(ChiEn1) from Coprinopsis cinerea and its Synergism in the Deg

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Heterologous Expression and Characterization of a Novel Chitinase (ChiEn1) from Coprinopsis cinerea and its Synergism in the Degradation of Chitin Xin Niu, Jiang-Sheng Zhou, Yan-Xin Wang, Cuicui Liu, Zhonghua Liu, and Sheng Yuan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02278 • Publication Date (Web): 19 Jul 2017 Downloaded from http://pubs.acs.org on July 21, 2017

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Journal of Agricultural and Food Chemistry

Heterologous Expression and Characterization of a Novel Chitinase (ChiEn1) from Coprinopsis cinerea and its Synergism in the Degradation of Chitin

Xin Niu§, Jiang-Sheng Zhou§, Yan-Xin Wang, Cui-Cui Liu, Zhong-Hua Liu*, Sheng Yuan* Jiangsu Key Laboratory for Microbes and Microbial Functional Genomics, Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources, College of Life Science, Nanjing Normal University, Nanjing, PR China 210023

Running Title: Chitinase from Coprinopsis cinerea

§ Co-first authors * To whom correspondence should be addressed: Dr Sheng Yuan, College of Life Science, Nanjing Normal University, 1 Wenyuan Rd, Xianlin University Park, Nanjing, 210023, PR China. Tel: 86-25-85891067 (O), Fax: 86-25-85891067 (O), E-mail: [email protected] Dr Zhong-hua Liu, College of Life Science, Nanjing Normal University, 1 Wenyuan Rd, Xianlin University

Park,

Nanjing,

210023,

PR

China.

Tel:

[email protected],

1

ACS Paragon Plus Environment

86-13584093709,

E-mail:


1

Abstract

2

Chitinase ChiEn1 did not hydrolyze insoluble chitin but showed hydrolysis and transglycosylation

3

activities towards chitin-oligosaccharides. Interestingly, the addition of ChiEn1 increased the amount of

4

reducing sugars released from chitin powder by endochitinase ChiIII by 105.0%, and among the

5

released reducing sugars the amount of (GlcNAc)2 was increased by 149.5% whereas the amount of

6

GlcNAc was decreased by 10.3%. The percentage of GlcNAc in the products of chitin powder with the

7

combined ChiIII and ChiEn1 was close to that in the products of chitin-oligosaccharides with ChiEn1,

8

rather than that with ChiIII. These results indicate that chitin polymers are first degraded into chitin

9

oligosaccharides by ChiIII and the latter are further degraded to monomers and dimers by ChiEn1, and

10

the synergistic action of ChiEn1 and ChiIII is involved in the efficient degradation of chitin in cell

11

walls during pileus autolysis. The structure modeling explores the molecular base of ChiEn1 action.

12 13

Keywords: Coprinopsis cinerea; chitin; chitinase; hydrolysis; transglycosylation

14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 2


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31

Introduction

32

Chitin, an insoluble β-1,4-linked N-acetyl-D-glucosamine polysaccharide, is one of the primary

33

structural components of the fungal cell wall.

34

cell walls, such as cell division and separation in yeast,

35

branching of hyphae, and hyphal autolysis in filamentous fungi

36

autolysis of fruiting bodies in basidiomycetes, 4,5 chitin polymers in cell walls need to be continuously

37

remodeled, presumably through the enzymatic digestion of chitin polymers by chitinases. 4-6 Chitinases

38

are divided into endochitinases, exochitinases and β-1, 4-N-acetyl-glucosaminidases. Endochitinases

39

randomly

40

chitinoligosaccharides. Exochitinases progressively release chitinbiose from the non-reducing or

41

reducing end of chitin chains; β-1,4-N-acetyl-glucosaminidases catalyze the release of terminal,

42

non-reducing N-acetylglucosamine residues from chitin or split chitinbiose into two monomers. 6-8

degrade

chitin

from

any

1

point

In physiological processes requiring the digestion of

along

2

the germination of spores, tip growth and

the

3

as well as morphogenesis and

polymer

chain

to

varying-length

43

Autolysis of the fruiting body of basidiomycete coprinoid mushrooms exhibits a remarkable feature:

44

the complete disintegration and liquefaction of the mature pileus for the efficient dispersal of

45

basidia.9,10 The disintegration and liquefaction of the mature pileus were suggested to result from the

46

degradation of cell walls by various glycoside hydrolases, such as chitinases and glucanases.

47

common fungal cell walls, the cell wall of basidiomycete fruiting bodies is primarily composed of

48

β-1,3-glucans with β-1,6-linked branches, chitin and proteins,

49

architecture. 15,16 In the central core of the fungal cell wall architecture, chitin chains are attached to the

50

non-reducing end of β-1,3/1,6-glucans.

51

involved in the cell wall autolysis of basidiomycete fruiting bodies.

52

and purified a group of glycoside hydrolases, including a β-1,3-glucosidase, an exo-β-1,3-glucanase, an

53

endo-β-1,3-glucanase, and partially purified a β-glucosidase, from the pileus extraction of C. cinerea

54

fruiting bodies; these enzymes showed highly efficient and synergistic action on the β-1,3-glucan

55

backbones of the C. cinerea cell wall during fruiting body autolysis.

56

exochitinase (ChiB1) gene and a putative class III endochitinase (ChiIII) gene were dominantly

57

expressed among eight predicted chitinase genes in the genome of Coprinopsis cinerea, and their

58

expression levels increased with the maturation of the fruiting bodies.

59

the exochitinase ChiB1 from the pilei of C. cinerea fruiting bodies 22 and heterologously expressed and

60

characterized the recombinant chitinase ChiIII from Coprinopsis cinerea.

1,17,18

12-15

9,11

As

together forming a fine wall

Therefore, glucanases and chitinases are suggested to be 4,5,10,11,19-22

22

3


23

Recently, we isolated

We also found that an

We purified and characterized

21

We found that removal of


61

the chitin chains by chitinases could release β-1,3/1,6-glucan molecules to promote the degradation of

62

the cell walls to work in synergy with β-1,3-glucanases for pileus autolysis. 22

63

In this study, we further report that the gene expression of a putative endochitinase (ChiEn1) also

64

increased with the maturation of fruiting bodies. Characterization of chitinase ChiEn1 shows that

65

ChiEn1 acts synergistically with the previously reported endochitinase ChiIII to efficiently hydrolyze

66

insoluble chitin polymers, elucidating the role of ChiEn1 in the degradation of chitin components in

67

cell walls for the autolysis of C. cinerea fruiting bodies.

68 69

Materials and methods

70

Chemicals—Chitin powder, 85% deacetylated chitosan, sodium carboxymethyl cellulose (CMC-Na),

71

glycol chitosan, laminarin, and (GlcNAc)1-3-pNP were purchased from Sigma-Aldrich Co. LLC (USA).

72

N-acetylglucosamine, chitinbiose, chitintriose, chitintetraose, chitinpentose, chitinhexaose, and

73

chitinheptaose were purchased from Elicityl Oligotech (France). Chitohexaose hydrochloride was

74

purchased from Zzstandard Shanghai Zzbio Co., Ltd. (China). Colloidal chitin and glycol chitin were

75

prepared from chitin powder according to the methods described by Sandhya et al.24 and Li et al.,25

76

respectively.

77

Strains, plasmid and culture conditions—C. cinerea (5026+5172) ATCC 56838 was purchased from

78

ATCC (U. S. A.). Fruiting bodies were cultivated as described by Zhou et al. 23 Pichia pastoris GS115

79

and the expression vector pPICZαA were purchased from Invitrogen (U. S. A.).

80

Cloning, expression, and purification of chitinase ChiEn1—The sequence of chitinase ChiEn1 was

81

obtained from the genome of C. cinerea okayama7 #130 in GenBank at NCBI (accession: EAU81461).

82

The signal peptide was analyzed using the Signal P 4.1 server (http://www.cbs.dtu.dk/services

83

/SignalP). The isolation and purification of total RNA from the pilei of C. cinerea fruiting bodies,

84

synthesis of first-strand cDNA from DNA-free RNA, and PCR amplification of cDNA coding the

85

mature ChiEn1 with the forward primer AGAGAGGCTGAAGCTGAATTCATGGCCTACGTCCC

86

TGTCG and reverse primer TGTTCTAGAAAGCTGGCGGCCGCCTGCCCCATACCCCTCGA were

87

performed as described by Niu et al.

88

were digested respectively with EcoR I and Not I and then ligated to generate the plasmid

89

pPICZαAChiEn1 using the ClonExpressTM II/One Step Cloning Kit (Vazyme, China). Transformation

90

of P. pastoris spheroplasts with pPICZαAChiEn1 and the selection of transformants with

21

The PCR products of chiEn1 cDNA and the plasmid pPICZαA

4


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91 92

pPICZαAChiEn1 were performed as described by Niu et al. 21 Cultivation of transformant cells, inducing the expression of recombinant ChiEn1, and the isolation

93

and purification of recombinant ChiEn1 were performed as described by Niu et al. 21

94

Protein analysis of the recombinant enzyme—The purity and molecular size of purified ChiEn1

95

were analyzed by SDS-PAGE.

96

Bradford method using bovine serum albumin as the standard.

97

peptide fragments from purified ChiEn1 by trypsin digestion was analyzed using the UltrafleXtreme

98

MALDI-TOF/TOF MS (Bruker Daltonics, Germany) as described by Zhou et al. 23

99

Chitinase activity assays—The amount of N-acetylglucosamine or reducing sugars released from the

100

chitin or the related polysaccharides by ChiEn1 was analyzed using the 3,5-dinitrosalicylic acid (DNS)

101

method. 28 A volume of 200 µL of a reaction mixture containing 2 µM ChiEn1 and 1% chitin or related

102

polysaccharides in 50 mM NaAc-HAc (pH 5.0) was incubated at 800 rpm, 37 °C for 4 h. After

103

incubation, the reaction mixtures were combined with 200 µL of a DNS reagent, heated at 100 °C for

104

15 min, and then placed on ice for 2 min. After centrifugation, the absorbance of the supernatants at

105

520 nm was measured. To determine the synergistic action of ChiEn1 and ChiIII, 200-µL aliquots of

106

reaction mixtures that contained 1% chitin powder and 1.0 µM of ChiEn1, ChiIII, ChiEn1 + ChiIII, or

107

heat-inactivated ChiEn1 + ChiIII in 50 mM NaAc-HAc (pH 5.0), with other parameters as listed above,

108

were used. To determine the effect of temperature on the hydrolysis activity of ChiEn1 towards glycol

109

chitin, the reaction mixtures were incubated at 10–90 °C, with all other parameters identical to those

110

listed above. To determine the effect of the pH on the hydrolysis activity of ChiEn1 towards glycol

111

chitin, reaction mixtures (as above) with pH in the range of 3–9 (using 50 mM NaAc-HAc buffer (pH

112

3.0–6.0), 50 mM Na2HPO4-NaH2PO4 buffer (pH 6.0–7.5), and 50 mM Tris-HCl buffer (pH 7.5–9.0))

113

were used. To determine the stability of ChiEn1, the reaction mixtures (as above) were first incubated

114

at 10–90 °C or at pH 6.0–9.0 in the absence of substrate for 1 h and then combined with glycol chitin to

115

react. To determine the effect of metal ions or the metal ion-chelator EDTA on the hydrolysis activity of

116

ChiEn1 towards glycol chitin, ChiEn1 was first incubated with 1 mM of the indicated metal ion salt or

117

1 mM or 2 mM EDTA in 50 mM NaAc-HAc (pH 5.0) in the absence of substrate at 37 °C for 1 h and

118

then combined with glycol chitin to react under the same conditions as listed above. One unit of

119

chitinase activity was defined as the amount of enzyme that liberates the reducing sugar corresponding

120

to 1 µmol of N-acetyl-D-glucosamine per minute.

26

The protein concentration of purified ChiEn1 was measured by the 27

The amino acid sequence of partial

5



121

The amount of p-nitrophenol that was released from pNP derivatives of chitin oligosaccharides by

122

ChiEn1 was analyzed using the chitinase assay kit (Sigma CS0980, USA). A volume of 100 µL of a

123

reaction mixture containing 0.067 µM ChiEn1 and 1 mg/mL of (GlcNAc)3-pNP, (GlcNAc)2-pNP, or

124

GlcNAc-pNP in assay buffer was incubated at 37 °C at 800 rpm for 20 min. Then, the reaction mixture

125

was combined with 200 µL of stop solution and the absorbance was measured at 405 nm. The enzyme

126

unit was defined as the amount of enzyme that released 1.0 µmol of p-nitrophenol from the indicated

127

substrate per min. To determine the reaction kinetics, reaction mixtures containing 0.022 µM chitinase

128

ChiEn1 and varying concentrations of (GlcNAc)3-pNP or (GlcNAc)2-pNP (0.125 to 3 mg/mL) were

129

analyzed. The reaction rate V was plotted directly against the substrate concentration. OriginPro 8 SR0

130

(OriginLab Corporation, Northampton, USA) was used to fit a hyperbola to the data and to determine

131

Km and Vmax. 29

132

All above assays reported in this study were done in triplicate in each of three independent

133

experiments.

134

HPLC analysis—For the HPAEC-PAD analysis of products released from chitin powder by ChiEn1

135

alone or in cooperation with ChiIII, a volume of 200 µL of a reaction mixture containing 1% chitin

136

powder and 1.2 µM ChiEn1, ChiIII, ChiEn1 + ChiIII, or heat-inactivated ChiEn1 + ChiIII in 50 mM

137

NaAc-HAc (pH 5.0) was incubated at 37 ˚C for 4 h, then combined with boiled water at 100 °C to a

138

final volume of 1 mL and heated at 100 °C for 10 min to stop the reaction. For the HPAEC-PAD

139

analysis of the products released from chitin oligosaccharides by ChiEn1, 20 µL of a reaction mixture

140

containing 100 nmol of chitin oligosaccharide and the appropriate amount of ChiEn1 (see results for

141

details) in 50 mM NaAc-HAc (pH 5.0) was incubated at 37 ˚C for the indicated times, then were

142

combined with boiled water at 100 °C to a final volume of 1 mL to stop the reaction. For the

143

HPAEC-PAD analysis of the products released from the pNP derivatives of chitin oligosaccharide by

144

ChiEn1, 100 µL of a reaction mixture containing 100 nmol of (GlcNAc)3-pNP, or (GlcNAc)2-pNP, or

145

GlcNAc-pNP, and 0.055 µM of ChiEn1 50 mM NaAc-HAc (pH 5.0) was incubated at 37 ˚C for 15 min

146

and then combined with boiled water (100 °C) to a final volume of 1 mL to stop the reaction. After

147

centrifugation and filtration, the above reaction mixtures were loaded on and eluted from a CarboPac

148

PA-1 column (4×250 mm, Dionex) with a PA-1 guard column equipped on a 940 Professional IC Vario

149

system with an IC Amperometric detector (Metrohm), as described by Niu et al. 21

150

For the TSKgel Amide-80 HPLC analysis of the anomeric configurations of products released from 6


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151

the chitin oligosaccharides by ChiEn1, 50 µL of a reaction mixture containing 100 nmol of chitin

152

oligosaccharide and 0.8 µM ChiEn1 in 50 mM NaAc-HAc (pH 5.0) was incubated on ice for 5 min and

153

then combined with ice-cooled water to 200 µL. After filtration, the reaction mixtures were loaded on a

154

TSK gel Amide 80 column (4.6×250 mm, Tosch) and eluted with 75% acetonitrile in ultrapure water at

155

a flow rate of 0.8 mL·min-1 at 25 °C. Chitin oligosaccharides in the eluate were detected by their UV

156

absorbance at 210 nm and quantified by measuring their peak areas with an Agilent 1100 HPLC system

157

(Agilent, USA). 30

158

MALDI-TOF MS analysis of transglycosylation products—The transglycosylation products in the

159

reaction mixture were analyzed by mass spectrometry. 1 µL samples from the reaction mixture was

160

mixed with 2 µL of a matrix solution (15 mg/mL 2,5-dihydroxybenzoic acid, in 30% acetonitrile), and

161

then spotted directly on a target plate and immediately dried using a heat gun. MS spectra of the

162

products were acquired using an UltrafleXtreme MALDI-TOF/TOF MS (Bruker Daltonics, Germany)

163

with gridless ion optics under control of Flexcontrol 4.1. The experiments were conducted using an

164

accelerating potential of 24 kV in the reflector mode. 31

165

Protein sequence analysis—The conserved domains in ChiEn1 were analyzed using NCBI's

166

conserved domain database.32 A phylogenetic tree was generated based on catalytic domain amino acid

167

sequences of chitinase proteins by the neighbor-joining method using MEGA v.6.06 software.33 ChiEn1

168

catalytic domain amino acid sequences and other chitinases catalytic domain amino acid sequences

169

were aligned to determine their sequence identity using DNAMAN software (version 7.212, Lynnon

170

Corp., Quebec, Canada).

171

Protein structure analysis—The three-dimensional model structure of ChiEn1 was predicted using

172

I-TASSER.

173

C-score was chosen for further accuracy analysis and identified by TM-align (http://zhang.

174

bioinformatics.ku.edu/I-TASSER/). Structures were visualized using Chimera.37

175

Insoluble chitin binding assays—Insoluble chitin binding studies were conducted as described by

176

Neeraja et al.38 using 1 mg of chitin powder or colloidal chitin incubated with 20-100 µg of ChiEn1 in

177

1 ml of 50 mM sodium acetate buffer (pH 5.0) for 1 h, in a gel shaker at 450 rpm at 4°C. After

178

incubation, reactions were stopped by centrifugation (12000 g, 10 min, 4 °C). Unbound protein in the

179

supernatant was determined by measuring the absorbance at 280 nm in spectrophotometer. The bound

180

protein was calculated as the total protein minus the unbound protein measured.

34-36

Among five models produced by I-TASSER, the I-TASSER model with the highest

7



181

Results

182

Cloning, heterologous expression and purification of enzyme

183

In the previous report, we showed that the gene transcription of a putative endochitinase (ChiEn1)

184

exhibited an increasing trend with the maturation of fruiting bodies, but there was no statistical

185

significance because the gene expression abundance of this enzyme was dramatically lower than that of

186

exochitinase ChiB1 and endochitinase (ChiIII).

187

cDNA template for DNA amplification, we found that chitinase ChiEn1 indeed expressed highly in the

188

pileus of fruiting bodies and the gene expression of chitinase ChiEn1 peaked when the pileus opened

189

90° during maturation of fruiting bodies (Fig. 1A), indicating that ChiEn1 plays a role in the

190

disintegration and liquefaction of the mature pileus. Thus, we cloned, heterologously expressed and

191

characterized the chitinase ChiEn1 from C. cinerea.

22

In this study, by increasing of the amount of the

192

The endochitinase ChiEn1 from C. cinerea okayama7 (#130) consisted of 465 amino acids with a

193

calculated molecular weight of 48808 Da. Analysis via the Signal 4.1 server (http://www.cbs.dtu.dk

194

/services/ SignalP) showed that the N-terminal 1-63 nucleotides sequence encodes a signal peptide

195

(MQFKTSFFALLAGFLASSTLA). The nucleotide sequence for the signal peptide was removed from

196

the chiEn1 gene such that the extracellular secretion of the expression product was mediated by the

197

N-terminal α-factor signal peptide of the plasmid pPICZαAChiEn1. Recombinant mature ChiEn1

198

formed a fusion protein with the C-terminal 6×histidines of pPICZαAChiEn1, and the mature protein

199

has a calculated molecular weight of 50038 Da. After six days of cultivation for inducing expression,

200

the hydrolysis activity of the culture medium towards the glycol chitin of the recombinant ChiEn1

201

expression strain reached 0.151 U mL-1.

202

The recombinant ChiEn1 was purified from the culture medium by Ni-affinity chromatography with

203

the yield of 0.05 mg mL-1 culture medium, and the specific activity of 0.451 U mg-1 protein towards

204

glycol chitin. SDS-PAGE analysis showed that there was an extra protein band at approximately 50

205

kDa in the culture medium of the recombinant ChiEn1 expression strain compared to the control strain

206

with an empty plasmid. The recombinant ChiEn1 purified from the culture medium by Ni-affinity

207

chromatography appeared at this position as a single band on SDS-PAGE (Fig. 1B). When this protein

208

band was analyzed using MALDI TOF/TOF MS, the amino acid sequences of the trypsinized protein

209

fragments were confirmed as consistent with the amino acid sequence of the putative endochitinase

210

(ChiEn1) (GenBank accession: EAU81461) (Fig. 1C, D) 8


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211 212

Enzymatic features

213

The hydrolysis activity of the chitinase ChiEn1 against several chitin-derived polysaccharide substrates

214

showed that ChiEn1 did not act on insoluble chitin powder and colloidal chitin but did act on soluble

215

glycol chitin and 85% deacetylated chitosan (Table 1). In addition, ChiEn1 did not hydrolyze soluble

216

glycol chitosan. However, a synergistic action of ChiEn1 and the previously reported endochitinase

217

ChiIII from C. cinerea 21 on chitinous polysaccharides was observed. As shown in Table 2, the amount

218

of reducing sugars released from chitin powder, colloidal chitin and glycol chitin by a combination of

219

ChiEn1 and ChiIII increased by 105.0%, 21.0%, 31.3%, respectively, compared with the sum of the

220

amount of reducing sugars released by ChiIII or ChiEn1 alone; however, the amount of reducing sugars

221

released from 85% deacetylated chitosan by the combination of ChiEn1 and ChiIII decreased by 26.4%

222

compared with the sum of the amount of reducing sugars released by ChiIII or ChiEn1 alone. The

223

hydrolysis activity of chitinase ChiEn1 on several chitin oligosaccharide substrates showed that

224

ChiEn1 did not degrade chitinbiose, whereas it degraded chitintriose or longer chitin oligosaccharides

225

(Table 1). The capacity of ChiEn1 to degrade (GlcNAc)4-6 was more than three times higher than its

226

ability to degrade (GlcNAc)3. The hydrolysis activity of chitinase ChiEn1 on several nitrophenyl

227

derivatives of chitin oligosaccharides showed that ChiEn1 hydrolyzed (GlcNAc)3-pNP and

228

(GlcNAc)2-pNP to release the colored pNP but did not hydrolyze GlcNAc-pNP. The hydrolysis activity

229

towards (GlcNAc)2-pNP was higher than that towards (GlcNAc)3-pNP (Table 1).

230

Some metal ions affected the hydrolysis activity of ChiEn1 towards glycol chitin (Table 3).

231

Compared with the control that lacked the addition of metal ions, the ChiEn1 activity was enhanced to

232

295% and 170% by the presence of 1 mM Mn2+ and Co2+, respectively, whereas the ChiEn1 activity

233

was inhibited to 9.6% and 27.3% by the presence of 1 mM Al3+ and Cu2+, respectively. The ChiEn1

234

activity was inhibited to 79.7% and 0% by the presence of 1 mM and 2 mM chelator EDTA,

235

respectively. Therefore, Mn2+ is essential for ChiEn1 activity.

236

At pH 5.0, the maximum activity of ChiEn1 towards glycol chitin occurred at 40˚C, whereas below

237

or above 40˚C, the hydrolysis activity quickly decreased (Fig. 2A1). The temperature stability of

238

ChiEn1 is poor, and its hydrolysis activity after being incubated for 30 min at 20–70 ˚C decreased with

239

increasing incubation temperature (Fig. 2A2). At 37 ˚C, the optimal pH for the hydrolysis activity of

240

ChiEn1 towards glycol chitin was pH 5.0, whereas at below or above 5.0, the hydrolysis activity of 9



241

ChiEn1 decreased (Fig. 2B1). The pH stability of ChiEn1 is good, and after incubation of ChiEn1 over

242

a broad range of pH (4.0–9.0) for 30 min, its hydrolysis activity did not significantly change (Fig.

243

2B2).

244

The effect of substrate concentration on the hydrolysis activity of ChiEn1 towards (GlcNAc)3-pNP

245

and (GlcNAc)2-pNP was analyzed at 37˚C and pH 5.0 (Fig. 2C). ChiEn1 exhibited a Km of 2342 µM, a

246

kcat of 1.13 s-1 and a Vmax of 1.35 µM min-1 mg protein-1 for (GlcNAc)3-pNP and a Km of 1254 µM, a

247

kcat of 2.28 s-1 and a Vmax of 2.74 µM min-1 mg protein-1 for (GlcNAc)2-pNP.

248 249

Hydrolysis products

250

The high performance anion-exchange chromatography with pulsed amperometric detection

251

(HPAEC-PAD) was used to analyze the hydrolysis products of chitin and chitin oligosaccharides by

252

ChiEn1. As shown in Fig. 3 and Table 4, although ChiEn1 itself did not release any chitin

253

oligosaccharides from chitin powder after 4 h of reaction, the addition of ChiEn1 increased the amount

254

of (GlcNAc)2 released from the chitin powder by 149.5% but decreased the amount of GlcNAc by

255

10.3%, compared with products released by ChiIII alone. It is noteworthy that the amount of GlcNAc

256

accounted for 40.5% of the products (GlcNAc and (GlcNAc)2) of the reaction of chitin powder with

257

ChiIII alone, whereas it only accounted for 19.9% of the products of the reaction of chitin powder with

258

the combination of ChiIII and ChiEn1. Fig. 4A shows that in a 15 min incubation of ChiEn1,

259

(GlcNAc)2 was not degraded, whereas (GlcNAc)3 was degraded to GlcNAc and (GlcNAc)2, and the

260

amount of GlcNAc only accounted for 30.7% of the products (GlcNAc and (GlcNAc)2). As seen in Fig.

261

4A, (GlcNAc)4 was hydrolyzed by ChiEn1 to 2×(GlcNAc)2 or (GlcNAc)3 without GlcNAc.

262

Interestingly, the minor transglycosylation products (GlcNAc)6 and (GlcNAc)5 were observed in the

263

reaction mixture of (GlcNAc)4 with ChiEn1; therefore, the (GlcNAc)3 was produced from the

264

(GlcNAc)6 or (GlcNAc)5. The time course of the hydrolysis of the (GlcNAc)4 by the ChiEn1 (Fig 4B)

265

showed that with the prolongation of the reaction time, after (GlcNAc)4 was completely degraded,

266

(GlcNAc)3 was gradually degraded to (GlcNAc)2 and GlcNAc rather than only (GlcNAc)2 and the

267

amount of GlcNAc accounted for 20.1% of the final products (GlcNAc and (GlcNAc)2). Fig. 4A shows

268

that the (GlcNAc)5 was degraded by ChiEn1 to (GlcNAc)3 and (GlcNAc)2; it also was degraded to

269

(GlcNAc)4 but not GlcNAc. Correspondingly, two minor transglycosylation products, (GlcNAc)7 and

270

(GlcNAc)6, also appeared in the reaction mixture. Therefore, the (GlcNAc)4 was produced from the 10


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271

(GlcNAc)6 or (GlcNAc)7. The time course of the hydrolysis of (GlcNAc)5 by ChiEn1 (Fig. 4C) showed

272

that with the prolongation of the reaction time, after (GlcNAc)5 was completely degraded, the

273

intermediates (GlcNAc)4 and (GlcNAc)3 were further degraded and the final products were only

274

(GlcNAc)2 and GlcNAc; the amount of GlcNAc accounted for 21.7% of the final products at the end of

275

720-min reaction. From Fig. 4A, the hydrolysis products of (GlcNAc)6 by ChiEn1 included (GlcNAc)5,

276

(GlcNAc)4, (GlcNAc)3, and (GlcNAc)2, as well as minor amounts of transglycosylation products

277

(GlcNAc)7 and (GlcNAc)8, but not GlcNAc. Apparently, (GlcNAc)6 was degraded to 2×(GlcNAc)3 or

278

(GlcNAc)2 and (GlcNAc)4, but it was not cleaved directly to (GlcNAc)5 and GlcNAc; (GlcNAc)5 was

279

produced from transglycosylation product (GlcNAc)8 or (GlcNAc)7. The time course of the hydrolysis

280

of (GlcNAc)6 by ChiEn1 (Fig. 4D) showed that with the prolongation of reaction time, the

281

intermediates (GlcNAc)5 and (GlcNAc)4 gradually degraded, as described above, to (GlcNAc)3 and

282

(GlcNAc)2, whereas (GlcNAc)3 also finally degraded to (GlcNAc)2 and GlcNAc. At the end of the

283

reaction, the amount of GlcNAc accounted for 23.8% of the total final products (GlcNAc +

284

(GlcNAc)2).

285

The hydrolytic products of the pNP derivatives of the chitin oligosaccharides by ChiEn1 were

286

analyzed to determine the direction of the reaction and the mode of action. ChiEn1 did not cleave

287

GlcNAc-pNP to release GlcNAc (Fig. 5A). ChiEn1 degraded (GlcNAc)2-pNP to (GlcNAc)2 and pNP

288

(here, pNP was not detected by HPAEC-PAD); however, the hydrolysis products contained (GlcNAc)3

289

and GlcNAc-pNP (Fig. 5B), which supports a transglycosylation mechanism, i.e., ChiEn1 transfers one

290

(GlcNAc)2 residue from a (GlcNAc)2-pNP to another (GlcNAc)2-pNP to produce (GlcNAc)4-pNP and

291

pNP, and the resulting (GlcNAc)4-pNP was subsequently cleaved to a (GlcNAc)3 and GlcNAc-pNP.

292

ChiEn1 not only degraded (GlcNAc)3-pNP to produce (GlcNAc)3 and pNP but also produced

293

(GlcNAc)2 and GlcNAc-pNP (Fig. 5C). Furthermore, an apparent peak of (GlcNAc)2-pNP occurred in

294

the HPAEC-PAD pattern, but a peak of GlcNAc did not appear (Fig. 5C), suggesting that ChiEn1

295

transferred one (GlcNAc)2 from a (GlcNAc)3-pNP to another (GlcNAc)3-pNP to produce

296

(GlcNAc)5-pNP and GlcNAc-pNP; after this reaction, (GlcNAc)5-pNP was cleaved to form (GlcNAc)3

297

and (GlcNAc)2-pNP.

298

There are α- and β-anomeric forms of chitin oligosaccharides. The mutarotation of α- and

299

β-anomers spontaneously occurs in solution in a non-enzymatic way to finally reach the equilibrium

300

between two anomers.

39

After mutarotation, the ratio between the α- and β-anomeric forms of chitin 11



301

oligosaccharides is approximately 60:40 at equilibrium. When the reaction was conducted at 0 ˚C on

302

ice, the mutarotation of the α- and β-anomeric forms of the hydrolysis products of chitin

303

oligosaccharides by chitinases was prohibited such that anomeric forms of the newly formed reducing

304

ends and those originating from the preexisting reducing end of substrates could be discriminated by

305

TSKgel Amide 80 column HPLC analysis.

306

GlcNAc in the ChiEn1 hydrolysis products of (GlcNAc)3 contained 84% and 82% β-anomer,

307

respectively, and the remaining (GlcNAc)3 exhibited a lower proportion (28%) of β-anomer than the

308

initial proportion (approximately 40%) of β-anomer that was present in the reaction before incubation,

309

suggesting that the β-anomer of (GlcNAc)3 was preferably degraded. Notably, the Amide 80 column

310

HPLC also detected less GlcNAc than (GlcNAc)2 in the hydrolysis products of (GlcNAc)3. In the

311

ChiEn1 hydrolysis products of (GlcNAc)4 (Table 5, Fig. 6B), (GlcNAc)2 contained 92% β-anomer, and

312

the proportion of β-anomer in the remaining (GlcNAc)4 was reduced to 20% from approximately 40%,

313

suggesting that the β-anomer of (GlcNAc)4 was preferably degraded to 2×β-(GlcNAc)2 by ChiEn1;

314

(GlcNAc)3 contained 53% β-anomer, which is consistent with the predicted combinations of

315

approximately 70% of the β-anomer of 2×(GlcNAc)3 resulting from (GlcNAc)6 and 40% of the

316

β-anomer of (GlcNAc)3 resulting from (GlcNAc)5 (see below). In the ChiEn1 hydrolysis products of

317

(GlcNAc)5 (Table 5, Fig. 6C), (GlcNAc)2 and (GlcNAc)3 contained 92% and 46% β-anomers,

318

respectively, indicating that β-(GlcNAc)2 was released only from the non-reducing end of (GlcNAc)5

319

and that the degradation of α- or β-(GlcNAc)5 by ChiEn1 was not selective; the transglycosylation

320

products (GlcNAc)6 and (GlcNAc)7 contained approximately 40% β-anomer, indicating that ChiEn1

321

transferred a (GlcNAc)2 residue from the non-reducing end of a (GlcNAc)5 to the non-reducing end of

322

another (GlcNAc)5 or intermediate (GlcNAc)4 to form a transglycosylation product; (GlcNAc)4 from

323

the transglycosylation product (GlcNAc)7 contained 66% β-anomer, suggesting that ChiEn1 cleaves

324

either (GlcNAc)3 or (GlcNAc)4 from the non-reducing end of (GlcNAc)7. In the ChiEn1 hydrolysis

325

products of (GlcNAc)6 (Table 5, Fig. 6D), (GlcNAc)2 and (GlcNAc)4 contained 96% β-anomer and

326

43% β-anomer, respectively, indicating that ChiEn1 cleaved the (GlcNAc)2 residue from the

327

non-reducing end of (GlcNAc)6 in a non-anomer selective manner; (GlcNAc)3 contained 63%

328

β-anomer, suggesting that ChiEn1 could symmetrically cleave (GlcNAc)6; the transglycosylation

329

products (GlcNAc)7 and (GlcNAc)8 contained approximately 40% β-anomer, also supporting the

330

hypothesis that ChiEn1 transferred a (GlcNAc)2 residue from the non-reducing end of a (GlcNAc)6 to

39,40

As shown in Table 5 and Fig. 6A, (GlcNAc)2 and

12


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331

the non-reducing end of another (GlcNAc)6 or intermediate (GlcNAc)5 to form a transglycosylation

332

product; (GlcNAc)5 contained approximately 46% β-anomer, indicating that ChiEn1 primarily cleaves

333

(GlcNAc)3 from the non-reducing end of the transglycosylation product (GlcNAc)8.

334 335

Analysis of the Sequence and Structure of ChiEn1

336

The analysis of NCBI conserved domains shows that ChiEn1 has only a catalytic domain but no

337

carbohydrate-binding domain (CBD). Thus, a phylogenetic tree of ChiEn1 and three reported

338

single-catalytic domain chitinases, ChiCH from Bacillus cereus,41 Chi18aD from Streptomyces

339

coelicolorA3(2),42 and SpChiD from Serratia proteamaculans,43 as well as two confirmed chitinases

340

(ChiIII and ChiB1) from C. cinerea and three typical chitinases (ChiA, ChiB, ChiC) from Serratia

341

marcescens was produced with the MEGA program by the neighbor-joining method, which indicates

342

that C. cinerea ChiEn1 and S. proteamaculans SpChiD are categorized in a subgroup (Fig. 7A). The

343

alignment of these chitinase catalytic domain sequences shows that ChiEn1 has 23.45% identity to

344

SpChiD from S. proteamaculans which is higher than its identities to other analyzed chitinases.

345

The protein structure of ChiEn1 was predicted using I-TASSER (Iterative Threading ASSEmbly

346

Refinement, http://zhanglab.ccmb.med.umich.edu/ I-TASSER/) based on the amino acid sequence of

347

ChiEn1; this prediction shows that ChiEn1 adopts the TIM (α/β)8 barrel fold with an active site

348

containing three catalytic acidic residues, Asp172, Asp174 and Glu176 (Fig. 7B1, B2). Like the typical

349

exo-acting enzyme ChiA (Fig. 7B3) and ChiB (Fig. 7B4) from S. marcescens,

350

domain insertion between strand 7 and helix 7 of the TIM barrel in the catalytic domain; therefore, it

351

exhibits a deeper substrate-binding cleft than the endo-acting enzyme ChiC2 from S. marcescens (Fig.

352

7B5).

353

exo-acting enzymes ChiA and ChiB from S. marcescens, similar to that of a single-domain chitinase,

354

SpChiD from S. proteamaculans (Fig. 7B6).43 Furthermore, there are fewer aromatic amino acids that

355

directly contact the substrate that is exposed on the surface of the substrate-binding cleft of ChiEn1

356

than there are in exo-acting chitinase ChiA and ChiB from S. marcescens (Fig. 7B3, B4), but there are

357

more than are found in the endo-acting chitinases ChiC2 from S. marcescens (Fig. 7B5) and SpChiD

358

from S. proteamaculans (Fig. 7B6). Among these aromatic amino acids W-255 and W-252 in the +3

359

and +5 positions of the subsite of ChiEn1 come from the α + β insertion domain that does not exist in

360

ChiC2 (comparing Fig. 7B2 to 7B5), similar to the W-290 in the positive subsite of SpChiD

44

44

ChiEn1 has an α+β

However, the substrate-binding cleft of ChiEn1 is more shallow and open than those of

13



361

(comparing 7B6), and Phe94 and Trp424 are at -3 to -1 position of ChiEn1. A distinct feature of the

362

structure is that two small protruding loops, a Ser71-His77 loop between the B1 β-strand and A1

363

α-helix and a Gln181-His187 loop between the B4 β-strand and A4 α-helix, are situated in the

364

entrances of the two sides of the substrate-binding cleft of ChiEn1. Furthermore, though a small

365

peptide fragment consisting of the N-terminal Ala1-Thr16 residues extends beyond the globular

366

structure of ChiEn1, it is not collinear with the substrate-binding cleft and does not expose any

367

aromatic amino acids on the surface; therefore, it is not a binding domain.

368 369

Insoluble chitin binding of ChiEn1

370

As shown in Table 6, after 1 h of incubation, almost all of ChiEn1 proteins were bound to the insoluble

371

chitin substrates, chitin powder and colloidal chitin, at low protein concentration, while only a part of

372

ChiEn1 proteins were bound to the insoluble chitin substrates at the high concentration due to high

373

concentration of ChiEn1 exceeding chitinase binding capacity of chitin substrate though ChiEn1 lacks

374

carbohydrate-binding domain and does not hydrolyze chitin powder and colloidal chitin. Interestingly,

375

the binding affinity of ChiEn1 for powder chitin is higher than that for colloidal chitin.

376 377

Discussion

378

This study shows that ChiEn1 can only hydrolyze soluble chitin or chitin oligomeric substrates but not

379

insoluble chitin polymeric substrates, despite being annotated previously as an endochitinase. In

380

contrast, known chitinases, either endochitinases or exochitinases, essentially could act on the insoluble

381

chitin polymers.

382

processive action or non-processive action) on chitin polymers 44,50 were not determined in this study. A

383

distinctive feature is that the hydrolysis pattern of ChiEn1 shifted with the length of the chitin

384

oligosaccharides: when the substrate was (GlcNAc)3-5, ChiEn1 released (GlcNAc)2 residues from the

385

non-reducing end of (GlcNAc)3-5; when the substrate was (GlcNAc)6, ChiEn1 cleaved (GlcNAc)6 to

386

2×(GlcNAc)3 except cleaved (GlcNAc)2 residues from the non-reducing end of (GlcNAc)6; when the

387

substrate was (GlcNAc)7, ChiEn1 could randomly cleave (GlcNAc)3 or (GlcNAc)4 residues from the

388

non-reducing end of (GlcNAc)7; when the substrate was (GlcNAc)8, ChiEn1 only cleaved (GlcNAc)3

389

from the non-reducing end of (GlcNAc)8. We propose that the GlcNAc moieties at the non-reducing

390

end of (GlcNAc)3-5 are bound preferentially to the -2 and -1 subsites in the substrate-binding site of

45-49

Thus, the modes of action of ChiEn1 (the endo-action or exo-action and the

14


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Page 15 of 41


391

ChiEn1; the GlcNAc moiety at the non-reducing end of (GlcNAc)6 is bound to the -3, -2 and -1

392

subsites or the -2 and -1 subsites in the substrate-binding site of ChiEn1; the GlcNAc moiety in the

393

non-reducing end of (GlcNAc)7 is bound randomly to the -3, -2, and -1 subsites, or the -4, -3, -2, and -1

394

subsites; the extending GlcNAc moiety at the non-reducing end of (GlcNAc)8 beyond the -4, -3, -2, and

395

-1 subsites in the substrate-binding site of ChiEn1 is unstable, so it preferentially bound to the -3, -2,

396

and -1 subsites (Fig. 8).

397

This study shows that ChiEn1 possesses a transglycosylation activity, transferring a cleaved

398

(GlcNAc)2 residue from the non-reducing end of one chitin oligosaccharide (GlcNAc)n (n=3–6) to

399

another (GlcNAc)n or intermediate (GlcNAc)n-1 to form (GlcNAc)n+2 and (GlcNAc)(n-1)+2. Although no

400

other product of transglycosylation except for the degraded products GlcNAc and (GlcNAc)2 was

401

observed in the reaction of (GlcNAc)3 with ChiEn1, the amount of generated monomer only accounted

402

for 30.7% rather than approximately 50% of the products (GlcNAc and (GlcNAc)2). This result

403

indicates that one part of the cleaved dimers was released as products and the other part of the cleaved

404

dimers was transferred to (GlcNAc)3 to form (GlcNAc)5; the resulting (GlcNAc)5 was cleaved much

405

faster than the (GlcNAc)3 and will likely never be observed. Since the amount of GlcNAc in the

406

reaction mixture of (GlcNAc)5 with ChiEn1 accounts for 21.69% of the total final products (GlcNAc +

407

(GlcNAc)2), the calculated theoretical value of the percentage of the monomer in the total final

408

products (GlcNAc + (GlcNAc)2) in the reaction of trimers with ChiEn1 should be approximately 35%,

409

which is close to the experimentally measured amount of monomer generated from the trimer in this

410

study. The fact that the ChiEn1 hydrolysis products of (GlcNAc)2-pNP contained (GlcNAc)3 and

411

GlcNAc-pNP also suggests that ChiEn1 transfers one (GlcNAc)2 residue from the (GlcNAc)3 analog

412

(GlcNAc)2-pNP to another (GlcNAc)2-pNP to produce (GlcNAc)4-pNP and pNP, and the resulting

413

(GlcNAc)4-pNP is subsequently cleaved to (GlcNAc)3 and GlcNAc-pNP. Thus, this study explores that

414

by the synergy of its random hydrolysis and transglycosylation activities, ChiEn1 can convert chitin

415

oligosaccharides into trimers that are further degraded to dimers and monomers, even though in the

416

reaction mixture of (GlcNAc)4 with ChiEn1, the amount of GlcNAc accounted for 20.05% of the total

417

final products.

418

This study explores that although ChiEn1 did not act on insoluble chitin polymers, ChiEn1 acted

419

synergistically with another endochitinase, ChiIII, from C. cinerea 21 to efficiently hydrolyze insoluble

420

chitin polymers. We previously reported that the C. cinerea endochitinase ChiIII could randomly bind 15



Page 16 of 41

421

to any site on the chitin chain in an endo-action mode and degrade chitin chains to various lengths of

422

chitin oligosaccharides. After each cleavage, ChiIII may disassociate from the cleavage site of the

423

substrate and bind to another site on the remaining chitin chain, to a new chitin chain, or to the chitin

424

oligosaccharide to continue hydrolysis. When the substrate has a polymerization degree of five or

425

fewer of oligosaccharides, ChiIII cleaves monomers rather than dimers from the chitin oligosaccharides

426

in a processive, exo-action mode until it obtains a dimer. However, the specific activities of ChiIII

427

towards chitintriose and chitintetraose are, 0.02 U mg-1 and 0.09 U mg-1,

428

much lower than those of ChiEn1 (0.99 U mg-1 and 3.48 U mg-1, respectively). Thus, ChiEn1 may

429

compensate for the deficiency of ChiIII in its capability to degrade chitintriose and chitintetraose to

430

enhance the hydrolysis of ChiIII towards chitin polymers. Consistent with this point, the addition of

431

ChiEn1 only increased the amount of (GlcNAc)2 released from chitin powder by 149.5% compared

432

with the control (ChiIII alone), whereas it decreased the amount of GlcNAc by 10.3%, which only

433

accounted for approximately 20% of products; this value is closer to the percentage (approximately

434

20–23%) in the products that was released from chitin oligosaccharides by ChiEn1 alone than to the

435

percentage (approximately 40%) in the products released from chitin powders by ChiIII alone. This

436

indicates that the further degradation of the oligosaccharides released from the chitin polymers in the

437

combined reaction mixture by ChiIII was mainly conducted by ChiEn1. As to the fact that the amount

438

of reducing sugars released from 85% deacetylated chitosan by the combination of ChiEn1 and ChiIII

439

was 26.4% lower than the sum of the amount of reducing sugars released by ChiIII or ChiEn1 alone, it

440

may be resulted from partially acetylated chito-oligosaccharides (CHOS) released from chitosan which

441

may bind nonproductively and serve as an inhibitor because its preferred binding mode places a

442

deacetylated unit in subsite −1.

443

ChiA

444

degradation of insoluble chitin substrates have been reported and were attributed to their different and

445

complementary activities (endo- vs. exo- for ChiA and ChiC) and directionalities (reducing- vs.

446

non-reducing for ChiA and ChiB).

447

chitinases also applies to hydrolysis products of one chitinase being used as substrates for another

448

chitinase. Since being similar to ChiIII, the expression level of ChiEn1 in the pileus increased with the

449

maturation of the fruiting body, the synergistic action of ChiEn1 and ChiIII is involved in the efficient

450

degradation of chitin in cell walls during pileus autolysis of the fruiting body of C. cinerea.

49

51

21

respectively, which are

The synergistic actions of endochitinase ChiC and exochitinases

or exochitinase ChiA and exochitinase ChiB

46

47,49

from bacterium S. marcescens on the

Our results first indicate that the synergistic action of two

16


Page 17 of 41


451

This study shows that ChiEn1 has only a catalytic domain and no carbohydrate-binding domain

452

(CBD). It is usually suggested that the presence of a carbohydrate-binding domain in chitinases enables

453

them to bind more tightly to substrates and enhances the processivity of chitinases. 7,45 Tjoelker et al.52

454

even reported that the human chitinase, chitotriosidase, contains a chitin-binding domain. Deleting this

455

domain has no effect on enzyme activity toward soluble chitin oligosaccharides, but activity is lost

456

against insoluble chitin polymers. It seems that the lack of degradation of the insoluble chitin may be

457

due to the lack of a chitin-binding domain of ChiEn1. However, known single-domain bacterial

458

chitinases that lack the chitin-binding domain, such as the exochitinase ChiCH from B. cereus,

459

chitinase Chi18aD from S. coelicolorA3(2),

460

43

461

domain in ChiEn1 does not explain the cause of the loss of activity against the insoluble chitin

462

polymers. The analysis of the molecular structure of SpChiD from S. proteamaculans indicates that

463

chitinases lacking the carbohydrate-binding domain often bind to substrates by the exposed aromatic

464

residues on the surface of substrate-binding cleft. 53,54 This study also shows that more aromatic amino

465

acids were exposed on the surface of the substrate-binding cleft of ChiEn1 than that of ChiC (which

466

has a carbohydrate-binding domain), but it is similar to the SpChiD from S. proteamaculans, which

467

could efficiently hydrolyze various insoluble chitin and soluble chitin.

468

chitin binding capacity of ChiEn1 has been proved in this study too. Therefore, we presume that the

469

reason that ChiEn1 could hydrolyze soluble chitin or chitin oligomeric substrates but not insoluble

470

chitin polymeric substrates may be due to the steric hindrance of two specific small loops situated in

471

the two sides of the binding site cleft, which is different than the case of SpChiD.

472

chitin could be bound to the aromatic residues-containing surface of substrate-binding cleft, the two

473

small protruding loops may impede the access of insoluble crystalline chitin chains to the active site of

474

the putative catalytic center of ChiEn1, in which case ChiEn1 could not act on an insoluble chitin

475

substrate. In contrast, the soluble chitin derivatives such as glycol chitin and 85% deacetylated chitosan

476

may have some flexibility of molecular structure so that they can overcome the steric hindrance to

477

correctly position in the active site in the cleft as substrates. Furthermore, the aromatic residues W255

478

and W252, located at the +2 and +3 subsites of the substrate-binding cleft may correspond to the

479

transglycosylation activity of ChiEn1 because the aromatic residues at the binding sites of sugar

480

acceptors have been demonstrated to be important to transglycosylation activity.31,55,56 The introduction

42

41

the

and the endochitinase SpChiD from S. proteamaculans,

can hydrolyze insoluble chitin powder and/or colloidal chitin. Apparently, the lack of a chitin-binding

17


54

Furthermore, the insoluble

53

Though insoluble


481

of a Trp at position +2 in the binding cleft of ChiA caused the enzyme to develop apparent

482

transglycosylation activity.

483

chitinases are W-W, while they are F94-W424 in ChiEn1. The W to F exchange in ChiEn1 allows for a

484

decrease in the aromatic surface area where the GlcNAc moieties at the non-reducing end of chitin

485

oligosaccharides can interact; thus, for the transglycosylation activity of ChiEn1, it can be predicted

486

that (GlcNAc)2 binds more tightly to the substrate-binding site than (GlcNAc)3 does. 31,57 Furthermore,

487

the catalytic domain of ChiEn1 contains an α + β insertion domain. Usually, a chitinase with an α+β

488

insertion domain in the catalytic domain is considered to be an exochitinase because the α+β insertion

489

domain leads to a deep substrate-binding cleft

490

proteamaculans, containing the so-called α/β insertion domain in the catalytic domain, shows a deeper

491

but more open catalytic cleft and was suggested to function as an endochitinase rather than an

492

exochitinase. 54 We found that although the substrate binding cleft of ChiEn1 is deeper than that of the

493

endo-acting enzyme ChiC2 from S. marcescens,

494

exo-acting enzymes ChiA and ChiB from S. marcescens,

495

hydrolytic patterns on different lengths of chitin oligosaccharides (although it does not act on the chitin

496

polymers). Certainly, the remarkable structural features and structure-function relationships of ChiEn1

497

explored by structure modeling above need to be supported further by a high-resolution

498

crystallographic analysis in the future.

31

Usually, the amino acids at the -3 to -1 sugar-donating subsites of

44

. However, the bacterial chitinase SpChiD from S.

44

it is more shallow and open than those of the 44

which may be related to its random

499 500

Acknowledgments

501

This work was supported by the National Natural Science Foundation of China (No. 31570046), the

502

Priority Academic Development Program of Jiangsu Higher Education Institutions and the Scientific

503

Innovation.

504 505

Supporting Information

506

The Supporting Information is available ：Fig S1. MALDI-TOF MS spectra of the reaction mixture of

507

(GlcNAc)5 (A) and (GlcNAc)6 (B) incubated with ChiEn1 for 5 min as described in Fig 4C, D.

508 509 510 18


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511

Reference

512

(1) Latgé, J. P., The cell wall: a carbohydrate armour for the fungal cell. Mol. Microbiol. 2007, 66,

513

279-290.

514

(2) Kuranda, M. J.; Robbins, P. W., Chitinase is required for cell separation during growth of

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Saccharomyces cerevisiae. J. Biol. Chem. 1991, 266, 19758-19767.

516

(3) Takaya, N.; Yamazaki, D.; Horiuchi, H.; Ohta, A.; Takagi, M., Cloning and characterization of a

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chitinase-encoding gene (chiA) from Aspergillus nidulans, disruption of which decreases germination

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frequency and hyphal growth. Biosci., Biotechnol., Biochem. 1998, 62, 60-65.

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(4) Lim, H.; Choi, H. T., Enhanced expression of chitinase during the autolysis of mushroom in

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Coprinellus congregatus. J. Microbiol. 2009, 47, 225-228.

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(5) Sakamoto, Y.; Nakade, K.; Konno, N.; Sato, T., Senescence of the Lentinula edodes fruiting body

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after harvesting. no. 1 ed.; INTECH Open Access Publisher: Rijeka, Croatia, 2012.

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(6) Li, D. Review of Fungal Chitinases. Mycopathologia 2006, 161, 345-360.

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(7) Seidl, V., Chitinases of filamentous fungi: a large group of diverse proteins with multiple

525

physiological functions. Fungal Biol. Rev. 2008, 22, 36-42.

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(8) Tzelepis, G. D.; Melin, P.; Stenlid, J.; Dan, F. J.; Karlsson, M., Functional analysis of the C-II

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subgroup killer toxin-like chitinases in the filamentous ascomycete Aspergillus nidulans. Fungal

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Genet. Biol. 2014, 64, 58-66.

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(9) Kües, U., Life history and developmental processes in the basidiomycete Coprinus cinereus. Mol.

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Biol. Rev. 2000, 64, 316-353.

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(10) Liu, Z.; Niu, X.; Wang, J.; Zhang, W.; Yang, M.; Liu, C.; Xiong, Y.; Zhao, Y.; Pei, S.; Qin, Q.,

532

Comparative study of nonautolytic mutant and wild-type strains of coprinopsis cinerea supports an

533

important role of glucanases in fruiting body autolysis. J. Agric. Food Chem. 2015, 63, 9609-9614.

534

(11) Bush, D. Autolysis of Coprinus comatus sporophores. Experientia 1974, 30, 984-985.

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(12) Bowman, S. M.; Free, S. J., The structure and synthesis of the fungal cell wall. BioEssays 2006,

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28, 799-808.

537

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completely removes transglycosyl activity associated with the Aspergillus fumigatus AfChiB1.

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Glycoconjugate J. 2009, 26, 525-534.

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chitinase A enhances transglycosylation. Biosci., Biotechnol., Biochem. 2006, 70, 243-251.

651 652 653 654 655 656 657 658 659 660 23



661

Figure legends

662

Figure 1A. The transcript levels of chitinase ChiEn1 gene in the pileus with different open angles.

663

β-tubulin was used to standardize the mRNA levels. All data were obtained using at least three

664

independent experiments with three replicates. B. SDS-PAGE analysis of the recombinant chitinase

665

ChiEn1. Lanes: M, standard protein molecular weight markers; 1, culture medium of control stain; 2,

666

culture medium of recombinant expression strain; 3, purified recombinant ChiEn1. C. The partial

667

amino acid sequences (shadowed in grey) of the partial peptide fragments from the recombinant

668

ChiEn1 with trypsin determined using MALDI-TOF/TOF MS analysis were consistent with that in the

669

entire amino acid sequence that was predicted from the gene sequence of a putative ChiEn1 annotated

670

in the C. cinerea genome (locus/protein identifier accession EAU81461 in GenBank): 1, the sequence

671

of the trypsinized peptide fragments 1 with Mascot ion score of 64 and m/z of 2675.1425; 2, the

672

sequence of the trypsinized peptide fragments 2 with Mascot ion score of 65 and m/z of 2332.0039. D.

673

The spectra of MALDI TOF/TOF MS of the trypsinized peptide fragments 1 (D1) and peptide

674

fragments 2 (D2) in ChiEn1 protein sequence in C.

675 676

Figure 2A. Temperature effect (1) and thermostability (2) of ChiEn1 activity towards glycol chitin at

677

pH 5.0. B. The pH effect and pH stability of ChiEn1 activity towards glycol chitin at 37 °C. C. Effect

678

of (GlcNAc)2-pNP(1) and (GlcNAc)3-pNP (2) concentration on ChiEn1 activity.

679 680

Figure 3. The HPAEC-PAD analysis of the products released from chitin powder by ChiEn1, ChiIII,

681

ChiEn1+ChiIII, or heat-inactivated ChiEn1+ChiIII. The chitin oligosaccharides (GlcNAc)1-6 were used

682

as standards.

683 684

Figure 4. The HPAEC-PAD analysis of the products released from chitin oligosaccharides by ChiEn1.

685

(A) 100 nM (GlcNAc)2-3 was incubated with 0.89 µM ChiEn1 or 100 nM (GlcNAc)4-6 was incubated

686

with 0.45 µM ChiEn1 for 15 min. (B) 100 nM (GlcNAc)4, (C) (GlcNAc)5, or (D) (GlcNAc)6 was

687

incubated with 0.56 µM ChiEn1 for different times. The chitin oligosaccharides (GlcNAc)1-6 were used

688

as standards. (GlcNAc)7 and (GlcNAc)8 in the reaction mixture were identified by their mass-to-charge

689

(m/z) of molecular ions in MALDI-TOF MS spectra (Supporting materials Fig S1).

690 24


Page 24 of 41

Page 25 of 41


691

Figure 5. The HPAEC-PAD analysis of the products released from (A) GlcNAc–pNP, (B)

692

(GlcNAc)2–pNP, or (C)(GlcNAc)3–pNP by ChiEn1. The identities of the peaks was determined using a

693

comparison to the standards (D) (GlcNAc)1–3–pNP and (E) (GlcNAc)1–6. pG1, pG2, and pG3 represent

694

GlcNAc–pNP, (GlcNAc)2–pNP, and (GlcNAc)3–pNP, respectively, and dp2, dp3, dp4, dp5, and dp6

695

represent (GlcNAc)2, (GlcNAc)3, (GlcNAc)4, (GlcNAc)5, and (GlcNAc)6, respectively.

696 697

Figure 6. The TSKgel Amide-80 HPLC analysis of the anomeric configurations of products released

698

from the chitin oligosaccharides by ChiEn1. (A) 100 nmol of (GlcNAc)3, (B) (GlcNAc)4, (C)

699

(GlcNAc)5, and (D) (GlcNAc)6 were reacted with 0.80 µM U of ChiEn1 in 50 µL of 50 mM

700

NaAc-HAc (pH 5.0) on ice for 5 min. The identity of the peaks of the α- and β-anomers of the chitin

701

oligosaccharides was determined by a comparison to (GlcNAc)1–6 standards (OligoTech GLU432-436)

702

(E).

703 704

Figure 7. A. The phylogenetic relationship of chitinase ChiEn1, ChiB1 and ChiIII from C. cinerea,

705

ChiA, ChiB and ChiC from S. marcesens, and three single-catalytic domain chitinases, SpChiD from S.

706

proteamaculans, ChiCH from Bacillus cereus, and Chi18aD from Streptomyces coelicolorA3. Analysis

707

was conducted using neighbour-joining with JTT substitution model and complete deletion of missing

708

data, based on CLUSTAL W alignment of catalytic domain amino acid sequences of these chitinases.

709

Branch support is indicated by bootstrap values. The scale bar corresponds to 0.2 amino acid

710

substitutions per site. Each sequences are marked with its locus/protein identifier, name and organism

711

source as found in GenBank. B. The predicted structure of ChiEn1 (B1), which shows a TIM (α/β)8

712

barrel fold consisting of eight α-helices (indicated by a blue arrow) and eight parallel β-strands

713

(indicated by a red arrow), an α+β insertion domain (indicated by a yellow arrow), two small

714

protruding loops (indicated by grey arrows) situated in the entrances of the two sides of the

715

substrate-binding cleft. The predicted substrate-binding clefts of ChiEn1 (B2) from C. cinerea, ChiA

716

(B3), ChiB (B4), and ChiC2 (B5) from S. marcesens, and SpChiD (B6) from S. proteamaculans, which

717

show the DXDXE catalytic motif and the aromatic amino acids that interact with the substrate.

718 719

Figure 8. A diagram showing the predicted products from different binding modes of chitin

720

oligosaccharides (GlcNAc)3-8 in the substrate-binding cleft of the chitinase ChiEn1. ChiEn1 has at least 25



721

seven subsites (numbered -4 to +2) in which the non-reducing end of the chitin oligosaccharides binds

722

to the negatively labeled subsites, the reducing end binds to positively labeled subsites, and the

723

glycosidic bond between the sugar residues at subsites -1 and +1 is enzymatically cleaved (indicated by

724

an arrow). The original reducing end of chitin oligosaccharides (partially shaded) is an equilibrium

725

mixture of α/β-anomers, and the newly formed reducing end of the glycosyl fragments retains the

726

β-anomeric configuration. The β-anomer of the reducing end of (GlcNAc)3-4 (fully shaded) preferably

727

bound to the +1 and +2 subsites of the substrate-binding cleft, whereas the reducing end of (GlcNAc)5-8

728

(partially shaded) extended beyond the +2 subsite so that the preference for the β-anomer disappeared.

729

The non-reducing end of (GlcNAc)3-5 preferably bound to the -2 subsite of the substrate-binding cleft,

730

whereas the non-reducing end of (GlcNAc)6-8 extended beyond the -2 subsite to bind to the -3, or even

731

-4, subsite.

732 733

26


Page 26 of 41

Page 27 of 41


Table 1. Chitinase ChiEn1 activity towards various substrates Substrate

Specific activity

polysaccharide

U/mg ×10-3

Colloidal chitin (made in the laboratory

0±0.3

by Sandhya et al. 24) Chitin powder (Sigma C7170)

0±0.2

Glycol chitin (made in the laboratory

50.7±0.2

by Li et al.25) 85% Deacetylated chitosan (Sigma C3646)

44.6±0.3

CMC-Na (Sigma C5013)

0±0.2

Glycol chitosan (Sigma G7753)

0±0.6

Laminarin (Sigma 9634 )

0±0.6

oligosaccharides

U/mg

Chitinbiose (OligoTech GLU432)

0

Chitintriose (OligoTech GLU433)

0.99±0.03

Chitintetraose (OligoTech GLU434)

3.48±0.01

Chitinpentose (OligoTech GLU435)

3.58±0.11

Chitinhexaose (OligoTech GLU436)

4.90±0.04

Chitohexaose (ZB-10010)

0

(GlcNAc)1-3-pNP

U/mg

4-Nitrophenyl N-acetyl-β-D-glucosaminide (Sigma, N9376) (GlcNAc-pNP) 4-Nitrophenyl N,N’-diacetyl-β-D-chitobioside

(Sigma,

N6133) ((GlcNAc)2-pNP) 4-Nitrophenyl-β-D-N,N’,N’’-triacetylchitotriose

0 2.39±0.05 1.01±0.05

(Sigma, N8638) ((GlcNAc)3-pNP)

27



Page 28 of 41

Table 2. Effect of Chitinase ChiEn1 on hydrolysis of chitinous polysaccharides by ChiIII

Chitinase in reaction

Reducing sugar released from chitinous polysaccharides (µmol/mL) 85% Deacetylated

Chitin powder

Colloidal chitin

Glycol chitin

ChiIII

0.582±0.007

2.805±0.057

0.645±0.006

0.662±0.009

ChiEn1

0±0.001

0±0.006

0.986±0.009

0.790±0.005

ChiIII+ChiEn1

1.194±0.013

3.395±0.009

2.142±0.028

1.068±0.011

105.0 %

21.0 %

31.3 %

- 26.4 %

Synergsim (%)*

chitosan

*increased or decreased amount (%) of reducing sugars released from chitin powder by combination of ChiEn1 and ChiIII compared to the summary of reducing surgars released respectively by ChiIII or ChiEn1 alone.

28


Page 29 of 41


Table 3. Effect of metal ions and ion chelator EDTA on chitinase ChiEn1activity Enzyme activity(U/mg ×10-3)

Relative activity (%)

161±0.3

100

474±7.7

295

273±1.3

170

44±0.3

27.3

1 mM Ni2+

202±1.4

126

2+

199±1.0

123

Reagent None 1 mM Mn

2+

1 mM Co2+ 1 mM Fe

3+

1 mM Ca

1 mM Mg2+ 1 mM Al

170±1.3

106

3+

15±0. 5

9.6

2+

170±1.0

106

46±1.3

28.5

1 mM Ba

1 mM Cu2+ 1 mM MoO42-

189±2.2

117

-

130±2.9

80.9

1 mM EDTA

128±0.7

79.7

2 mM EDTA

0

0

1 mM Cr2O7

29



Table 4. The chitin oligosaccharides released from chitin powder by ChiEn1, or ChiIII, or ChiEn1+ChiIII, respectively. Released chitin oligosaccharides (µM/mL) Treatment GlcNAc

Chitinbiose

GlcNAc in products (%)

ChiIII

0.193±0.019

0.283±0.017

40.5 %

ChiEn1

0

0

0

ChiIII+ChiEn1

0.175±0.006

0.706±0.012

19.9 %

Synergism (%)*

-10.3%

149.5%

*increased or decreased amount (%) of GlcNAc or (GlcNAc)2 released from chitin powder by combination of ChiEn1 and ChiIII compared to the summary of GlcNAc or (GlcNAc)2 released respectively by ChiIII or ChiEn1 alone.

30


Page 30 of 41

Page 31 of 41


Table 5. Percentage of β-anomer of the produts degraded by chitinase ChiEn1 from (GlcNAc)3-6 Products (% β-anomer) Substrates GlcNAc

(GlcNAc)2

(GlcNAc)3

82

84

28

(GlcNAc)4

92

53

20

(GlcNAc)5

92

46

(GlcNAc)6

96

63

(GlcNAc)3

(GlcNAc)4

(GlcNAc)5

(GlcNAc)6

(GlcNAc)7

66

35

43

38

43

46

36

38

31


(GlcNAc)8

37


Page 32 of 41

Table 6. Insoluble chitin binding capacity of ChiEn1 Total protein

Protein bound to chitin (% total protein)

(µg/mL)

Chitin powder

Colloidal chitin

20

100

100

40

100

83

60

83

50

80

62

37

100

50

30

32


Page 33 of 41


Figure 1

33



Figure 2

34


Page 34 of 41

Page 35 of 41


Figure 3

35



Figure 4

36


Page 36 of 41

Page 37 of 41


Figure 5

37



Figure 6

38


Page 38 of 41

Page 39 of 41


Figure 7

39



Figure 8

40


Page 40 of 41

Page 41 of 41


TOC Graphic

41


(ChiEn1) from Coprinopsis cinerea and its Synergism in the Deg

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