separation of target bacteria. Freshly cultured E. coli

191

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

192

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

193

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

194

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

195

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

196

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

197

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

198

was determined by following equation:

%CE=

Ncon − Nunbound ×100 Ncon

199

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

200

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

201

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

202

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

203

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

204

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

205

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

206

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

10


Page 10 of 34

Page 11 of 34


207

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

208

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

209

tested through three successive recycling of the same material.

210

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

211

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

212

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

213

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

214

significance was accepted for P-value of

Molecular Rearrangement of Glucans from Natural ... - ACS Publications

Recommend Documents