Coimmobilization of Î²-Agarase and Î±-Neoagarobiose Hydrolase for

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Biotechnology and Biological Transformations

Co-immobilization of #-agarase and #-neoagarobiose hydrolase for enhancing the production of 3,6-anhydro-L-galactose Qidong Wang, Jianan Sun, Zhen Liu, Wen-Can Huang, Changhu Xue, and Xiangzhao Mao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01974 • Publication Date (Web): 12 Jun 2018 Downloaded from http://pubs.acs.org on June 14, 2018

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Journal of Agricultural and Food Chemistry

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Co-immobilization of β-agarase and α-neoagarobiose hydrolase for enhancing

2

the production of 3,6-anhydro-L-galactose

3

Qidong Wang1, Jianan Sun1, Zhen Liu1, Wencan Huang1, Changhu Xue1,2, Xiangzhao

4

Mao1,2 *

5 1

6

College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China

7 8 9

2

Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China

10 11

* Corresponding author: Professor Xiangzhao Mao

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Address: College of Food Science and Engineering, Ocean University of China,

13

Qingdao 266003, China

14

Tel.: +86-532-82032660

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Fax: +86-532-82031789

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E-mail: [email protected]

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1

ACS Paragon Plus Environment


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ABSTRACT

19

Here

we

report

a

simple

and

efficient

method

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to

produce

20

3,6-anhydro-L-galactose (L-AHG) and agarotriose (AO3) in one step by a

21

multi-enzyme system with co-immobilized β-agarase AgWH50B and α-neoagarobiose

22

hydrolase K134D. In which K134D was obtained by AgaWH117 mutagenesis and

23

showed an improved thermal stability when immobilized via covalent bonds on

24

functionalized magnetic nanoparticles. The obtained multi-enzyme biocatalyst was

25

characterized by FTIR. Compared with free agarases, the co-immobilized agarases

26

exhibited a relatively higher agarose-to-L-AHG conversion efficiency. The yield of

27

L-AHG obtained by the co-immobilized agarases was 40.6%, which was 6.5% higher

28

than that obtained by the free agarases. After eight cycles, the multi-enzyme

29

biocatalyst still preserved 46.4% of the initial activity. To the best of our knowledge,

30

this is the first report where two different agarases were co-immobilized. These results

31

demonstrated the feasibility of the new method to fabricate a new multi-enzyme

32

system onto magnetic nanoparticles via covalent bonds to produce L-AHG.

33

words:

Co-immobilization;

34

Key

35

3,6-Anhydro-L-Galactose; Agarotriose

β-agarase;

α-Neoagarobiose

36

2


Hydrolase;

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37

■INTRODUCTION

38

The main component of red macroalgae (Rhodophyta) cell wall is agar, which is

39

made up of two components: agarose and a charged agaropectin. Agarose, which is a

40

heteropolysaccharide, is composed of equal molar amounts of L-AHG and

41

D-galactose.1 L-AHG is linked to D-galactose via a α-1,3-glycosidic bond, and the

42

resulting

43

3,6-anhydro-L-galactosyl-α-1,3-D-galactoside) are linked to form agarose via a

44

β-1,4-glycosidic linkage.1 Agarose can be biologically degraded into oligosaccharides

45

by β-agarases or α- agarases, which have special molecular structures, and thus have

46

different functional characteristics, such as anti-tumor, anti-oxidant, hepatoprotective,

47

whitening,

48

anti-inflammatory properties.2-4 Neoagarobiose can be used as a novel moisturizer

49

with

50

mitogen-activated protein kinases and nuclear factor-kB signaling pathways in

51

lipopolysaccharide-stimulated macrophages to weaken the inflammatory responses.2

52

Agaro-oligosaccharides and neo-agaroligosaccharides have been widely applied in the

53

food and medical industries .6

heterodimers

apoptosis-inducing,

whitening

effect.5

(i.e.,

α-neoagarobiose:

immunoregulatory,

Neoagarotetraose

(NA4)

anti-allergic,

may

downregulate

and

the

54

L-AHG which constitutes 50% of agarose exhibits significant skin whitening and

55

anti-inflammatory activities.7 L-AHG plays a key role in the whitening effect of

56

agar-derived sugars.8 However, L-AHG with high purity is hardly available in the

57

market even as a reagent. Currently, L-AHG is mainly prepared by chemical methods

58

or the combined saccharification method which is acid prehydrolysis and enzymatic

3



59

saccharification of agarose. However, the resulting type of oligosaccharide products is

60

uncontrollable and their structures are easily destroyed when using acid method.9

61

L-AHG and galactose can be overdegraded to generate toxic byproducts by strong

62

acid or high concentration of acid, such as 5-hydroxy-methyl-furfural.10-12 Although

63

some researchers have reported that the combined saccharification method has higher

64

yield of L-AHG, a small amount of L-AHG was still degraded into

65

5-hydroxy-methyl-furfural.13

66

α-Neoagarobiose hydrolase (NABH), which belongs to the glycoside hydrolases

67

family (GH117), can cleave the α-1,3-glycosidic bond of neoagarobiose to produce

68

L-AHG and D-galactose with similar molecular weight.14 Yun et al.7 reported that

69

weak acids can pre-hydrolyze agarose into oligosaccharides, which were hydrolyzed

70

into L-AHG and D-galactose successively by an exo-type β-agarase (Aga50D)10, 15

71

and a NABH extracted from Saccharophagus degradans 2-40 (SdNABH, formerly

72

AgaJ)14, 16. And the purity of L-AHG obtained by combining the acid and enzymes

73

was 95.6%, with a final yield of 4.0% based on the initial agarose.7 However,

74

production of some byproducts is still unavoidable.7 Neoagarobiose hydrolysis is

75

beneficial to the increase of ethanol production by fermenting both L-AHG and

76

galactose.17, 18 However, it is not conducive for the preparation of L-AHG with high

77

purity because of the difficulty to separate the two monosaccharides. In the same

78

group, Yun et al.19 also reported that L-AHG was obtained from agarose by β-agarases

79

I and II and NABH sequentially. Although the toxic by-products can be avoided, the

80

reaction steps are complex and the final products are still L-AHG and D-galactose.

4


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81

They need two steps for purification: size exclusion chromatography and silica gel

82

column chromatography to separate them. And silica gel column chromatography

83

requires toxic organic reagents, bringing about less than welcoming environment.

84

Although Koti et al.7, 20 reported that oligosaccharides or L-AHG were prepared

85

by agarases, they still used some acids to pre-treat agarose, causing a non

86

environmentally friendly process, and the reaction steps are complex.

87

In our previous research, β-agarase AgWH50B from Agarivorans gilvus

88

WH0801, which can directly cleave the β-1,4-glycosidic bond of agarose, was utilized

89

to produce NA4 efficiently without use of acids to pre-treat agarose.6 Then, NA4 was

90

hydrolyzed into L-AHG and AO3, which is also an important bioactive

91

oligosaccharide, by NABH from A. gilvus WH0801.21 These two sugars are easily

92

separated

93

chromatography. The purity of L-AHG obtained via one-step purification by biologic

94

enzyme catalysis without use of acids was above 95.0%, with recovery yield of

95

4.9%.21 This showed an obvious advantage for the preparation of L-AHG. Meanwhile,

96

we also obtained another AO3 (with a purity level higher than 98.0%) (Figure.S1),

97

which exhibited protective effects against alcoholic liver injury.22

by

size

exclusion

chromatography

without

silica

gel

column

98

The simultaneous preparation of L-AHG and AO3 can be achieved by a one-step

99

reaction if these two enzymes are coupled. However, free enzymes have many

100

shortcomings. For example, they can easily pollute products and cannot be reused. On

101

the contrary, immobilized enzymes have many benefits over soluble enzymes in many

102

biotechnological applications. The number of applications of immobilized enzymes is

5



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103

increasing steadily.23 Easy separation of immobilized enzymes from the products

104

simplifies the application of biocatalysts. In addition, reuse of immobilized enzymes

105

provides advantages in terms of costs of biologic enzyme catalysis. Su et al.24 reported

106

that magnetic Fe3O4 nanoparticles like the carrier skeleton can improve the

107

operational stability of the Combi-CLEAs. Meanwhile, co-immobilization allows the

108

obtainment of the product(s) by one-step, which omits some intermediate reactions

109

and improves the final yield. Yang et al.25 reported that double enzyme

110

co-immobilization can increase the thermal stability of enzymes and it can be better

111

adapted to the process of industrial biocatalysis. To date, studies on the

112

immobilization of agarase are still few. Koti et al.26 have recently shown that the

113

agarase system

114

agar-oligosaccharides from agarose. Some researchers also used magnetic

115

nanoparticles to immobilize agarases. For example, carboxyl-functionalized magnetic

116

nanoparticles were used to immobilize agarase from marine Vibrio.27, 28 However, to

117

the best of our knowledge, co-immobilization of two different agarases has not been

118

reported.

119

was

immobilized

on an amberlite

IRA-900

to

produce

In this study, first, β-agarase and α-neoagarobiose hydrolase (AgWH50B and

120

AgaWH117,

respectively)

were

121

(TCT)-functionalized

122

separately. The optimal immobilization conditions were studied. The immobilized

123

enzyme was characterized by using a FTIR. Enzymatic properties, including optimum

124

temperature, optimum pH, and thermal stability, were studied. In order to further

silica-coated

immobilized

magnetite

on

trichlorotriazine

nanoparticles

(CC-Fe3O4@SiO2)

6


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125

improve the optimum reaction temperature and thermal stability of α-neoagarobiose

126

hydrolase, we obtained an AgaWH117 mutant (K134D) that showed a higher optimal

127

reaction temperature and thermal stability by site-directed and site-saturation

128

mutagenesis. This mutant and AgWH50B were co-immobilized on the supports. The

129

production of L-AHG obtained by co-immobilized agarases was compared with that

130

obtained by free agarases.

131

■MATERIALS AND METHODS

132

Materials. Agarose (low gelling temperature), ferric chloride hexahydrate

133

(FeCl3ˑ6H2O),

134

3-(triethoxysilyl)-propylamine (APTES) were obtained from Sigma-Aldrich Co.

135

(USA). Bovine serum albumin (BSA), Coomassie Brilliant Blue G-250, and

136

Ampicillin were purchased from Solarbio Co. (China). Ethanol, tetraethylorthosilicate

137

(TEOS), tetrahytrofuran (THF), sodium chloride (NaCl), and ammonium hydroxide

138

(NH4OH) were obtained from Sinopharm Chemical Reagent Co. Ltd. (China), while

139

tryptone

140

3,6-anhydro-D-galactose was purchased from Dextra Laboratories Ltd (England). The

141

recombinant strains pET21a-E. coli BL21 (DE3), containing the encoding gene of

142

AgWH50B and AgaWH117, were previously constructed in our laboratory.21 All

143

enzymes used for molecular cloning were obtained from Takara (Dalian, China). PCR

144

primers and DNA sequencing were finished by BGI (Shanghai, China). Other

145

analytical reagents, unless otherwise noted, were obtained from Sigma-Aldrich Co.

146

(USA).

and

ferrous

yeast

chloride

extract

tetrahydrate

were

(FeCl2ˑ4H2O),

obtained

7


from

Oxoid

TCT,

and

(England).


147

Construction of AgaWH117 mutants. The mutation sites were selected based

148

on the TK-SA protein model29-35, which could analyze the thermal stability of protein

149

by calculating the electrostatic interaction between internal ionizable protein amino

150

acid residues. Negative Gibbs free energy (△Gqq) plays an active role in protein

151

stability and contributes to protein folding. To get rid of the amino acid residues at the

152

loop and active site, the amino acids with △Gqq>2.0e+00 were transformed to

153

alanine (Ala) through mutagenesis. The selected amino acid residues are shown in

154

Table S1.

155

Amino acid substitutions were introduced by using the Quick-ChangTM

156

Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to supplier

157

instructions. The recombinant plasmid pET21a/AgaWH117 was used as the template

158

for the mutagenesis reaction. Moreover, site-saturation mutagenesis was carried out at

159

the selected positions for further study. All the primers containing the appropriate base

160

changes are listed in Table S2.

161

Expression and purification of three recombinant enzymes. Three E. coli

162

strains of engineered bacteria, namely, BL21 (DE3)-pET21a-agWH50B, BL21

163

(DE3)-pET21a-agaWH117, BL21 (DE3)-pET21a-K134D, were cultured using a

164

similar method. The E. coli strains were cultured in Luria-Bertani medium (1.00%

165

tryptone, 0.50% yeast extract, and 1.00% NaCl) with shaking (180 rpm) at 37.0°C for

166

12 h with 100 µg/mL Ampicillin. Thereafter, the activated strains were sub-cultured in

167

the auto-inducing ZYP-5052 medium (1.00% tryptone, 0.50% yeast extract, 0.71%

168

Na2HPO4, 0.68% KH2PO4, 0.33% (NH4)2SO4, 0.024% MgSO4, 0.50% glycerin,

8


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169

0.05% glucose, and 0.20% α-lactose) supplemented with 100 µg/mL Ampicillin and

170

incubated in a shaker (220 rpm) at 20.0°C for 48 h.6 The culture broth was centrifuged

171

at 8000×g for 10 min at 4°C. The resulting cell pellet was re-suspended in 20 mM

172

phosphate buffer (pH 7.6). Next, total cellular protein was obtained by sonication (3 s

173

at 300 W and 3 s off cooling on ice for 40 min) and centrifugation (16000×g for 45

174

min at 4oC). The crude extract was filtered and purified with Ni2+-NTA resin in

175

accordance with the manufacturer’s instructions (TransGen Biotech, China).6 Finally,

176

the purified protein was analyzed by SDS-PAGE, and its concentration was

177

determined using Coomassie Brilliant Blue G-250 with BSA as the standard. The

178

purified enzyme was then used for further enzyme activity assay.

179

Preparation of functionalized silica-coated modified magnetite nanoparticles.

180

Fe3O4 nanoparticles and silica-coated magnetite nanoparticles were synthesized

181

according to the literature.36 The surface of the MNPs was modified by TCT

182

according to the method of Wang and Liu et al.37

183

Optimization

of

immobilization

conditions

of

agarases.

The

184

triazine-functionalized MNPs (10.0 mg) were dispersed in 800 µL of buffer solution

185

(50 mM, pH 4.0-10.0). Then, various amounts of the agarases (50-2000 µg) were

186

added into the suspension and the mixture was shaken at 10.0-60.0oC and 180 rpm for

187

0-5 h. The immobilized agarases were removed by using an external magnetic field

188

and washed three times with the same buffer solution used for immobilization. The

189

amount of agarases immobilized on MNPs was determined by measuring the initial

190

and final concentration of agarases by the Bradford method38 in the supernatant

9



191

obtained after separation of the nanoparticles. For the co-immobilization, AgWH50B

192

was first immobilized onto the carrier (0-300 µg), then K134D was subsequently

193

immobilized onto the AgWH50B-bound carrier (200 µg). Other conditions were the

194

optimal conditions for the single enzyme immobilization procedure.

195

Enzyme assay. AgWH50B activity assays were performed using the

196

3,5-dinitrosalicylic acid (DNS) method as previously described, with some

197

modifications.39 Each reaction of 400 µL contained 10.0 mg immobilized AgWH50B,

198

20 mM phosphate buffer (pH 7.0) and 0.30% (w/v) low gelling temperature agarose.

199

After incubation at the optimum temperature for 20 min, 200 µL of reaction solution

200

was mixed with 300 µL of DNS reagent, boiled immediately for 5 min, and then

201

cooled in a cold water bath. Samples were subsequently diluted with 1 mL water, and

202

the absorbance was determined at 540 nm. Heat-inactivated enzyme was used as a

203

control. One unit of enzymatic activity (U) was defined as the amount of enzyme that

204

produced 1 µmol of reducing sugar per min by hydrolyzing agarose under the assay

205

conditions.

206

NABH activity was measured using high-performance liquid chromatography

207

(HPLC).40 Standard assay conditions were as follows: The enzyme was added to 400

208

µL of 20 mM phosphate buffer (pH 7.0) containing 0.20% (w/v) NA4 as the substrate.

209

After incubating at 30oC for 30 min, the enzyme reaction was stopped by boiling for 5

210

min. The amount of product was inferred from the peak area of the HPLC analysis

211

(eluent, water containing 50.0 mg/L ethylenediaminetetraacetic acid calcium disodium

212

salt hydrate; flow rate, 0.5 mL/min; detector, refractive index) with a Waters

10


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213

Sugar-Pak I Column (300 × 6.5 mm2; Waters, Milford, MA, USA) after comparison

214

with a standard curve (Figure.S4). One unit of enzyme activity was defined as the

215

amount of the enzyme required to produce 1 µmol of L-AHG per minute under

216

standard assay conditions.

217

Effects of pH and temperature on the activity of immobilized enzymes. The

218

activity and stability of immobilized agarases were assayed at different temperatures

219

and pH values by the enzyme assay. The effect of temperature on the agarase activity

220

was evaluated at temperature range of 20.0-50.0oC. The effect of pH on the activity of

221

the immobilized agarase was investigated at seven different pH levels (4.0, 5.0, 6.0,

222

7.0, 8.0, 9.0, and 10.0).

223

Thermal stability. Thermal stability was determined by measuring the residual

224

activities of the immobilized AgWH50B within 0-720 min of incubation in a

225

phosphate buffer (50 mM, pH 7.6) at specific temperature. Similarly, thermal stability

226

of the immobilized AgaWH117 was also determined within 0-120 min.

227

Catalyst recycling and yield comparison. The stability of the co-immobilized

228

agarase was evaluated by reusing it eight times. A 10-mL volume of 0.30% (w/v) low

229

gelling temperature agarose in 20 mM phosphate buffer (pH 7.0) was added to the

230

co-immobilized enzymes and incubated for 60 min under constant shaking for each

231

cycle. At the end of the reaction, the co-immobilized enzymes were taken and washed

232

with 20 mM phosphate buffer (pH 7.0) and then a substrate solution was added to

233

start a new cycle. The supernatant was assayed for L-AHG activity.

234

The 100.0 mg co-immobilized agarases were reacted with 0.30% (w/v) low

11



235

gelling temperature agarose for 36 h at the optimal reaction conditions. The free

236

agarases, which were equal in amounts to the co-immobilized agarases, were reacted

237

with 0.30% (w/v) low gelling temperature agarose separately at 27.5oC and 35.0oC for

238

36 h at 20 mM phosphate buffer solution (pH 7.0) . The yields of L-AHG were

239

compared.

240

The hydrolysates were analyzed by HPLC-ESI-Q-TOF-MS. The eluent is ultra

241

pure water. The flow rate of eluent and the temperature of the column were consistent

242

with the method mentioned in HPLC determination. The MS instrument was Bruker

243

maXis II (Bruker, Germany) equipped with an ESI source in negative ion mode. End

244

plate offset-500 V, capillary 2200 V, nebulizer 2.8 bar, flow rate of nitrogen 6 L/min,

245

dry temperature 180 oC, funnel RF 200.0 Vpp, ion energy 4.0 eV, collision energy 8.0

246

eV. The scanning range was from 50 to 1500 (m/z).

247

Characterization. Presence of surface functional groups and the binding of

248

agarases onto CC-Fe3O4@SiO2 were analyzed by a Fourier transform Infrared

249

Spectroscopy (Nicolet is10-FTIR, USA).

250

■RESULTS AND DISCUSSION

251

Characteristics of CC-Fe3O4@SiO2. The modified magnetite nanoparticles

252

CC-Fe3O4@SiO2 were well-dispersed in the aqueous solution and aggregated only

253

when a permanent magnet or any appropriate magnetic separator was applied in the

254

test. This phenomenon demonstrated that agarase-CC-Fe3O4@SiO2 could be separated

255

from the reaction mixture by an external magnetic field during initial use and reuse

256

(Figure.S2).

12


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257

The absorption band around 579.46 cm-1 in the FTIR spectra of Fe3O4,

258

Fe3O4@SiO2 and CC- Fe3O4@SiO2 nanoparticles (Figure.1a) corresponds to Fe-O

259

bonds. The peaks at 801.27 cm-1 and 1083.80 cm-1 correspond to the symmetric

260

stretching of Si-OH and Si-O-Si, respectively, and the broad bands at around 3400.11

261

cm-1 and 1622.51 cm-1 can be assigned to O-H stretching vibrations. Instead, those at

262

around 3388.69 cm-1 and 1635.77 cm-1 can be assigned to -NH2. In the FTIR spectra

263

of CC-Fe3O4@SiO2 (Figure.1a), in addition to the above-mentioned vibrations, the

264

C=N band at 1580.00-1603.00 cm-1 is a good indication for the presence of triazine

265

fragments on the magnetic nanoparticles. Due to the presence of stretching vibration

266

of the Si-O band, the C-Cl band of CC at 1010.00 cm-1 was masked.41 The binding of

267

agarases to modified MNPs was confirmed by FTIR analysis. Figure.1b shows the

268

FTIR spectra of the modified MNPs with AgWH50B, AgaWH117, and K134D.

269

However,

270

CC-Fe3O4@SiO2 were similar. The characteristic bands of proteins were at 1635.00

271

cm-1, 1456.00 cm-1, and 1090.00 cm-1. The peaks of 1635.00 cm-1 and 1456.00 cm-1

272

after immobilization of agarases on CC-Fe3O4@SiO2 correspond to agarases-

273

CC-Fe3O4@SiO2. This showed the presence of agarases in the samples, confirming

274

the binding of agarases to modified MNPs.

the

characteristic

bands

of

different

agarases

immobilized

on

275

Optimization of immobilization conditions. In order to obtain the optimum

276

immobilization efficiency and activity of biocatalysts, the immobilization conditions

277

have to be well controlled in the covalent immobilization method, because of the

278

formation of a chemical bond between the support and the agarases, which can

13



279

potentially cause variations in the structure of the enzyme, changing its catalytic

280

activity.42 Enzyme activity and enzyme recovery ratio are widely used as indicators to

281

measure the possibility of industrial applications of immobilized enzymes.43

282

Therefore, in order to improve the activity and enzyme recovery rate of the

283

immobilized enzyme, four parameters (i.e., the amount of enzyme added,

284

immobilization time, temperature, and pH) affecting the enzyme immobilization were

285

optimized in the present study.

286

Different amounts of AgWH50B (50-2000 µg) were used for immobilization on

287

10.0 mg of the modified magnetite nanoparticles. As shown in Figure.2a, the amount

288

of the immobilized AgWH50B increased with increasing initial amount of AgWH50B,

289

and the relative activity reached 91.0% at 283.50 µg of AgWH50B. Then, the relative

290

activity did not significantly increase with increasing initial amount of AgWH50B.

291

The amount of immobilized AgWH50B and the relative activity of AgWH50B versus

292

reaction time are shown in Figure.2b. It was found that, with the reaction time

293

increasing from 0 h to 5 h, the amount of immobilized agarase increased and remained

294

constant after approximately 4 h. This may be because the amino-group of agarase

295

blocked most of the TCT groups on the surface of magnetic nanoparticles after that

296

time period. However, the relative activity of the immobilized AgWH50B increased

297

with reaction time up to 0.5 h, then it remained constant. This behavior can be related

298

to some unfavorable protein-protein interactions. Thus, the optimal time was

299

considered to be 0.5 h. Figure.2c shows that the temperature of immobilization of

300

AgWH50B, between 10.0-30.0oC, had similar effectiveness of immobilization.

14


Page 14 of 49

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301

Thereafter, the relative activity sharply decreased with increasing temperature. This

302

behavior can be related to agarase denaturation. As shown in Figure.2d, the

303

immobilization of AgWH50B increased from pH 4.0 to 8.0, and above this value

304

decreased. However, a maximum value of the relative activity was reached at pH 8.0

305

in the phosphate buffer solution.

306

Similar

to

AgWH50B,

the

α-neoagarobiose

hydrolase

AgaWH117

307

immobilization conditions were also optimized. Figure.2e shows that the amount of

308

the immobilized AgaWH117 increased with increasing initial amount of AgaWH117.

309

Although the initial amount of AgaWH117 was 2000 µg, the TCT groups on the

310

surfaces of MNPs were not saturated; the relative activity reached 85.8% at 200.0 µg

311

of AgaWH117. Thereafter, the relative activity did not significantly increase with

312

increasing initial amount of AgaWH117. As shown in Figure.2f, the relative activity

313

of AgaWH117 reached 78.7% with reaction time of up to 15 min, but the relative

314

activity of AgaWH117 dropped to 76.7% with reaction time of up to 0.5 h. Thus,

315

within 0.5 h, the reaction was unstable. The relative activity of AgaWH117 almost

316

reached 92.9% after 1 h of immobilization. Temperature of immobilization of

317

AgaWH117 was studied in the range10.0-60.0oC (Figure.2g). It can be observed that

318

the relative activity of AgaWH117 decreased gradually with the increase of

319

temperature of the immobilization reaction. The optimum temperature is 10.0oC.

320

Concerning the pH effect, the maximum value of relative activity was reached at pH

321

8.0 in the phosphate buffer (Figure.2h).

322

Study on the properties of immobilized enzymes. pH is one of the important

15



323

criteria for industrial application of agarases. As shown in Figure.3a, immobilized

324

AgWH50B maintained high relative activity in a wider range of pH conditions. The

325

optimal pH of the immobilized AgWH50B was 7.0, and the immobilized AgWH50B

326

had above 90.0% relative activity in the pH range of 6.0-8.0. Compared with the free

327

AgWH50B,6 the immobilized AgWH50B showed better pH tolerance. Figure.3b

328

shows that the optimal pH of AgaWH117 was also 7.0, shifting from pH 6.0 for the

329

free AgaWH117. Other researchers43, 44 also covalently immobilized some enzymes

330

on nanoparticles and observed shifts in optimal pH after immobilization. The results

331

of the present study showed that the immobilized AgWH50B and AgaWH117 had the

332

optimal activity at the same pH. It further can improve the catalytic efficiency when

333

co-immobilized enzymes catalyze multiple reactions in a pot.

334

The optimal temperature of the free enzyme was 40.0oC and 27.5oC for

335

AgWH50B and the free AgaWH117, respectively. The results showed that the

336

agarases immobilization on CC-Fe3O4@SiO2 reduced the temperature of optimal

337

catalytic activity. The optimum of activity was observed at 35.0oC (Figure.4a) and

338

22.5oC (Figure.4c), for the immobilized form of AgWH50B and AgaWH117,

339

respectively. The structures of the agaro-oligosaccharides can easily change at a

340

higher temperature, producing by-products, so immobilization keeps structural

341

stability of products at a lower optimum reaction temperature. Figure.4a and Figure.4c

342

also shows that the immobilized AgWH50B and AgaWH117 had high activity and

343

thermal stability below 35.0oC. In addition, AgWH50B was incubated for 12 h at

344

30.0oC and 35.0oC separately, and the relative activity of AgWH50B was still more

16


Page 16 of 49

Page 17 of 49


345

than 90.0% (Figure.4b). However, the thermal stability of AgaWH117 was poor

346

(Figure.4d).

347

Enhancing the thermostability of α-neoagarobiose hydrolase. In order to

348

enhance the thermal stability of AgaWH117, mutants (56 Lys, 61 Tyr, 62 His, 94 His,

349

134 Lys, 136 Tyr, 139 Tyr, 201 His, 216 Tyr, 218 Tyr, and 268 His) were constructed

350

by site-directed mutagenesis. Only the mutant 134 Lys exhibited both higher residual

351

activities than AgaWH117 after incubating at 40.0oC for 10 and 20 min, revealing that

352

the mutant 134 Lys had better thermal stability (Figure.S3a). Therefore, we attempted

353

to construct and characterize AgaWH117 mutant proteins using site-saturation

354

mutagenesis of the 134th residue (Lys) for further study. After incubating at 40.0oC for

355

20 min, 19 mutants with a 134th Lys substitution retained AgaWH117 activity, in

356

which K134D indicated the highest activity (115.0%) and the best thermal stability

357

(Figure.S3b). To further indicate the potential of K134D, the thermal stability at

358

different temperatures (25.0-50.0oC) was tested (Figure.S3c). As shown, after 30 min

359

of pre-incubation at 40.0oC, AgaWH117 almost completely lost its activity, while

360

K134D retained about 25.0% of the maximum activity. K134D still kept a detectable

361

activity even after incubation at 40.0oC for 40 min. Enhancement of both activity and

362

thermal stability of K134D clearly shows its industrial potential.

363

The catalytic properties of K134D, including optimal temperature and pH, were

364

also studied. As shown in Figure.5a, the activity of K134D increased with increasing

365

temperature and reached its peak at 30.0oC shifting from 27.5oC for AgaWH117. The

366

change in the trend of K134D activity with pH was consistent with that of AgaWH117

17



367

Page 18 of 49

(Figure.S3d), and the optimal pH was found to be pH 6.0.

368

K134D which had better thermal stability was immobilized on CC-Fe3O4@SiO2

369

using the same method used for immobilizing AgaWH117. Figure.5b shows that the

370

immobilized K134D and the immobilized AgaWH117 had a optimum of activity at

371

the same temperature. However, compared with the immobilized AgaWH117, the

372

immobilized K134D had a higher relative activity at the same temperature. When

373

reaction temperature was below 30.0oC, the relative activity of the immobilized

374

K134D was approximately 10.0% higher than that of the immobilized AgaWH117.

375

The relative activity of the immobilized K134D was approximately 20% higher than

376

that of the immobilized AgaWH117 at 35.0oC.

377

Study on the co-immobilization of AgWH50B and K134D. Co-immobilized

378

enzymes reaction enables the combination of cascade reactions in one step. As shown

379

in Figure.2a and Figure.2e, when the relative activity of agarase reached a peak value,

380

the TCT groups on the surface of MNPs were not saturated. Therefore, we considered

381

that two agarases were co-immobilized on the same support to achieve a one-step

382

reaction to produce L-AHG.

383

Due to over saturation of enzyme molecules on MNPs, causing some

384

unfavorable

protein-protein

interactions,

the

catalytic

385

co-immobilized enzymes will decrease. When co-immobilized enzymes have high

386

catalytic efficiency, the catalytic efficiency of AgWH50B is expected to be equivalent

387

to that of K134D. The specific enzyme activity of a fresh enzyme solution is constant.

388

The performance ratio and the recovery ratio of enzyme activity show certain values

18


efficiency

of

the

Page 19 of 49


389

at certain added enzyme amount. In order to improve the catalytic performance of the

390

co-immobilized enzymes, the amount of AgWH50B added was optimized. The

391

relative activity of the co-immobilized enzymes versus the added amount of

392

AgWH50B is shown in Figure.6a. It was found that, by increasing the added amount

393

of AgWH50B from 0 to 206 µg, the relative activity of the co-immobilized enzymes

394

increased, and then decreased thereafter. The relative activity reached a maximum

395

value at 206.0 µg of AgWH50B. Figure.6b shows that the optimum activity of the

396

co-immobilized enzymes was observed at 27.5oC, and it remained similar up to

397

30.0oC, then it showed a sharp decline.

398

The operational stability of the co-immobilized agarases was determined by

399

reusing it eight times. According to Figure.7, the co-immobilized agarases were found

400

to be active during reuse, and 46.4% of its initial hydrolytic activity was obtained

401

even after eight cycles, which also showed that the residual activity fell slowly and

402

remained almost constant after six cycles. Although the activities of the

403

co-immobilized agarases decreased slightly, the co-immobilized agarases had better

404

durability and practicability than the high-cost pure free agarases.

405

Comparisons between the co-immobilized and free agarases. The

406

co-immobilized and free agarases were equal in amounts to the co-immobilized

407

agarases reacted with agarose separately. The reaction temperature of the

408

co-immobilized enzymes and agarose was 27.5oC. The reaction temperatures of free

409

agarases were 27.5oC and 35.0oC, respectively. According to Table 1, the yield of

410

L-AHG, which is the co-immobilized agarases reacted with agarose to obtain L-AHG,

19



411

was 145964.85 µg. However, the yields of L-AHG when the free agarases were used

412

with agarose to obtain L-AHG were 102141.99 µg and 100642.87 µg at 27.5oC and

413

30.0oC, respectively. Obviously, under the same condition, the co-immobilized

414

enzymes had a higher catalytic performance than the free enzymes.

415

In addition, the HPLC result of the hydrolysates obtained when the agarases

416

hydrolyzed agarose is shown in Figure.S5. The hydrolysates obtained by free agarases,

417

except NA4 and AO3, were complex. The peak at 7.09 min was not identified;

418

therefore, it may be a derivative of NA4 or AO3 produced at a higher temperature

419

(above 35oC). The small peaks between 8.00-10.00 min (Figure.S5a) were identified

420

by high resolution HPLC-Mass Spectra (HPLC-MS) as some compounds containing

421

nitrogen element (Figure.S7); they may be some amino acids that are not completely

422

removed. Therefore, this showed that the hydrolysates were polluted by the free

423

agarases. The peak of the standard 3,6-anhydro-D-galactose (D-AHG) was at 11.05

424

min (Figure.S6), which should be similar to L-AHG. And HPLC-MS confirmed that

425

the peak of the products at 11.05 min is L-AHG (Figure.S8). On the basis of the

426

retention time,

427

but may be a derivative of L-AHG produced at a higher temperature (above 35oC) as

428

observed using high resolution HPLC-MS. Figure.S5b showed that there was only a

429

small peak between 8.00-10.0 min. A large amount of L-AHG was obtained. In

430

summary, the co-immobilized enzyme showed great advantages.

it can be suggested that the peak at 10.61 min might be not L-AHG,

431

In summary, we successfully employed a covalent method to co-immobilize

432

β-agarase AgWH50B and α-neoagarobiose hydrolase mutant K134D onto

20


Page 20 of 49

Page 21 of 49


433

CC-Fe3O4@SiO2 nanoparticles to fabricate a two-enzyme system, which was an

434

efficient biocatalyst in the one-pot conversion of agarose to L-AHG and AO3. The

435

co-immobilized agarases exhibited a relatively high agarose-to-L-AHG and AO3

436

conversion efficiency. The yield of L-AHG obtained by the co-immobilized agarases

437

was 40.6%, which was 6.5% higher than that obtained by the free agarases, and had a

438

considerable reusability. To our knowledge, this is the first report to co-immobilize

439

two agarases to produce L-AHG, which cannot be obtained commercially. Compared

440

with free agarases, the co-immobilized agarases exhibited greater advantages,

441

providing a novel method to improve the yield of L-AHG.

442

■ABBREVIATIONS

443

L-AHG:

3,6-anhydro-L-galactose;

agarotriose;

neoagarotetraose;

NABH:

FTIR:

444

fourier transform infrared;

445

hydrolase;

446

silica-coated

447

trichlorotriazine-functionalized

448

3-(triethoxysilyl)-propylamine; BSA: Bovine serum albumin; tetraethylorthosilicate;

449

THF:

450

trichlorotriazine-functionalized

451

3,5-dinitrosalicylic

452

HPLC-ESI-Q-TOF-MS: HPLC-Electrospray ionization-Quadrupole-Time of Flight

453

Mass Spectrometry; D-AHG: 3,6-anhydro-D-galactose; SDS: sodium dodecyl sulfate;

454

PAGE: polyacrylamide gel electrophoresis;

TCT:

NA4:

AO3:

trichlorotriazine;

AP-Fe3O4@SiO2:

magnetite

nanoparticles

△Gqq:

tetrahytrofuran;

acid;

silica-coated

Gibbs

silica-coated

HPLC:

magnetite

free

magnetite

high-performance

21


α-neoagarobiose

amino-functionalized CC-Fe3O4@SiO2: nanoparticles;

APTES:

energy;

MNPs:

nanoparticles;

DNS:

liquid

chromatography;


455 456

■FUNDINGS This work was supported by the National Natural Science Foundation of China

457

(31471607) and Applied Basic Research Program of Qingdao (16-5-1-17-jch).

458

Notes

459

The authors declare that they have no competing interests.

22


Page 22 of 49

Page 23 of 49


460

Supporting Information

461

Figure S1. HPLC analysis of agarotriose.

462

Figure S2. Demonstration of the magnetic separation of magnetic CC-Fe3O4@SiO2 nanoparticles.

463 464

Figure S3. The thermal stability of AgaWH117 and mutants. (a) The thermal stability

465

of mutants 56 Lys, 61 Tyr, 62 His, 94 His, 134 Lys, 136 Tyr, 139 Tyr, 201 His,

466

218 Tyr, and 268 His, (b) the thermal stability of saturation mutagenesis strains

467

of 134th site, (c) the thermal stability of AgaWH117 and K134D after

468

pre-incubation at 25°C to 50°C for 10-40 min, (d) effects of pH on the activity of

469

AgaWH117 and K134D. (The abbreviation for AgaWH117 is 117.)

470

Figure S4. The standard curve of D-AHG.

471

Figure S5. HPLC analysis of the agarose hydrolysates.

472

Figure S6. HPLC analysis of D-AHG standard substance.

473

Figure S7. HPLC-MS analysis of the agarose hydrolysates with retention time

474

between 8-10 min.

475

Figure S8. HPLC-MS analysis of the agarose hydrolysate with retention time at 11.05

476

min.

477

Table S1. The selected amimo acids.

478

Table S2. Primers of the mutagenesis.

479

23



480

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481

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30


Page 30 of 49

Page 31 of 49


618

Figure legends

619

Figure 1. FTIR spectra of (a) immobilized materials and (b) immobilized enzymes.

620

Figure 2. The effect of the immobilization parameters on the immobilized enzyme

621

activity. (a) The initial amount of AgWH50B, (b) time, (c) temperature, (d) pH,

622

(e) the initial amount of AgaWH117, (f) time, (g) temperature, (h) pH. (■)

623

Relative activity; (□) mg immobilized/g NP. pH4.0-6.0: Citric acid-sodium

624

citrate buffer; pH6.0-8.0: Phosphate buffer; pH7.0-9.0: Tris-HCl buffer;

625

pH9.0-10.0: Glycine-NaOH buffer, the same below.

626 627

Figure 3. The effects of pH on immobilized agarase activity. (a) Immobilized AgWH50B and (b) immobilized AgaWH117.

628

Figure 4. The effects of temperature on immobilized agarase activity and stability. (a)

629

Optimum temperature of the immobilized AgWH50B, (b) thermal stability of the

630

immobilized AgWH50B, (c) optimum temperature of the immobilized

631

AgaWH117, (d) thermal stability of the immobilized AgaWH117.

632 633

Figure 5. The optimum temperature of (a) free and (b) immobilized α-neoagarobiose hydrolases.

634

Figure 6. (a) The effect of different amounts of AgWH50B on the activity of the

635

co-immobilized enzymes and (b) the optimum reaction temperature of

636

co-immobilized enzymes.

637

Figure 7. Catalyst recycling of the co-immobilized agarases.

31



Page 32 of 49

638

Table

639

Table 1. Yield of L-AHG obtained by co-immobilized and free enzymes. co-immobilized

free agarases

free agarases

agarases (27.5oC)

(27.5oC)

(35.0oC)

360

300

300

146±3

102±3

101±2

40.6

34.1

33.6

initial amount of agarose (mg) yield of L-AHG (mg) productivity (%) 640 641

32


Page 33 of 49


642

Figures

643 644

Figure 1. FTIR spectra of (a) immobilized materials and (b) immobilized enzymes.

33



645 646

Figure 2. The effect of the immobilization parameters on the immobilized enzyme

647

activity. (a) The initial amount of AgWH50B, (b) time, (c) temperature, (d) pH,

648

(e) the initial amount of AgaWH117, (f) time, (g) temperature, (h) pH. (■) 34


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Page 35 of 49


649

Relative activity; (□) mg immobilized/g NP. pH4.0-6.0: Citric acid-sodium

650

citrate buffer; pH6.0-8.0: Phosphate buffer; pH7.0-9.0: Tris-HCl buffer;

651

pH9.0-10.0: Glycine-NaOH buffer, the same below.

652

35



653 654

Figure 3. The effects of pH on immobilized agarase activity. (a) Immobilized

655

AgWH50B and (b) immobilized AgaWH117.

656

36


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Page 37 of 49


657 658

Figure 4. The effects of temperature on immobilized agarase activity and stability. (a)

659

Optimum temperature of the immobilized AgWH50B, (b) thermal stability of the

660

immobilized AgWH50B, (c) optimum temperature of the immobilized AgaWH117, (d)

661

thermal stability of the immobilized AgaWH117.

662

37



663 664

Figure 5. The optimum temperature of (a) free and (b) immobilized α-neoagarobiose

665

hydrolases.

666

38


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Page 39 of 49


667 668

Figure 6. (a) The effect of different amounts of AgWH50B on the activity of the

669

co-immobilized

670

co-immobilized enzymes.

enzymes

and

(b)

the

optimum

671

39


reaction

temperature

of


672 673

Figure 7. Catalyst recycling of the co-immobilized agarases.

674

40


Page 40 of 49

Page 41 of 49


675 676

TOC Graphic

677 678

41



Figure 1. FTIR spectra of (a) immobilized materials and (b) immobilized enzymes. 150x53mm (300 x 300 DPI)


Page 42 of 49

Page 43 of 49


Figure 2. The effect of the immobilization parameters on the immobilized enzyme activity. (a) The initial amount of AgWH50B, (b) time, (c) temperature, (d) pH, (e) the initial amount of AgaWH117, (f) time, (g) temperature, (h) pH. (■) Relative activity; (□) mg immobilized/g NP. pH4.0-6.0: Citric acid-sodium citrate buffer; pH6.0-8.0: Phosphate buffer; pH7.0-9.0: Tris-HCl buffer; pH9.0-10.0: Glycine-NaOH buffer, the same below. 106x150mm (300 x 300 DPI)



Figure 3. The effects of pH on immobilized agarase activity. (a) Immobilized AgWH50B and (b) immobilized AgaWH117. 150x53mm (300 x 300 DPI)


Page 44 of 49

Page 45 of 49


Figure 4. The effects of temperature on immobilized agarase activity and stability. (a) Optimum temperature of the immobilized AgWH50B, (b) thermal stability of the immobilized AgWH50B, (c) optimum temperature of the immobilized AgaWH117, (d) thermal stability of the immobilized AgaWH117. 150x106mm (300 x 300 DPI)



Figure 5. The optimum temperature of (a) free and (b) immobilized α-neoagarobiose hydrolases. 150x53mm (300 x 300 DPI)


Page 46 of 49

Page 47 of 49


Figure 6. (a) The effect of different amounts of AgWH50B on the activity of the co-immobilized enzymes and (b) the optimum reaction temperature of co-immobilized enzymes. 150x53mm (300 x 300 DPI)



Figure 7. Catalyst recycling of the co-immobilized agarases. 150x106mm (300 x 300 DPI)


Page 48 of 49

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TOC 85x49mm (300 x 300 DPI)


Coimmobilization of Î²-Agarase and Î±-Neoagarobiose Hydrolase for

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