Efficient Immobilization of Bacterial GH Family 46 Chitosanase by

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Biotechnology and Biological Transformations

Efficient immobilization of bacterial GH family 46 chitosanase by carbohydrate-binding module fusion for the controllable preparation of chitooligosaccharides Si Lin, Zhen Qin, Qiming Chen, Liqiang Fan, Jiachun Zhou, and Liming Zhao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01608 • Publication Date (Web): 27 May 2019 Downloaded from http://pubs.acs.org on May 28, 2019

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Journal of Agricultural and Food Chemistry

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Efficient immobilization of bacterial GH family 46 chitosanase by carbohydrate-

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binding module fusion for the controllable preparation of chitooligosaccharides

3

Si Lina†, Zhen Qinab†, Qiming Chenab, Liqiang Fanab, Jiachun Zhouab, and Liming

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Zhaoab*

5 6

a

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Center of Separation and Extraction Technology in Fermentation Industry, East China

8

University of Science and Technology, Shanghai 200237, China

9

b

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School of Biotechnology, State Key Laboratory of Bioreactor Engineering, R&D

Shanghai Collaborative Innovation Center for Biomanufacturing Technology

(SCICBT) , Shanghai 200237, China

11 12

† These

13

*

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E-mail address: [email protected] (L.M. Zhao)

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Fax: +86 021-64250829

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No. 130 Meilong Road, Shanghai 200237, China

authors contributed equally in this study.

Corresponding author.

17 18 19 20 21 22

ACS Paragon Plus Environment


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Abstract

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Chitooligosaccharide has been reported to possess diverse bioactivities. The

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development of novel strategies for obtaining optimum degree of polymerization (DP)

26

chitooligosaccharides has become increasingly important. In this study, two glycoside

27

hydrolase family 46 chitosanases were studied for immobilization on curdlan (insoluble

28

β-1,3-glucan) using a novel carbohydrate binding module (CBM) family 56 domain

29

from a β-1,3-glucanase. The CBM56 domain provided a spontaneous and specific

30

sorption of the fusion proteins onto a curdlan carrier, and two fusion enzymes showed

31

increased enzyme stability in comparison with native enzymes. Furthermore, a

32

continuous packed-bed reactor was constructed with chitosanase immobilized on a

33

curdlan

34

chitooligosaccharide products with different molecular weights were prepared in

35

optimized reaction conditions. This study provides a novel CBM tag for the

36

stabilization and immobilization of enzymes. The controllable hydrolysis strategy

37

offers potential for the industrial-scale preparation of chitooligosaccharides with

38

different desired DPs.

carrier

to

control

the

enzymatic

hydrolysis

of

chitosan.

Three

39 40

Keywords: Chitosanase; Carbohydrate binding module; Chitooligosaccharide; One-

41

step immobilization-purification; Packed-bed reactor

42 43 44


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INTRODUCTION

46

Chitooligosaccharides (COS), the hydrolyzed product of chitosan, are oligomers of

47

β-1,4-linked D-glucosamine with high solubility in neutral aqueous solutions.

48

Chitooligosaccharide has various beneficial biological activities, such as antimicrobial

49

activity

50

activity 5 and immunostimulatory activity 6. As a result of these biological activities,

51

chitooligosaccharides are widely applied in various fields, including functional foods,

52

agriculture and pharmacy.

1-2,

antioxidant activity 3, anti-inflammatory activity 4, anti-tumor/anticancer

53

A variety of techniques have thus far been used for the production of

54

chitooligosaccharides from chitosan, including chemical 7-8, physical 9-10 and enzymatic

55

methods 11-12. In terms of high safety levels and low toxicity, the enzymatic degradation

56

method has shown great potential in industrial applications. Chitosanase (E C 3.2.1.132)

57

is a specific chitosan degrading enzyme. Most of the reported chitosanases are endo-

58

type glycoside hydrolases, which hydrolyze the β-1,4-linked glycosidic bond inside the

59

chitosan chain, releasing a mixture of chitooligosaccharides with uncontrollable

60

degrees of polymerization (DP)

61

biological activities of chitooligosaccharides are related to their DP 15. Although there

62

are many advantages in the enzymatic preparation of chitooligosaccharides, obtaining

63

chitooligosaccharides with the desired DP is generally time consuming and challenging.

64

The persistent instability and the high cost of the process restricts the application of

65

chitosanases in large-scale industrial applications

66

highly efficient bioconversion process for the industrial-scale production of

13-14.

However, previous studies have shown that the

16

and, therefore, a controllable and



67

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chitooligosaccharides is sought.

68

Carbohydrate binding modules (CBMs) are independent domains existing in

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carbohydrate active enzymes. The majority of such domains exhibit carbohydrate

70

binding activity, which enhances the catalytic efficiency of carbohydrate active

71

enzymes 17. Due to this highly specific binding ability towards insoluble carbohydrate,

72

CBMs have been used as efficient immobilization tools of recombinantly fused proteins

73

for different applications 18-20. Mai-Lan et al. 21 reported the construction of three fusion

74

enzymes composed of the chitin-binding domain and β-galactosidase. The fusion β-

75

galactosidases were immobilized on chitin beads to provide an approach for lactose

76

hydrolysis and the production of prebiotic galacto-oligosaccharides. Chang et al.

77

fused six different CBMs to the double-site variant of β-glucosidase to effectively

78

hydrolyze soybean isoflavone glycosides. Furthermore, other CBM-fusion strategies

79

enhance the properties (such as catalytic efficiency, organic solvent tolerance and

80

stability) of target enzymes 23-25.

81

22

In this study a novel CBM family 56 β-1,3-glucan binding module, derived from 26,

82

the Paenibacillus barengoltzii β-1,3-glucanase (PbBgl64A)

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glycoside hydrolase (GH) family 46 chitosanases. The catalytic properties of the fusion

84

proteins were then evaluated for the preparation of immobilized enzymes. On this basis,

85

the fusion chitosanase was immobilized into curdlan support and then loaded in a

86

continuous packed-bed reactor with the potential to control the enzymatic hydrolysis

87

process of chitosan. This study thereby provides a novel CBM tag for the stabilization

88

and immobilization of enzymes. The subsequent controllable hydrolysis strategy offers


was fused to two

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the potential for the industrial-scale preparation of different desired DPs

90

chitooligosaccharides products.

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MATERIALS AND METHODS

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Materials and microorganisms

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The DNA amplification was performed with EasyPfu DNA Polymerase (TransGen

95

Biotech, Beijing, China). Escherichia coli DH5α and BL21 (DE3) strains were

96

purchased from Cwbio (Beijing, China) and used as cloning and protein expression

97

hosts, respectively. Both hosts were cultivated in Luria-Bertani (LB) medium.

98

Kanamycin was purchased from Biosharp (Beijing, China) and used as a selectable

99

marker at 50 μg/mL. pET-28a(+) vector was used as an expression vector. Restriction

100

enzymes were purchased from Takara (Otsu, Japan). Chitosan (degree of

101

deacetylation≥95 %, viscosity < 200 mPa.s) was purchased from Aladdin (Beijing,

102

China). Curdlan was purchased from Shandong Zhongke (Shandong, China). All other

103

chemicals were analytically pure.

104

Construction of fusion enzymes

105

In our previous study, two GH family 46 chitosanases (GsCsn46A and BaCsn46A)

106

from different bacteria were identified and expressed. GsCsn46A was a cold-adapted

107

chitosanase from Gynuella sunshinyii

108

amyloliquefaciens, which showed high specific activity towards chitosan (1031.2 U/mg)

109

to yield a series of chitooligosaccharides 29. These two chitosanases were typical GH

110

family 46 chitosanases, which are widely used in the industrial preparation of

27.

And BaCsn46A was from Bacillus



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111

chitooligosaccharides. Additionally, in our previous structural study of a GH family 64

112

β-1,3-glucanase from Paenibacillus barengoltzii (PbBgl64A)

113

family 56 β-1,3-glucan binding module (CBM56) was detected in the N-terminus of

114

PbBgl64A. Genes used in this study are listed in Table 1 and gene sequence information

115

are listed in supplemental Table S1.

26,

a potential CBM

116

Two fusion proteins were constructed, based on the nucleotide sequences of

117

GsCsn46A, BaCsn46A and CBM56. CBM56 was linked at the respective N-terminus

118

of two chitosanases, and a natural loop linker, ‘-SGQPDPEPS-’ from PbBgl64A, was

119

inserted between CBM56 and the chitosanases in the fusion protein. A total of seven

120

PCR primers were used in this study, as listed in Table 2. Both fusion genes (CBM56-

121

GsCsn46A and CBM56-BaCsn46A) were constructed by overlapping PCR, and

122

engineering the CBM56 gene into the 5’-terminus of GsCsn46A gene and BaCsn46A

123

gene, respectively. The detailed protocol of overlapping PCR is described as follows.

124

The gene fragment of CBM56-1 and GsCsn46A, for the construction of CBM56-

125

GsCsn46A, were amplified by PCR using primers (F1, R1-Gs) and primers (F2-Gs, R2-

126

Gs). And the fragments of CBM56-2 and BaCsn46A, for the construction of CBM56-

127

BaCsn46A, were amplified by PCR using primers (F1, R3-Ba) and primers (F4-Ba, R4-

128

Ba). The template were pUC57-CBM56 plasmid, pET28a(+)-GsCsn46A plasmid and

129

pET28a(+)-BaCsn46A plasmid respectively. Using the amplified CBM56-1,

130

GsCsn46A as templates and F1, R2-Gs as primers, overlapping of PCR for the entire

131

fusion gene, CBM56-GsCsn46A, was carried out as follows: 30 cycles at 94 °C for 5

132

min, 55 °C for 30 s, and 72 °C for 2 min. Similarly, another entire fusion gene, CBM56-


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BaCsn46A, was amplified using the PCR products CBM56-2 and BaCsn46A, along

134

with the primers F1- and R4-Ba, under the same overlapping conditions. The amplified

135

target PCR products were gel-purified using a Gel Extraction Kit (Sangon Biotech,

136

Shanghai, China), digested with NheI and XhoI, and inserted into the pET-28a(+) vector,

137

5 μL recombinant plasmid was added into 100 uL E. coli DH5α, 37 °C at 200 rpm for

138

45 min, and the transformants were cultured on LB agar plates containing 50 μg/mL

139

Kanamycin at 37 °C overnight.

140

Expression and purification of fusion enzymes

141

The constructed plasmids were transformed into E. coli BL21 (DE3) and cultured

142

on LB-agar plates containing Kanamycin (50 μg/mL). The expression of fusion

143

enzymes was induced at OD600 of about 0.6–0.8 by adding isopropyl β-D-1-

144

thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM and further incubating

145

at 30°C at 200 rpm overnight.

146

The fusion enzyme was purified, as described previously 27. The E. coli cells were

147

harvested using centrifugation at 10000 ×g for 5 min. The cells were then re-suspended

148

in buffer A (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 20 mM imidazole). The target

149

proteins were released from the cells by sonication (the diameter of ultrasonic horn is

150

6 mm, ultrasonic 2 s, pause 3 s, repeat 10 min, power 650 W). The debris was removed

151

by centrifugation at 10000 ×g for 10 min, and the supernatant was pumped into a Ni-

152

IDA column (GE life Sciences) pre-equilibrated with buffer A. Then the recombinant

153

protein was eluted by step gradient of 20-200 mM imidazole. The resulting samples



154

were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-

155

PAGE) using 12.5% separation gel. The protein concentration was determined by

156

means of a Bradford protein assay kit (Solarbio, Beijing, China), using BSA as the

157

standard.

158

Chitosanase activity assay

159

Chitosanase activity was determined by the 3,5-dinitrosalicylic acid (DNS) method,

160

as described previously 27. The content of reducing sugar was determined by measuring

161

OD at 540 nm, using D-glucosamine as standard. The chitosanase activity of CBM56-

162

BaCsn46A was determined using the same method, except that the substrate mixture

163

was prepared in a 100 mM sodium acetate buffer pH 6.0, and the reaction mixture was

164

incubated at 50 °C for 10 min. All reactions were done in triplicate and their mean and

165

standard deviation values were used for analysis. One unit (U) of chitosanase activity

166

is defined as the amount of enzyme liberating 1 μmol D-glucosamine-equivalent

167

reducing sugars per minute under the above conditions.

168

Characterization of two fusion enzymes  

169

Chitosan was used as the substrate to determine the characterization of the fusion

170

enzymes. With respect to the enzymatic characterization of CBM56-GsCsn46A, the

171

optimal pH was determined by measuring the activity levels from 3.0 to 9.0 using the

172

following 0.2 M buffers: Mcllvaine buffer (pH 2.5–5.0), sodium acetate buffer (pH 4.5–

173

6.0), phosphate buffer (pH 6.0–7.5), Tris-HCl buffer (pH 7.5–9.0) and glycine-NaOH

174

buffer (pH 9.0–11.0). To determine the pH stability, residual activity was measured


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175

after incubation of the enzyme at 25 °C for 30 min in the various 0.2 M buffers. The

176

optimal temperature was determined at 15–60 °C in 0.2 M sodium acetate buffer, pH

177

5.5. Enzymatic activity was determined under standard conditions. Thermostability of

178

the enzyme was determined by measuring residual activity after incubation of the

179

enzyme at various temperatures (15–60 °C) for 30 min in 0.2 M sodium acetate buffer,

180

pH 5.5. For CBM56-BaCsn46A, the optimal pH was measured in various 0.2 M buffers,

181

pH (4.0–8.0). The pH stability of CBM56-BaCsn46A was measured by incubation of

182

the enzyme in the range of pH (2.5–10.5) at 25 °C for 30 min. Optimal temperature of

183

CBM56-BaCsn46A was measured at temperatures of 30–70 °C in 0.2 M sodium acetate

184

buffer, pH 6.0. The thermal stability was investigated at 30–70 °C and determined in

185

0.2 M sodium acetate buffer, pH 6.0.

186

Immobilization efficiency of fusion protein onto curdlan

187

Food grade curdlan, washed three times by sodium acetate buffer (50 mM, pH 6.0),

188

was employed as the carrier for immobilization. The curdlan (1 g) was dispersed in 20

189

mL sodium acetate buffer (50 mM, pH 6.0), to which 5 mL diluted cell-free crude

190

extracts (containing 1000 U of the fusion chitosanases) was added. The immobilization

191

experiment was carried out at 20 °C with gentle agitation for 10 min. The amount of

192

the immobilized chitosanases was calculated according to the residual chitosanase

193

activity in the supernatant after treating with curdlan. After affinity sorption, the curdlan

194

carrier was washed three times with 50 mM sodium acetate buffer (pH 6.0) and eluted

195

with 1 mL of SDS-PAGE solubilizing buffer (100 mM Tris-HCl buffer pH 8.8



196

containing 1% SDS, 1% β-mercaptoethanol, 30% glycerol, and 0.01% bromphenol blue)

197

at boiling temperature for 5 min. The curdlan beads were removed by centrifugation,

198

and the supernatant was subjected to SDS-PAGE under reducing conditions.

199

Reuse of the immobilized enzyme

200

In order to evaluating the reuse of the immobilized enzyme, 5 mL of suspended

201

immobilized enzyme (chitosanase activity 100 U/mL) were incubated with 100 mL

202

chitosan substrate solution (100 mg/mL, pH 5.5) for 3 hours at 45 °C. Supernatant

203

samples withdrawn at different times were immediately analyzed by DNS method to

204

evaluate the reducing sugar content of chitooligosaccharides products. The relative

205

reducing sugar content of the products represents the catalytic activity of the

206

immobilized enzyme resin. After each reaction, the immobilized enzyme was washed

207

with 50 mL 0.1 M sodium acetate buffer pH 5.5 to remove residual substrate, then

208

stored at 4°C until the next repetition of the batch reaction. The final concentration of

209

reducing sugar in the first cycle reaction was defined as 100 % relative reducing sugar

210

content. The immobilized enzyme reuse experiment was repeated six times.

211

Application of immobilized chitosanase in packed-bed reactor

212

In order to achieve control in the preparation of chitooligosaccharides with different

213

DPs, a packed-bed reactor was built to evaluate the application of the immobilized

214

chitosanase. In consideration of the catalytic efficiency and stability of the immobilized

215

chitosanase, CBM56-BaCsn46A was selected for further research into the controllable

216

preparation of chitooligosaccharides. Briefly, 10 g curdlan was dispersed in 100 mL


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217

sodium acetate buffer (50 mM, pH 6.0), incubated with 25 mL cell-free crude extracts

218

at 20 °C with gentle agitation for 20 min. After affinity sorption, the curdlan carrier was

219

washed three times with 50 mM sodium acetate buffer (pH 6.0). The curdlan-

220

immobilized enzyme was separated from the supernatant by centrifugation and

221

resuspended in 250 mL sodium acetate buffer (50 mM, pH 6.0). The immobilized

222

chitosanase activity was determined using suspension by the standard enzyme assay

223

conditions mentioned above.

224

The resuspended curdlan immobilized enzyme was placed in a water-jacketed glass

225

column (diameter: 45 mm, height: 300 mm) and silica sand (5 g, particle size 100–150

226

μm) was mixed with the curdlan powder to ensure flux. The enzyme amount, flow rate

227

and concentration of the chitosan substrate were all optimized as follows to regulate the

228

chitosan hydrolysis reaction process:

229

(1) The effect of the enzyme amount: Different amounts (immobilized chitosanase

230

range from 50 U to 1000 U) of the abovementioned resuspended curdlan (containing

231

3888 U protein/g) were placed into the column. The substrate solution (1 % (w/v)

232

chitosan, 50 mM sodium acetate buffer, pH 5.0) was pumped into the packed-bed

233

reactor from top to bottom by peristaltic pump at a flow rate of 2.0 mL/min.

234

(2) The effect of the flow rate: Resuspended curdlan (immobilized 250 U chitosanase)

235

and silica sand were placed into the column. The substrate solution (containing 1 %

236

(w/v) chitosan and 50 mM sodium acetate buffer, pH 5.0) was pumped into the column

237

at different flow rates (0.5 to 4 mL/min) to control the reaction times.



238

(3) The effect of the concentration of chitosan: In order to evaluate the influence of

239

substrate solution concentrations on chitooligosaccharide production, four different

240

concentrations of chitosan solution (ranging from 0.5 % to 2.0 %, w/v) were used for

241

the chitosan hydrolysis.

242

All abovementioned reaction temperature was stabilized at 45 °C by circulating

243

thermostated water in the jackets surrounding the enzyme columns. The reaction

244

product was collected for further analysis.

245

Chitooligosaccharide analysis methods

246

The relative average molecular weight of chitosan hydrolysate is measured using

247

the acetylacetone method 28. The acetylacetone solution was prepared by transferring

248

3.5 mL of acetylacetone into 46.5 mL buffer solution (1 M sodium carbonate). A

249

dimethyl benzaldehyde solution was prepared using 0.8 g dimethyl benzaldehyde

250

mixed with 15 mL ethanol (AR, 100 %) and 15 mL hydrochloric acid (AR, 37 %). To

251

determine the relative average molecular weight of the reaction product, 1 mL

252

acetylacetone solution was added to the 5 mL chitooligosaccharide sample (1 mg/mL),

253

following by a boiling water bath for 25 min. After cooling to room temperature, 1 mL

254

dimethyl benzaldehyde solution and 3 mL ethanol (100 %) were added to the reaction

255

mixture. The mixture was incubated in a 60°C water bath for 1 h, then rapidly cooled

256

to room temperature. The relative average molecular weight was determined by

257

measuring OD at 525 nm using D-glucosamine as standards.

258

The reaction products withdrawn at different times were immediately boiled for 5


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259

min, and then analyzed by thin-layer chromatography (TLC). Samples (1 μL) were

260

spotted and developed on a silica gel plate (TLC aluminum sheets silica gel 60; Merck,

261

Darmstadt, Germany) with an isopropanol-water-28 % ammonia (75:10:15) mixture as

262

the developing solvent. The hydrolysis products were visualized by spraying them with

263

alcohol: p-anisaldehyde: sulfuric acid: acetic acid (89:5:5:1, v/v) and then heating the

264

plate at 130 °C in an oven for a few minutes.

265

The molecular weight distribution of the chitooligosaccharide products was

266

determined by gel permeation chromatography (GPC) (P230; Elite, Dalian, China).

267

Separation of chitooligosaccharides was achieved by PL aquagel-OH MIXED-H 8 μm

268

(7.5 mm×30 cm; Agilent, Santa Clara, California) at 40 °C. The sodium nitrate (0.5 M)

269

was used as mobile phase, with a flow rate of 1.0 mL/min.

270

271

RESULTS AND DISCUSSION

272

Plasmid construction and expression of fusion protein

273

In order to evaluate the novel carbohydrate binding domain (CBM56) for fusion,

274

two GH family 46 chitosanases with different characterization were selected to be fused

275

with the CBM56. The CBM56 was linked at the N-terminus of chitosanases, and a

276

nearby natural loop linker ‘-SGQPDPEPS-’ was inserted between it and the target

277

chitosanases in the fusion protein. The two fusion proteins (CBM56-GsCsn46A and

278

CBM56-BaCsn46A) were constructed in the plasmid pET28a(+) (Fig. 1), respectively,

279

and transformed into E. coli BL21 (DE3). The CBM56 encoding gene and the two



280

chitosanases genes were 255 bp (CBM56), 729 bp (GsCsn46A) and 729 bp

281

(BaCsn46A), respectively.

282

Results showed that the recombinant fusion enzymes were both expressed in the

283

soluble fraction. Two recombinant fusion enzymes were purified by one step of affinity

284

chromatography to electrophoretic homogeneity (Fig. 2). The molecular mass of

285

CBM56-GsCsn46A and CBM56-BaCsn46A were estimated to be about 37 kDa by

286

SDS-PAGE (Fig. 2), which matched its predicted molecular mass (37.5 kDa and 37.8

287

kDa).

288

Catalytic properties of native enzymes and fusion enzymes

289

In order to reveal the effect of the fusion CBM56 tag on the catalytic activity of the

290

native protein, the catalytic properties of the two fusion proteins were determined. The

291

fusion protein CBM56-GsCsn46A exhibited specific activity towards chitosan (303

292

U/mg) at 30 °C in 50 mM sodium acetate buffer, pH 5.5, which is slightly higher than

293

the specific activity of native GsCsn46A (260.87 U/mg)

294

conditions. CBM56-BaCsn46A exhibited highly specific activity towards chitosan

295

(1156.54 U/mg) at 50 °C in 50 mM sodium acetate buffer pH 6.0, which is slightly

296

higher than that of the native BaCsn46A (1031.2 U/mg)

297

conditions. Furthermore, the effects of pH and temperature on the chitosanase activity

298

of two fusion enzymes were examined. The range of optimal pH for the activity of

299

fusion chitosanase was found to be comparable to that of the original enzymes

300

expressed in the same host. CBM56-GsCsn46A displayed maximal chitosanase activity


27

29

under the same reaction

under the same reaction

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301

at pH 6.5 in 50 mM phosphate buffer (Fig. 3A) and exhibited good pH stability within

302

the range of pH 3.5–10.5, retaining over 95 % of its activity (Fig. 3B), while the native

303

chitosanase GsCsn46A showed an optimum pH at 5.5, and exhibited good pH stability

304

within the range of 4.0–8.5 27. CBM56-BaCsn46A also displayed maximal chitosanase

305

activity at pH 6.5 in 50 mM phosphate buffer (Fig. 4A) and exhibited good pH stability

306

within the range of pH 3.0–10.5, retaining over 95 % of its activity (Fig. 4B). The native

307

chitosanase BaCsn46A showed an optimum pH at 6.0, and exhibited good pH stability

308

within the range of 4.0–9.5

309

determined to be 40 °C (Fig. 3C). The fusion enzyme was stable up to 35 °C, with more

310

than 90 % of its activity remaining (Fig. 3D), while the native chitosanase GsCsn46A

311

showed an optimum temperature at 30 °C and maintained stability up to 35 °C, with

312

more than 80 % its activity remaining

313

BaCsn46A was determined to be 50 °C (Fig. 4C). The fusion enzyme was stable up to

314

55 °C, with more than 80 % of its activity remaining (Fig. 3D), while the native

315

chitosanase BaCsn46A showed an optimum temperature at 50 °C and was stable up to

316

45 °C, with more than 80 % of its activity remaining 29.

29.

The optimal temperature for CBM56-GsCsn46A was

27.

The optimal temperature for CBM56-

317

Fusion protein is a common protein engineering approach to improve the

318

performance of target proteins 30. Previous studies showed that the fusion of a small

319

domain can not only improve the thermal stability31-32, but also affect the optimal

320

temperature of target proteins

321

chemical reactions. Thus, increased stability helps the enzyme to catalyze at higher

322

reaction temperatures. The fusion of a small domain could enhance the rigidity of target

33.

In general, higher temperatures increase the rate of



323

protein, and reduce the flexibility of the highly flexible terminal loop, thus increase the

324

protein stability

325

stability, rigidity and activity of target enzymes 31, 34. In this study, the CBM56 domain

326

may increase the rigidity of the fusion protein and stabilize the terminus of the target

327

protein. Thus, the stability of the target chitosanases has been enhanced. However, in

328

other cases, the addition of a fusion protein to a CBM domain at the N- or C-terminus

329

of the enzymes produced a lower catalytic effect than that of the native enzyme 31, 35-36,

330

which suggests that only a specifically suitable CBM can produce synergistic

331

interactions with the catalytic module to enhance the activity and stability of an enzyme

332

31.

333

these two chitosanases.

334

Immobilization of fusion chitosanases on curdlan

30, 33.

Fusion of the CBM domain may, in some cases, enhance the

Thus, CBM56 could be the suitable module to improve the catalytic properties of

335

The immobilization of proteins generally requires time-consuming, multi-step

336

protocols, so there is a clear need for one-step methods to enable the purification and

337

immobilization of enzymes at a low cost. In this study, the CBM56 domain displayed

338

a specific affinity for β-1,3-glucan. One-step immobilization-purification of enzymes

339

may, therefore, be achieved by fusing the CBM56 domain in the recombinant protein,

340

thereby enabling the direct purification and immobilization of enzymes from E. coli

341

crude cell extracts using β-1,3-glucan carrier materials. Here, the suitability of

342

commercially available β-1,3-glucan (curdlan) was chosen for further study (Fig. 5).

343

The fusion proteins crude cell extracts were used in equal volume to study their affinity


Page 16 of 37

Page 17 of 37


344

to curdlan. SDS-PAGE showed that both CBM56-BaCsn46A and CBM56-GsCsn46A

345

were specifically absorbed on curdlan (Fig. 5C and 5D), indicating that this insoluble

346

polysaccharide is able to immobilize and purify enzymes from crude cell extracts.

347

Immobilized efficiency results showed that 896.94 U of CBM56-GsCsn46A and 901.25

348

U of CBM56-BaCsn46A fusion proteins bound to 1 g of curdlan powder from 1000 U

349

crude cell extracts after 20 minutes of incubation at 20 °C, indicating that the loading

350

efficiency of the CBM56-GsCsn46A and CBM56-GsCsn46A was 89.7% and 90.1 %,

351

respectively. This strategy utilizes a natural polysaccharide as the immobilized support

352

without any treatment, making it convenient to use and low cost as well as avoiding the

353

environmental effects of chemically modified supports. In order to evaluating the reuse

354

of the immobilized enzyme, reusability of the immobilized CBM56-BaCsn46A was

355

then investigated by batch hydrolysis of chitosan substrate. The catalytic activity of the

356

reused immobilized enzyme was characterized by the reducing sugars contents during

357

the reaction process. Result showed that immobilized CBM56-BaCsn46A could be

358

used to hydrolyze chitosan for at least six times, with retaining 77.5% of its initial

359

catalytic activity (Figure 5E). This means that the immobilized chitosanase could be

360

reused in batch reactions.

361

Many previous studies have used CBM-fused proteins for immobilization onto an

362

insoluble polysaccharide (mostly, cellulose and chitin). This study provided a novel

363

CBM56 domain for immobilizing enzymes onto curdlan (β-1,3-glucan), a naturally

364

insoluble polysaccharide, which can be used directly as an immobilization carrier.

365

Furthermore, curdlan is edible and, therefore, suitable for use in purifying and



366

immobilizing food enzymes, which can then be used directly for food processing.

367

Although the enzyme immobilization for the preparation of functional oligosaccharides

368

does have some advantages, the complicated process and high-cost immobilization

369

techniques limit their use in industrial application. Among the methods of chitosanase

370

immobilization, the fusion of a CBM56 domain to immobilize enzymes on a curdlan

371

carrier would be a simple, cost-effective strategy, which is worth a further investigate.

372

Controllable preparation of chitooligosaccharides by immobilized BaCsn46A

373

In order to control the enzymatic hydrolysis of chitosan, a continuous packed-bed

374

reactor with chitosanase immobilized on a curdlan carrier was constructed for the

375

controllable preparation of chitooligosaccharides with different DPs. We found that the

376

hydrolysis products and hydrolysis processes of the two chitosanases are similar in the

377

pre-experiment. The use of two enzymes in the experiment does not help to prepare

378

chitooligosaccharides with different degrees of polymerization. In consideration of the

379

catalytic efficiency and stability of the immobilized chitosanase, CBM56-BaCsn46A

380

was selected for the further research about the controllable preparation of

381

chitooligosaccharides. The amount and flow rate of the enzyme and the concentration

382

of chitosan substrate were optimized to regulate the reaction process of chitosan

383

hydrolysis. The DP/average molecular weight of different chitooligosaccharide

384

products obtained by various conditions were analyzed by TLC and the acetylacetone

385

method. Results showed that the ratio of the lower DP chitooligosaccharide product

386

increases as the amount of chitosanase increases (Fig. 6). Here, the relative average


Page 18 of 37

Page 19 of 37


387

weight of chitooligosaccharide product decreased from 4944 Da to 1211 Da along with

388

the increasing amount of chitosanase (Table S2). In contrast, higher DP

389

chitooligosaccharide products were accumulated during the increase in flow rate of the

390

chitosan substrate (Fig. 6) and, with an increase in the substrate pumping rate, a higher

391

molecular weight of COS was gradually accumulated. The relative average weight of

392

chitooligosaccharide product increased from 1156 Da to 2327 Da (Table S2). Thus,

393

changing the flow rate of the chitosan substrate could enable control of the contact times

394

between substrate and enzyme. Increasing the concentration of chitosan slightly

395

increased the average weight of the products (Table S2).

396

The optimized preparation conditions were then utilized in the packed-bed reactor.

397

According to the aforementioned results of the average weight of products, three

398

conditions were selected for the production of different DPs chitooligosaccharides: (1)

399

low molecular weight: 1000 U of immobilized chitosanase, 1 % (w/v) chitosan solution,

400

0.5 mL/min substrate pumping rate; (2) medium molecular weight: 250 U of

401

immobilized chitosanase, 1 % (w/v) chitosan solution, 2 mL/min substrate pumping

402

rate; and (3) high molecular weight: 250 U of immobilized chitosanase, 1 % (w/v)

403

chitosan

404

chitooligosaccharides produced under these three conditions were qualitatively

405

analyzed by means of TLC and HPLC (Fig. 7A and supplementary Fig. S2). As

406

expected, a higher enzyme activity or lower substrate pumping rate led to complete

407

chitosan hydrolysis. Under the first condition, mainly lower DP chitooligosaccharides

408

(DP 2–4) were produced in the continuous hydrolytic process, while higher DP

solution,

4

mL/min

substrate

pumping


rate.

The

different


Page 20 of 37

409

chitooligosaccharides (DP 4–6) were accumulated under the second condition. Under

410

the third condition, chitooligosaccharides with high DPs were the main products. The

411

average molecular weight of the three products was subsequently analyzed by GPC (Fig.

412

7). Chitooligosaccharides products with three different average molecular weights (Mw:

413

7050, 33500 and 79100) were prepared under optimized reaction conditions. Thus,

414

based on the abovementioned immobilized chitosanase (CBM56-BaCsn46A), the

415

continuous packed-bed reactor was able to control the enzymatic hydrolysis of chitosan.

416

Numerous previous studies have reported the value of chitosanases in the

417

preparation of chitooligosaccharides. However, only a few subsequent studies have

418

investigated processes suitable for controllable chitooligosaccharides production.

419

Industrial scale enzymatic preparation of chitooligosaccharides with different desired

420

DPs is still complicated. As the biological activities of chitooligosaccharides are related

421

to their DP, a low-cost method for the preparation of specific DP chitooligosaccharides

422

should be urgently explored. Enzyme membrane reactor is a proven effective method

423

for separating high DP chitooligosaccharides

424

pressurized membrane reactor limits its application on an industrial scale. Protein-

425

engineering of the product specificity of the chitosanase is another successful approach

426

to control the DP of chitooligosaccharide products

427

chitooligosaccharides are still hard to obtain due to the structure of the catalytic groove

428

of chitosanase. In immobilized forms, enzymes are easy to facilitate in continuous

429

production. In this study, a packed-bed reactor was constructed to enable the easy

430

monitoring and termination of the chitosan hydrolytic reaction, though its application

29, 37-38,

nevertheless, the high cost of a


39,

however, high DP

Page 21 of 37


431

is currently limited by preparation of chitooligosaccharides mixtures with definite DP.

432

Three types of chitooligosaccharide products with different ranges of DP were obtained

433

by controlling the reaction conditions, making this approach suitable for the separation

434

and purification of specific chitooligosaccharides in further studies.

435

In conclusion, this work describes an efficient method for immobilizing

436

chitosanases onto curdlan (insoluble β-1,3-glucan) via a β-1,3-glucan-binding domain

437

(CBM56) and using the immobilized enzyme for the controllable preparation of

438

chitooligosaccharides. Two fusion enzymes (CBM56-GsCsn46A and CBM56-

439

BaCsn46A) exhibited improved pH levels and temperature stability after CBM56

440

domain fusion. With respect to the specific sorption of the CBM56 domain, the

441

immobilization process incubated fusion chitosanases onto curdlan under mild

442

conditions, with no additional purification. Based on the above immobilized

443

chitosanase (CBM56-BaCsn46A), a continuous packed-bed reactor was constructed to

444

control the enzymatic hydrolysis of chitosan. Chitooligosaccharide products with

445

different DPs (Mw: 7050, 33500 and 79100) were prepared by optimizing the reaction

446

conditions. This study provides a promising and easy-control approach for controllable

447

chitosan hydrolysis the production of chitooligosaccharides with different DPs on an

448

industrial scale.

449 450

Funding

451

This work was financially supported by the National Natural Science Foundation of

452

China (31701537), “Shu Guang” (15SG28) and “Chen Guang” (17CG28) Project of



453

Shanghai Municipal Education Commission and Shanghai Education Development

454

Foundation, Shanghai Sailing Program (17YF1403500), Fundamental Research Funds

455

for the Central Universities (222201814031, 22221818014), The National Key

456

Research and Development Program of China (2018YFC0311200) and the 111 Project

457

(B18022).

458 459

Supporting Information

460

The Supporting Information is available free of charge on the ACS Publications website.

461 462

References

463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487

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24. Wang, S.; Cui, G.-Z.; Song, X.-F.; Feng, Y.; Cui, Q., Efficiency and Stability Enhancement of Cis-epoxysuccinic Acid Hydrolase by Fusion with a Carbohydrate Binding Module and Immobilization onto Cellulose. Appl. Biochem. Biotechnol. 2012, 168 (3), 708-717. 25. Reyes-Ortiz, V.; Heins, R. A.; Cheng, G.; Kim, E. Y.; Vernon, B. C.; Elandt, R. B.; Adams, P. D.; Sale, K. L.; Hadi, M. Z.; Simmons, B. A.; Kent, M. S.; Tullman-Ercek, D., Addition of a carbohydrate-binding module enhances cellulase penetration into cellulose substrates. Biotechnology for Biofuels 2013, 6. 26. Qin, Z.; Yang, D.; You, X.; Liu, Y.; Hu, S.; Yan, Q.; Yang, S.; Jiang, Z., The recognition mechanism of triple-helical beta-1,3-glucan by a beta-1,3-glucanase. Chem. Commun. 2017, 53 (67), 9368-9371. 27. Qin, Z.; Chen, Q.; Lin, S.; Luo, S.; Qiu, Y.; Zhao, L., Expression and characterization of a novel cold-adapted chitosanase suitable for chitooligosaccharides controllable preparation. Food Chem. 2018, 253, 139-147. 28. Liu Lin, Z. j., Li peng，Zeng fanjun., Determination of Average Degree of Polymerization of Chitosan Oligosaccharides by Spectrophotometry. food science 2010, 09, 318-320. 29. Qin, Z.; Luo, S.; Li, Y.; Chen, Q.; Qiu, Y.; Zhao, L.; Jiang, L.; Zhou, J., Biochemical properties of a novel chitosanase from Bacillus amyloliquefaciens and its use in membrane reactor. Lwt-Food Sci. Technol.2018, 97, 9-16. 30. Yu, K.; Liu, C.; Kim, B.-G.; Lee, D.-Y., Synthetic fusion protein design and applications. Biotechnol. Adv. 2015, 33 (1), 155-164. 31. Han, Y.; Gao, P.; Yu, W.; Lu, X., Thermostability enhancement of chitosanase CsnA by fusion a family 5 carbohydrate-binding module. Biotechnol. Lett 2017, 39 (12), 1895-1901. 32. Zhao, W.; Liu, L.; Du, G.; Liu, S., A multifunctional tag with the ability to benefit the expression, purification, thermostability and activity of recombinant proteins. J. Biotechnol. 2018, 283, 1-10. 33. Zhou, S.-H.; Liu, Y.; Zhao, Y.-J.; Chi, Z.; Chi, Z.-M.; Liu, G.-L., Enhanced exo-inulinase activity and stability by fusion of an inulin-binding module. Appl. Microbiol. Biotechnol. 2016, 100 (18), 8063-8074. 34. Zhang, Y.; Wang, L.; Chen, J.; Wu, J., Enhanced activity toward PET by site-directed mutagenesis of Thermobifida fusca cutinase-CBM fusion protein. Carbohydr. Polym. 2013, 97 (1), 124-129. 35. Zhang, J.; Moilanen, U.; Tang, M.; Viikari, L., The carbohydrate-binding module of xylanase from Nonomuraea flexuosa decreases its non-productive adsorption on lignin. Biotechnol. Biofuels 2013, 6. 36. Zhang, D.; Tu, T.; Wang, Y.; Li, Y.; Luo, X.; Zheng, F.; Wang, X.; Bai, Y.; Huang, H.; Su, X.; Yao, B.; Zhang, T.; Luo, H., Improving the Catalytic Performance of a Talaromyces leycettanus α-Amylase by Changing the Linker Length. J. Agric. Food. Chem. 2017, 65 (24), 5041-5048. 37. Kuroiwa, T.; Izuta, H.; Nabetani, H.; Nakajima, M.; Sato, S.; Mukataka, S.; Ichikawa, S., Selective and stable production of physiologically active chitosan oligosaccharides using an enzymatic membrane bioreactor. Process Biochem. 2009, 44 (3), 283-287. 38. Sinha, S.; Dhakate, S. R.; Kumar, P.; Mathur, R. B.; Tripathi, P.; Chand, S., Electrospun polyacrylonitrile nanofibrous membranes for chitosanase immobilization and its application in selective production of chitooligosaccharides. Bioresour. Technol. 2012, 115, 152-157. 39. Regel, E. K.; Weikert, T.; Niehues, A.; Moerschbacher, B. M.; Singh, R., Protein-engineering


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of chitosanase from Bacillus sp. MN to alter its substrate specificity. Biotechnol. Bioeng. 2018, 115 (4), 863-873.

578 579 580

Figure legends

581

Figure 1 Schematic presentation of the construction of vectors for the expression of

582

the fusion enzymes (left); schematic presentation of the fusion of CBM56 with

583

chitosanases.

584

Figure 2 SDS-PAGE analysis of purified fusion enzymes. Recombinant proteins were

585

purified by Ni-IDA column. (M) protein marker, (1) purified CBM56-GsCsn46A, (2)

586

purified CBM56-BaCsn46A.

587

Figure 3 The effects of temperature and pH on the activity of the purified native

588

GsCsn46A (black) and CBM56-GsCsn46A (red). (A) optimum pH; (B) pH stability;

589

(C) optimum temperature; (D) thermostability. For thermostability, the residual activity

590

was measured after incubating the enzyme at different temperatures for 30 min in 50

591

mM acetate buffer (pH5.5). The experiments were done in triplicate and their mean and

592

standard deviation values were used for analysis.

593

Figure 4 The effects of temperature and pH on the activity of the purified native

594

BaCsn46A (black) and CBM56-BaCsn46ACsn46A (red). (A) optimum pH; (B) pH

595

stability; (C) optimum temperature; (D) thermostability. For thermostability, the

596

residual activity was measured after incubating the enzyme at different temperatures

597

for 30 min in 50 mM acetate buffer (pH6.0). The experiments were done in triplicate



598

and their mean and standard deviation values were used for analysis.

599

Figure 5 The properties of the immobilized chitosanases. (A) photograph of curdlan

600

powder; (B) photograph of immobilized enzyme; (C) and (D) SDS-PAGE analysis of

601

one step immobilized CBM56-GsCsn46A (C) and CBM56-BaCsn46A (D) from E. coli

602

crude cell extracts. M: protein marker, 1: crude cell extract; 2: protein adsorbed by

603

curdlan carrier. (E) The reuse of the immobilized CBM56-BaCsn46A. The relative

604

reducing sugar content of the products represents the catalytic activity of the

605

immobilized enzyme resin.

606

Figure 6. TLC analysis of hydrolysates. (A) the effect of the enzyme amount; (B) the

607

effect of the flow rate; (C) the effect of the concentration of chitosan.

608

Figure 7 (A) TLC analysis of the three optimized different chitooligosaccharide

609

products; (M) chitooligosaccharide marker; (1) hydrolysis condition: 1000 U of

610

immobilized enzyme, 1 % (w/v) chitosan solution, 0.5 mL/min substrate pumping rate;

611

(2) hydrolysis condition: 250 U of immobilized enzyme, 1 % (w/v) chitosan solution,

612

2 mL/min substrate pumping rate; (3) hydrolysis condition: 250 U of immobilized

613

enzyme, 1 % (w/v) chitosan solution, 4 mL/min substrate pumping rate. B-D: GPC

614

analysis of three different chitooligosaccharide products. (B) the low molecular weight

615

chitooligosaccharide; (C) the medium molecular weight chitooligosaccharide; (D) the

616

high molecular weight chitooligosaccharide. Horizontal axis: molecular weight

617

logarithm; Vertical axis: the value of the vertical axis at a point on the curve is related

618

to the content of the sample molecules at each molecular weight.


Page 26 of 37

Page 27 of 37


Table 1 Gene sources in this study gene

gene source

reference

GsCsn46A

chitosanase (GsCsn46A) from G. sunshinyii YC6258

27

BaCsn46A

chitosanase (BaCsn46A) from B. amyloliquefaciens YX-01

29

CBM56

β-1,3-glucan binding module (CBM56) from P. barengoltzii

26

CAU904



Table 2 Primers used in this study Target gene

Prime

Sequence (5’-3’)

r CBM56

F1

CGCCATATGTTAGCTGATTTCACTCAAGGAGCGGA

R1-Gs

CTGCTGAGCGGTCAGCTGAGCCGAAGGTTCCGGATCGGGT T

GsCsn46

F2-Gs

A

CGAAGGTTCCGGATCGGGTTGGCTCAGCTGACCGCTCAGC A

R2-Gs

CCGCTCGAGTTAACGGATCGGCAGGATGAAAA

CBM56

R3-Ba

CGGCCCGACTTATTCCTAGTCCGAAGGTTCCGGATCGGGTT

BaCsn46

F4-Ba

CGAAGGTTCCGGATCGGGTTGCCGGGCTGAATAAGGATC

R4-Ba

CCGCTCGAGTTATTGAATAGTGAAATTACCGTATTCGC

A

The restriction sites of Nde I and Xho I are highlighted in the box, while the overlapping regions are underlined.


Page 28 of 37

Page 29 of 37




Figure 1


Page 30 of 37

Page 31 of 37


Figure 2



Figure 3


Page 32 of 37

Page 33 of 37


Figure 4



Figure 5


Page 34 of 37

Page 35 of 37


Figure 6



Figure 7


Page 36 of 37

Page 37 of 37


TOC Graphic


Efficient Immobilization of Bacterial GH Family 46 Chitosanase by

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