Chitinase Chi1 from Myceliophthora thermophila C1, a Thermostable

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Chitinase Chi1 from Myceliophthora thermophila C1, a thermostable enzyme for chitin and chitosan depolymerization Malgorzata Krolicka, Sandra W. A. Hinz, Martijn J. Koetsier, Rob Joosten, Gerrit Eggink, Lambertus A.M. van den Broek, and Carmen Gabriela Boeriu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04032 • Publication Date (Web): 23 Jan 2018 Downloaded from http://pubs.acs.org on January 24, 2018

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Page 1 of 52

Journal of Agricultural and Food Chemistry

1

Chitinase Chi1 from Myceliophthora thermophila C1, a

2

thermostable enzyme for chitin and chitosan depolymerization

3 4 5

Malgorzata Krolicka,† Sandra W.A. Hinz,§ Martijn J. Koetsier,§ Rob

6

Joosten,§ Gerrit Eggink,† Lambertus A.M. van den Broek,¥ and Carmen G.

7

Boeriu¥*

8 9 10 11 12

†

Department of Bioprocess Engineering, Wageningen University,

Wageningen, The Netherlands §

DuPont Industrial Biosciences, Wageningen, The Netherlands

¥

Wageningen Food & Biobased Research, Wageningen, The Netherlands

13

14

Running Head: Mode of action of chitinase Chi1 from M. thermophila C1

15 16

*Address correspondence to Carmen G. Boeriu: [email protected]

17

and +31 317 480168

18

1

ACS Paragon Plus Environment


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19

ABSTRACT

20

A thermostable chitinase Chi1 from Myceliophthora thermophila C1 was

21

homologously produced and characterized. Chitinase Chi1 shows high

22

thermostability at 40 °C (>140 h 90% activity), 50 °C (>168 h 90%

23

activity), and 55 °C (half-life 48 h). Chitinase Chi1 has broad substrate

24

specificity, and converts chitin, chitosan, modified chitosan and chitin

25

oligosaccharides. The activity of chitinase Chi1 is strongly affected by the

26

degree of deacetylation (DDA), molecular weight (Mw) and side chain

27

modification of chitosan. Chitinase Chi1 releases mainly (GlcNAc)2 from

28

insoluble chitin and chito-oligosaccharides with a polymerization degree

29

(DP) ranging from 2 to 12 from chitosan, in a processive way. Chitinase

30

Chi1 shows higher activity towards chitin oligosaccharides (GlcNAc)4-6 than

31

towards (GlcNAc)3 and is inactive for (GlcNAc)2. During hydrolysis,

32

oligosaccharides bind at subsites -2 to +2 in the enzyme’s active site.

33

Chitinase Chi1 can be used for chitin valorisation and for production of

34

chitin- and chito-oligosaccharides at industrial scale.

35

36

Keywords: Myceliophthora thermophila C1, chitinase, chitin, chitosan,

37

chito-oligosaccharides

38

39

2


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INTRODUCTION

41

Chitin consists of β-(1,4)-linked N-acetyl-D-glucosamine (GlcNAc) units

42

and is one of the most abundant polymers in nature. Chitin is a main

43

component found in shells of crabs, shrimps and lobsters which are

44

popular types of seafood. Every year approximately 6 to 8 million tons of

45

shell-waste are produced from the seafood industry globally.1 This waste

46

stream represents a cheap and renewable resource of chitin, which can be

47

used for production of value-added chemicals. Next to crustacean waste,

48

chitin can also be isolated from insects and fungi. Products obtained from

49

chitin and its deacetylated derivative chitosan can be used in medical

50

applications, packaging, food and nutrition, biotechnology, agriculture and

51

environmental protection.2,3 In recent years, special interest has been paid

52

to water-soluble chito-oligosaccharides, which can act as antimicrobial,4

53

antitumor and anti-inflammatory5 agents. Chito-oligosaccharides can be

54

produced by chemical or enzymatic depolymerization of chitin and

55

chitosan. Common procedures for the production of chito-oligosaccharides

56

rely on acid catalysis, which is characterized by a low yield and high

57

environmental impact. The use of enzyme catalysis for depolymerization

58

of chitin and chitosan is a promising alternative to the chemical methods

59

because

60

oligosaccharides

61

process.6 Nevertheless, development of an efficient enzymatic process

62

requires fundamental knowledge of the catalytic mechanisms of enzymes

63

and understanding the interactions with their substrate.

it

allows in

the a

production

controlled

of

way

specific and

chitin-

and

environmentally

chitofriendly

3



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In nature, chitin is degraded by three groups of enzymes: chitinases (EC

65

3.2.1.14), releasing water soluble chitin oligosaccharides from chitin, N-

66

acetylglucosaminidases (EC 3.2.1.52), degrading products released by

67

chitinases

68

monooxygenases (LPMOs; EC 1.14.99.53) that cleave chitin crystalline

69

chains in an oxidative way, yielding a lactone (C1-oxidation) and a

70

ketoaldose (C4-oxidation) product.8,9,10 The copper-dependent LPMOs act

71

in synergy with chitinases, and enhance the accessibility of chitin chains

72

for chitinases and N-acetylglucosaminidases by disrupting the crystal

73

structure of chitin and generation of more soluble polymer chains with

74

increased susceptibility for enzymatic hydrolysis. Based on amino acid

75

sequence, chitinases have been classified into the glycoside hydrolase (GH)

76

families 18 and 19, and N-acetylglucosaminidase into GH 20 and GH 3,

77

according to the carbohydrate active enzymes classification (CAZy),

78

(http://www.cazy.org/).

79

Chitinases are spread in nature and are involved in physiological processes

80

of bacteria, archea, fungi, animals, and plants.11,12 In recent years special

81

interest has been paid to thermostable chitinases from bacteria and fungi

82

due to their potential application in bioconversion of chitin waste and in

83

the industrial production of chitin oligosaccharides from chitin and chito-

84

oligosaccharides from chitosan. The advantage of thermostable enzymes

85

is that these enzymes do not lose their activity at higher temperatures

86

which are implemented in bioconversion of waste and in industrial

87

processes.6 A number of thermophilic chitinases have been described from

to

monomers,7

and

chitin-active

lytic

polysaccharide

4


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bacteria

including

89

Paenibacillus, Streptomyces, Microbispora, Bacillus, and Brevibacillus.6,13

90

However,

91

thermophilic

92

thermophilum15,

93

Thermoascus aurantiacus vs. levisporus15, Thermomyces lanuginosus19,20,

94

Trichoderma viridae21.

95

The thermophilic filamentous fungus Myceliophthora thermophila C1

96

(previously

97

International

98

homologous and heterologous protein expression.22 M. thermophila C1 has

99

been used before for production of different cell wall degrading enzymes

100

as described by Hinz and co-workers.23,24 We reported before that M.

101

thermophila C1 produces an endochitinase entitled chitinase Chi1.22 Dua

102

et al.25 published recently an exochitinase rMtChit from M. thermophila

103

BJA produced from the same gene sequence as chitinase Chi1 by

104

recombinant expression in Pichia pastoris, however, characteristics of

105

exochitinase rMtChit differed significantly from our chitinase Chi1. Since

106

chitinase Chi1 from M. thermophila C1 might have potential application in

107

bioconversion of chitin waste sources and in industrial production of chitin

108

and chito-oligosaccharides, a full characterization of the chitinase Chi1 and

109

understanding of the interactions of chitinase Chi1 with its substrates is

110

important. Here, we describe the production and detailed characterization

only

Chitinophaga,

a

few

chitinases

thermophilic like

Gliocladium

known B.V.,

as a

Alcaligenes,

fungi

Aspergillus

DuPont

have

been

company)

has

been

for

Chaetomium

Rhizopus

lucknowense

Massilia,

explored

fumigatus14,

catenulatum16,

Chrysosporium

Virgibaillus,

C1;

oryzae17,

Genencor

developed

for

5



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111

of chitinase Chi1 with focus on thermostability, catalytic properties and

112

mode of action on chitin, chitosan and chitin and chito-oligosaccharides.

113

MATERIALS AND METHODS

114

Chemicals

115

Chitin azure, chitin from shrimp shells, glycol chitosan, Schiff’s reagent, 4-

116

nitrophenyl–N-acetylglucosamine

117

diacetyl-β-D-chitobioside ((GlcNAc)2-pNP) and 4-nitrophenyl-β-D-N,N’,N’’-

118

triacetylchitotriose ((GlcNAc)3-pNP), were obtained from Sigma-Aldrich (St.

119

Louis, USA). Oxidized chitosan (Mw 100 kDa, DDA 84 %, degree of

120

oxidation 5%, containing C6-aldehyde and carboxyl groups in a ratio of

121

20:1)

122

(Wageningen, The Netherlands). Hydroxypropyl-chitosan was a kind gift

123

from Nippon Suisan (Japan). Chitin oligosaccharides (GlcNAc)2-6 were

124

obtained

125

purchased from Heppe Medical Chitosan GmbH (Halle, Germany) and

126

Nippon Suisan Kaisha LTD (Tokyo, Japan). The deacetylation degree (DDA

127

in %) and molecular weight (Mw in kDa) are: chitosan 88 DDA/3000 and

128

chitosan 90 DDA/100 (Nippon Suisan Kaisha LTD), chitosan 77 DDA/600,

129

chitosan 78 DDA/600, chitosan 91 DDA/600 and chitosan 94 DDA/600

130

(Heppe Medical Chitosan GmbH). All other chemicals were of the highest

131

purity available.

was

produced

from

at

Megazyme

(GlcNAc-pNP),

Wageningen

(Co.

Food

Wicklow,

&

4-nitrophenyl-N,N’-

Biobased

Ireland).

Research

Chitosans

were

6


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132

Swollen chitin preparation

133

Swollen chitin was prepared according to Monreal and Reese26 with some

134

modifications. Chitin from shrimp shells (1 g) was stirred in 25 mL 85%

135

(v/v) phosphoric acid and left at room temperature for 20 hours.

136

Subsequently it was precipitated by pouring the gelatinous mixture into an

137

excess

138

centrifugation at 3,000 × ց and washed with demineralized water up to

139

pH 6.0.

140

Overexpression of Chi1 gene in M. thermophila C1 and 2 L-scale fermentation for

141

production of chitinase Chi1

142

Overexpression of chitinase Chi1 in M. thermophila C1 and the 2 L-scale

143

fermentation for production of chitinase Chi1 have been carried out based

144

on the procedures previously reported: (i) the DNA sequence of the gene

145

encoding (Chi1) for chitinase Chi1 (GenBank accession number HI550986)

146

was described in a patent27, (ii) the homologous overexpression of

147

chitinase Chi1 in M. thermophila C1 (previously known as C. lucknowense

148

C1) was described in detail by Visser et al.22 and (iii) the preparation of a

149

monocomponent strain of M. thermophila C1 producing chitinase Chi1 and

150

the detailed conditions of the fed-batch fermentation resulting in high

151

production level of chitinase Chi1 (7.5 g/L) were also described by Visser

152

et al.22 In short, the Chi1 gene was amplified from genomic M.

153

thermophila C1 DNA and cloned into a M. thermophila C1 expression

154

vector. The expression cassette containing the chi1 promoter, gene and

155

the terminator obtained from the vector was transformed into a low

of

ice-cold

water.

The

swollen

chitin

was

separated

by

7



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156

protease/(hemi-)cellulase free M. thermophila C1-expression host. Ninety

157

six transformants were grown in a microtiter plate28 and screened for

158

chitinase Chi1 production levels in the culture broth using chitin azure as

159

substrate. The transformant showing the highest level of chitinase activity

160

was selected for fed-batch fermentation to produce chitinase Chi1 at 2-L

161

scale. The strain was grown aerobically in 2 L fermenters in mineral

162

medium, containing glucose as carbon source, ammonium sulfate as

163

nitrogen source and trace elements for the essential salts. The enzyme

164

was produced at pH 6.0 and 32 °C.27 The supernatant containing chitinase

165

Chi1 was centrifuged at 20,000 × ց for 20 minutes to remove the biomass

166

and concentrated (4 fold) using a 5 kDa PES membrane (Vivacell® 70,

167

Sartorius). The crude enzyme extract was subsequently dialyzed against

168

10 mM potassium phosphate buffer pH 6.0 and freeze-dried to obtain the

169

crude enzyme preparation. Freeze-drying was used to prevent microbial

170

decay of enzyme preparation and to avoid the use of preservatives.

171

Sequence analysis of chitinase Chi1

172

Nucleotide and deduced amino acid sequences were analyzed using Clone

173

Manager software. BLAST analysis of the deduced amino acid sequence of

174

chitinase

175

(https://blast.ncbi.nlm.nih.gov/Blast.cgi). Analysis for conserved domains

176

were performed using Conserved Domain Search and Conserved Domain

177

Database (containing information from databases including Pfam, SMART,

178

COG,

179

(https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).

Chi1

PRK,

was

performed

TIGRFAM)29

at

at

the

the

NCBI

NCBI The

server

server signal 8


Page 9 of 52


180

peptide

181

(http://www.cbs.dtu.dk/services/SignalP/) and theoretical isoelectric point

182

(pI)

183

(https://web.expasy.org/compute_pi/). Potential N-linked glycosylation

184

sites and potential O-linked glycosylation sites were predicted by NetOGlyc

185

4.0 Server and NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/).

186

To generate a 3D model of chitinase Chi1 the deduced amino acid

187

sequence of chitinase Chi1 was submitted to the Phyre2 web portal for

188

protein modelling.30 The Phyre2 generated the protein model on the basis

189

of its closest template.

190

Purification of chitinase

191

The freeze-dried crude enzyme preparation containing 142 mg total

192

protein was dissolved in 50 mL 0.05 M Bis-Tris buffer pH 7.0 and purified

193

by anion exchange chromatography using a HiPrep DEAE FF 16/10 column

194

(GE Healthcare Bio-Science AB, Uppsala, Sweden) and an ÄKTA™ pure

195

system (GE Healthcare Bio-Science AB, Uppsala, Sweden). The column

196

was equilibrated with five column volumes (CV) 0.05 M Bis-Tris buffer pH

197

7.0 (buffer A). A sample of 50 mL was loaded onto the column and eluted

198

using 0.05 M Bis-Tris buffer pH 7.0 followed by elution with 1 M NaCl in

199

0.05 M Bis-Tris buffer pH 7.0 (buffer B) as follows: 20% buffer B for 10

200

CV and 45% buffer B for 10 CV with a flow rate of 5 mL min-1. Fractions

201

were collected and screened for chitinase and N-acetylglucosaminidase

202

activity. The fraction containing (10 mL) the highest chitinase activity was

203

subjected to size exclusion chromatography and loaded onto a HiLoad

was

was

analyzed

calculated

at

the

SignalP

with Compute pI/Mw

tool on

4.0

ExPASy

server

server

9



Page 10 of 52

204

16/600 Superdex 75 pg column (GE Healthcare Bio-Science AB, Uppsala,

205

Sweden). Proteins were eluted isocratic with 0.05 M Bis-Tris buffer pH 7.0

206

containing 0.15 M NaCl with a flow rate of 0.5 mL min-1. The absorbance

207

was measured at 280 nm.

208

Enzyme assays and protein concentration

209

During

210

N-acetylglucosaminidase

211

determined using colloidal chitin azure31 as substrate. The enzyme

212

solution (0.05 mL) was incubated with 0.95 mL 5 % (w/v) colloidal chitin

213

azure in 50 mM Bis-Tris buffer pH 7.0, and the mixture was incubated at

214

50 °C for 30 min. After incubation, the reaction was terminated by heating

215

at 96 °C for 5 min, to inactivate the enzyme. The reaction mixture was

216

centrifuged at 20,000 × ց for 5 min and the absorbance of the supernatant

217

was measured at 560 nm. An enzyme-free mixture was used as negative

218

control, and each reported value was the average of duplicate tests. One

219

enzyme unit was defined as a change in the absorbance of 0.01 min-1.32

220

For N-acetylglucosaminidase activity, the reaction mixture contained 0.09

221

mL 2 mM GlcNAc-pNP in 0.1 M citrate-phosphate buffer pH 4 and 0.01 mL

222

enzyme solution. After 10 min incubation in a microtiter plate at 50 °C,

223

0.2 mL 0.25 M Tris/HCl buffer pH 8.8 was added to the mixtures and the

224

absorbance at 405 nm was measured using a Tecan Safire plate reader

225

(Grodig, Austria). One enzyme activity was defined as the amount of

226

enzyme

227

concentration was determined using the bicinchoninic acid assay (BCA)

the

that

purification

liberated

process were

1

µmol

the

activities

measured.

of

pNP

of

chitinase

and

activity

was

Chitinase

per

minute.

The

protein

10


Page 11 of 52


228

according to the recommendation of the supplier (Pierce) with bovine

229

serum albumin as standard.

230

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

231

identification of glycosylated proteins and Isoelectric point determination

232

Sodium dodecyl sulfate-polyacrylamide (10% (w/v)) gel electrophoresis

233

(SDS-PAGE) was performed as described by Laemmli.33 A NuPAGE Novex

234

System (ThermoFisher Scientific, Bleiswijk, The Netherlands) with 10%

235

(w/v) Bis-Tris gels was used. Prior to electrophoresis, all samples were

236

heated for 10 min at 70 °C in NuPAGE LDS Sample Buffer with NuPAGE

237

Sample Reducing Agent, according to the instructions of the manufacturer.

238

Gels

239

recommendation of the supplier (ThermoFisher Scientific). For detection of

240

glycosylated proteins, the SDS-PAGE gel was stained using periodic acid-

241

Schiff staining (PAS).34 The gel was incubated subsequently for 1 h in 12.5%

242

(w/v) trichloroacetic acid, 1 h in 1% (v/v) periodic acid/3% (v/v) acetic

243

acid, 1 h in 15% (v/v) acetic acid (replaced every 10 min), and 1 h at

244

4 °C in the dark in Schiff`s reagent (Sigma-Aldrich, Zwijndrecht, The

245

Netherlands). Hereafter, the gel was washed two times for 5 min in 0.5%

246

(w/v) sodium bisulfite and destained in 7% (v/v) acetic acid. The

247

isoelectric point (pI) of chitinase Chi1 was estimated by isoelectric

248

focusing (IEF) using PhastGel

249

(Pharmacia Biotech, Uppsala, Sweden) with a broad protein calibration kit

250

(pH 3-10, GE Healthcare) as standard. Proteins were stained with

251

Coomassie blue R-2.

were

stained

with

SimplyBlue™

TM

SafeStain

according

to

the

IEF on a Pharmacia LKB Phast System

11



Page 12 of 52

252

Mass spectrometry

253

The molecular weight of chitinase Chi1 was determined by matrix assisted

254

laser-desorption

255

Samples were prepared by the dried droplet method on a 600 µm

256

AnchorChip target (Bruker), using 5 mg mL-1 2,5-dihydroxyacetophenone,

257

1.5 mg mL-1 diammonium hydrogen citrate, 25% (v/v) ethanol and 3%

258

(v/v) trifluoroacetic acid as matrix. Spectra were derived from ten 500-

259

shots (1,000 Hz) acquisitions taken at non-overlapping locations across

260

the sample. Measurements were made in the positive linear mode, with

261

ion source 1, 25.0 kV; ion source 2, 23.3 kV; lens, 6.5 kV; pulsed ion

262

extraction, 680 ns. Protein Calibration Standard II (Bruker) was used for

263

external calibration.

264

Purity and identity of chitinase Chi1

265

To evaluate the identity and purity of chitinase Chi1, a sample containing

266

the purified chitinase Chi1 was sent to The Scripps Research Institute,

267

Proteomics Core (Jupiter, Florida, USA) for proteolytic digestion and HPLC-

268

ESI-MS/MS analysis.

269

Influence of temperature and pH on activity and stability of chitinase Chi1

270

Influence of temperature on activity of chitinase Chi1 was analyzed with

271

swollen chitin as substrate in the range of 30-80 °C. The enzyme solution

272

(0.05 mL) containing 1.6 µM chitinase Chi1 was incubated with 0.95 mL of

273

1% (w/v) swollen chitin in 0.1 M citrate-phosphate-borate buffer pH 6.0 at

274

50 °C while mixing at 800 rpm for 30 min. The reaction was terminated by

time-of-flight

mass

spectrometry

(MALDI-TOF-MS).

12


Page 13 of 52


275

heating at 96 °C for 5 min. The reaction mixture was centrifuged at

276

20,000 × ց for 10 min. The produced reducing sugars in the supernatant

277

were measured using the p-hydroxybenzoic acid hydrazide (PAHBAH)

278

assay.35 An enzyme-free mixture was used as negative control, and each

279

reported value was the average of duplicate tests. N-acetyl-D-glucosamine

280

was used as a standard and one enzyme unit (U) was defined as the

281

amount of enzyme that liberated 1 µmol reducing sugar per minute. The

282

thermostability was determined by pre-incubating the purified chitinase

283

Chi1 (1.6 µM) at pH 6.0 (0.1 M citrate-phosphate-borate buffer) at various

284

temperatures (40–60°C) for different time intervals up to 168 h. The

285

influence of pH on activity of chitinase Chi1 was determined by incubating

286

chitinase Chi1 (1.6 µM) at different pH levels (3.0–9.0) in 0.1 M citrate-

287

phosphate-borate buffer using swollen chitin as substrate.

288

Determination of kinetic parameters

289

The

290

90 DDA/100 kDa

291

(GraphPad

292

supernatant were measured using the p-hydroxybenzoic acid hydrazide

293

(PAHBAH) assay.35

294

Depolymerization of chitosans with different Mw and DDA

295

To elucidate the influence of Mw and DDA of chitosans on chitinase Chi1

296

activity a wide range of different chitosans were tested including: glycol

297

chitosan, hydroxypropyl chitosan, oxidized chitosan and chitosans with

Km,

Vmax

and

kcat

were

values

calculated

Software, USA). The

for

swollen

with

chitin

GraphPad

reducing

sugars

and

chitosan

Prism

software

produced

in

the

13



Page 14 of 52

298

different DDA and Mw. Chitosans were used in a concentration of 0.1%

299

(w/v) with 40.7 nM of purified chitinase Chi1 in 1 mL 0.05 M sodium

300

phosphate buffer pH 6.0. The mixture was incubated at 50 °C while

301

mixing at 800 rpm for 15 min. After incubation, the enzyme activity was

302

terminated by heating at 96 °C for 5 min. The reaction mixture was

303

centrifuged at 20,000 × ց for 5 min. The reducing sugars produced in the

304

supernatant were measured using the p-hydroxybenzoic acid hydrazide

305

(PAHBAH) assay.35

306

Hydrolysis of swollen chitin and chitosan

307

For the enzymatic hydrolysis of swollen chitin and chitosan 90 DDA/100

308

the reaction mixtures containing 1 mL of 0.45% (w/v) substrate in 0.05 M

309

sodium phosphate buffer pH 6.0 with 100 nM purified chitinase Chi1 were

310

incubated at 50 °C while mixing at 800 rpm. Aliquots were taken at

311

different time intervals and the hydrolysis products were analyzed by

312

High-Performance Anion-Exchange Chromatography (HPAEC) and MALDI-

313

TOF-MS.

314

Hydrolysis of chitin oligosaccharides and pNP-substrates

315

Hydrolysis of chitin oligosaccharides (GlcNAc)2-6 and pNP-substrates was

316

followed in time. Incubations were performed with 25 nM purified

317

chitinase Chi1 in 0.5 mL reaction volume containing 2 mM substrate

318

(GlcNAc)2-6, 1 mM (GlcNAc)2-pNP or (GlcNAc)3-pNP, and 50 mM sodium

319

phosphate buffer pH 6.0. Samples were incubated at 50 °C and aliquots of

320

60 µL were taken at different time intervals. The reaction was terminated

14


Page 15 of 52


321

by heating at 96 °C for 5 min and the hydrolysis products were analyzed

322

by HPAEC.

323

High Performance Anion-Exchange Chromatography (HPAEC)

324

An ICS-3000 Ion Chromatography HPLC system equipped with a CarboPac

325

PA-1 column (2×250 mm) in combination with a CarboPac PA-guard

326

column (2×25 mm) at 22 °C and a pulsed electrochemical detector (PAD)

327

in pulsed amperometric detection mode (Dionex) at 30 °C was used. A

328

flow rate of 0.25 mL min-1 was used and the column was equilibrated with

329

water. The following gradient was used: 0-25 min H2O, 25-65 min at

330

0-0.045 M NaOH, 65-70 min at 0.045 M NaOH-1 M sodium acetate in

331

0.1 M NaOH,

332

75-75.1 min 1 M sodium acetate in 0.1 M NaOH-0.1M NaOH, 75.1-80 min

333

0.1M NaOH, 80-95 min H2O. Post column addition was used for increasing

334

the PAD signal by 0.5 M NaOH at a flow rate of 0.15 mL min-1.

335

Identification of chitin and chito-oligosaccharides by MALDI-TOF-MS

336

MALDI-TOF-MS was performed on a Bruker UltraFlextreme (Bruker

337

Daltonics) in reflective mode and positive ions were examined. The

338

instrument was calibrated for positive ions with a mixture of maltodextrin

339

standards with known molecular masses. Samples were diluted in the

340

matrix solution containing 10 mg mL-1 2,5-dihydroxybenzoic acid in 50%

341

(v/v) acetonitrile. For analysis, 1 µL of the mixture was transferred to the

342

target plate and dried under a stream of dry air. The lowest laser intensity

70-75

min

at

1 M sodium

acetate

in

0.1 M

NaOH,

15



Page 16 of 52

343

needed to obtain a good quality spectrum was applied and 10 times 50

344

laser shots randomly obtained from the sample, were accumulated.

345

RESULTS

346

Sequence analysis of chitinase Chi1

347

The putative gene chi1 encoding for chitinase Chi1 had an ORF of 1,281-

348

bp that encoded a protein with 426 amino acids, in which a signal peptide

349

is predicted that consists of 23 amino acids in the N-terminal region of the

350

protein. The deduced molecular weight of chitinase Chi1 was 43.8 kDa and

351

a theoretical pI was at pH 4.95. Four potential O-linked glycosylation sites,

352

one potential N-linked glycosylation site, and 47 phosphorylation sites

353

were found in the sequence. Multiple sequence alignment of the active site

354

of the deduced protein sequence of chitinase Chi1 (Figure 1A) with

355

Chitinase A from Serratia marcescens (1NH6_A), Chitinase B from

356

Arthrobacter

357

(AAA83223),

358

revealed the presence of the conserved glycoside GH 18 domain in

359

chitinase Chi1. In total 10 conserved amino acids and one conserved

360

active-site motif consisting of aspartate (D) and glutamate (E) residues

361

forming the D-X-E motif were found in chitinase Chi1. Modeling of the

362

secondary structure of chitinase Chi1 revealed that chitinase Chi1 is

363

composed of 16 α-helixes and 14 β-sheets (Figure 1B). 3D modelling of

364

chitinase Chi1 was based on similarity with chitinase from the fungus

365

Clonostachys

sp.

TAD20

chitinase

rosea

from

(1KFW_A), Clostridium

belonging

to

GH

Janthinobacterium paraputrificum

18

(template

lividum

(BAD12045)

PDB

entry:

16


Page 17 of 52


366

3G6MA)(Figure 1C). Using this sequence 386 amino acids residues (equal

367

to 96 % of the whole amino acid sequence) have been modelled with 100 %

368

confidence by the single highest scoring template. This modeling revealed

369

the (β/α)8 barrel fold (TIM) of chitinase Chi1.

370

Purification of chitinase Chi1

371

The gene chi1 encoding for chitinase Chi1 was successfully cloned into the

372

M. thermophila C1-expression host. The transformant with the highest

373

production was used for the production of high amounts of chitinase Chi1,

374

(7.5 g/L), in 2-L fermentation. From the culture broth, 15 g of protein

375

containing about 60 % chitinase Chi1 (based on SDS-PAGE, Figure 2A)

376

was obtained. The crude enzyme preparation was further subjected to a

377

two-step purification process using anion exchange and size exclusion

378

chromatography. The first purification step (Figure 2A) resulted in the

379

separation of the extract in two main protein peaks of which the peak

380

eluting first showed mainly N-acetylglucosaminidase activity and the

381

second peak chitinase activity. However, in the second peak some N-

382

acetylglucosaminidase activity was detected. Therefore, in order to

383

remove this activity, Fraction I from the first purification step was

384

subjected to size exclusion chromatography using a Superdex 75 (Figure

385

2B). This step enabled a clear separation between chitinase Chi1 and the

386

remaining

387

purified to homogeneity as shown on SDS-PAGE as one single band

388

(Figure 3A) and was confirmed by HPLC-ESI-MS/MS analysis of the

389

proteolytic digest (Figure S1, Supporting Information), that identified only

N-acetylglucosaminidase.

Chitinase

Chi1

was

successfully

17



Page 18 of 52

390

peptides originating from chitinase Chi1. Staining with periodic acid-Schiff

391

(PAS) did not detect any glycosylation of chitinase Chi1 (Figure 3B). The

392

specific activity of the purified chitinase Chi1 was 3.5 U mg-1 for colloidal

393

chitin azure (Table 1).

394

Molecular weight and isoelectric point of chitinase Chi1

395

Chitinase Chi1 is a monomeric polypeptide and the molecular weight

396

predicted from the protein sequence is 43.8 kDa. The molecular weight of

397

the purified chitinase Chi1 was measured by MALDI-TOF-MS and was

398

shown to be 42.9 kDa (Figure 3D). The MALDI-TOF-MS spectrum of

399

chitinase Chi1 showed an intense signal of the single charged protein

400

[M+H]+ at m/z 42,882 and the signal of the double-charged protein

401

[M+2H]2+ at m/z 21,370. The SDS-PAGE revealed a molecular weight of

402

43 kDa (Figure 3A). The isoelectric point of chitinase Chi1 was found to be

403

3.95 (Figure 3C).

404

Influence of pH and temperature on activity and stability of chitinase Chi1

405

Chitinase Chi1 was active at pH 3.0 to 9.0 (Figure 4A), and exhibited the

406

highest activity at pH 6.0. Chitinase Chi1 showed activity from 30 to 70 °C

407

with the highest activity at 55 °C (Figure 4B) and it was remarkably stable

408

at 40 °C (>140 h 90% activity) and 50 °C (>168 h 90% activity) (Figure

409

4C). At 55 °C the enzyme reached its half-life after 48 h. Incubation at

410

60 °C resulted in fast inactivation of the enzyme, with a loss of 90% of

411

the initial catalytic activity after 1h incubation.

18


Page 19 of 52


412

Kinetic parameters for chitinase Chi1

413

Kinetic parameters of chitinase Chi1 were determined for swollen chitin

414

and for chitosan 90 DDA/100. Activities were determined based on

415

reducing sugars released during the reaction.

416

For swollen chitin the specific activity was 1.4 ±0.2 U mg-1, Vmax was 12.2

417

±0.5 µM min-1, Km was 2.0 ±0.2 mg mL-1, and kcat was 0.11 ±0.0 s-1.

418

For chitosan the specific activity was 10.0 ±0.6 U mg-1, Vmax was 194

419

±21.4 µM min-1, Km was 0.9 ±0.3 mg mL-1, and kcat was 1.9 ±0.2 s-1.

420

Influence of molecular weight and degree of deacetylation of chitosan on

421

chitinase Chi1 activity

422

To evaluate the influence of the Mw (100, 600 and 3,000 kDa) and DDA

423

(77, 78, 88, 90, 94) of chitosan on chitinase Chi1 activity, a range of

424

chitosans was tested (Figure 5). In case of chitosans with the same Mw

425

(600 kDa), chitinase Chi1 showed decreased activity when DDA was

426

increased. The highest activity was measured for chitosan with the lowest

427

Mw (chitosan 90 DDA/100). For chitosan with similar DDA but different

428

Mw (chitosan 88 DDA/3000 and chitosan 90 DDA/100), chitinase Chi1

429

showed higher activity for lower Mw chitosan. Chitinase Chi1 degraded

430

partially oxidized chitosan (i.e. chitosan with random oxidation at C6

431

positions), but showed 10% of activity as measured for the untreated

432

parent chitosan (chitosan 90 DDA/100). Chitinase Chi1 was not able to

433

degrade glycol chitosan and hydroxypropyl-chitosan, which are both fully

434

deacetylated.

19



Page 20 of 52

435

Degradation of chitin and chitosan by chitinase Chi1

436

Degradation of chitin incubated with chitinase Chi1 was followed in time

437

and products released were analyzed by HPAEC. The main products

438

formed were (GlcNAc)2 next to small amount of (GlcNAc)3 and GlcNAc

439

(Figure 6). After the first 30 min the rate of (GlcNAc)2 release gradually

440

decreased in time. The concentration of (GlcNAc)3 and GlcNAc increased

441

gradually up to 30 min of incubation and levelled off hereafter. At 90 min

442

incubation the concentration of (GlcNAc)3 started to decrease, when

443

degradation into (GlcNAc)2 and GlcNAc became the predominant reaction.

444

The ratio (GlcNAc)2 to GlcNAc at the end of the reaction was equal to 11.

445

Chitin conversion expressed as the amount of (GlcNAc)2 produced was 3.1%

446

after 6 h.

447

Products released from chitosan 90 DDA/100 were analyzed by MALDI-

448

TOF-MS. Chito-oligosaccharides were detected as potassium and/or

449

sodium adducts and are summarized in Table S1 (Supporting Information).

450

Chitinase Chi1 was able to release a whole spectrum of hetero-

451

oligosaccharides consisting of GlcNAc and GlcN units with a polymerization

452

degree (DP) ranging from 2 to 12. The chito-oligosaccharide composition

453

of the reaction mixture changed over time (Figure S2, Supporting

454

Information). At the early stages of the reaction, a large diversity of chito-

455

oligosaccharides were formed, containing 2 to 6 GlcNAc units and 1 to 9

456

GlcN residues in different combinations and with a ratio GlcNAc/GlcN

457

spanning the range between 0.5 to 4 (Table 1S, Figure S2, Supporting

458

Information). During the reaction, chito-oligosaccharides with more than

20


Page 21 of 52


459

three GlcNAc residues might be further degraded by chitinase Chi1, and

460

new GlcN-enriched chito-oligosaccharides containing up to 10 GlcN

461

residues and one or two GlcNAc residues are formed, that accumulate in

462

time (Table 1S, FigureS3, Supporting Information). Fully acetylated DP 2

463

((GlcNAc)2, 447.2 m/z [M+Na]+) was identified from the early stages of

464

the reaction and during the entire incubation. Fully acetylated DP 3 (650.3

465

m/z [M+Na]+) was detected only at 15 min incubation time, indicating

466

that it was subsequently degraded by the enzyme. Accumulation of the

467

heterologous dimer (DP 2) composed of GlcNAc and GlcN (405.2 m/z

468

[M+Na]+) at longer reaction times, in the slow phase of the reaction, may

469

indicate the ability of chitinase Chi1 to cleave glycosidic linkages between

470

GlcN and GlcNAc moieties in the chitosan chain, but only when GlcNAc is

471

positioned in -1 subsite of the enzyme active site.

472

Degradation of chitin oligosaccharides and pNP-substrates by chitinase Chi1

473

In order to study the binding preferences and to determine the shortest

474

possible

475

oligosaccharides (GlcNAc)2-6 was followed in time and the hydrolysis

476

products were analyzed by HPAEC (Figure 7). (GlcNAc)2 was not

477

hydrolyzed by chitinase Chi1 (data not shown), whereas (GlcNAc)3 was

478

cleaved to (GlcNAc)2 and GlcNAc (Figure 7A). (GlcNAc)4 was split only to

479

(GlcNAc)2 (Figure 7B). (GlcNAc)5 was degraded to (GlcNAc)2 and (GlcNAc)3,

480

however, after 25 h (GlcNAc)3 was degraded to GlcNAc and (GlcNAc)2

481

(Figure 7C). Depolymerization of (GlcNAc)6 resulted in the initial release of

482

(GlcNAc)4 and (GlcNAc)2 and small amounts of (GlcNAc)3 (Figure 7D). The

substrate

for

chitinase

Chi1

the

hydrolysis

of

chito-

21



Page 22 of 52

483

released (GlcNAc)4 was further degraded to (GlcNAc)2 and (GlcNAc)3 was

484

cleaved to (GlcNAc)2 and GlcNAc. The calculated rates for the degradation

485

of chitin oligosaccharides were 0.18 mM min-1 for (GlcNAc)6, 0.17 mM min-

486

1

for (GlcNAc)5, 0.20 mM min-1 for (GlcNAc)4, and 0.02 mM min-1 for

487

(GlcNAc)3.

488

Among pNP-labelled chitin oligosaccharides, activity of chitinase Chi1 was

489

detected for (GlcNAc)3-pNP and (GlcNAc)2-pNP with the highest specific

490

activity for (GlcNAc)2-pNP (Figure S3 A, Supporting Information). No

491

activity was found for GlcNAc-pNP, which is in agreement that no activity

492

was detected for (GlcNAc)2.

493

To investigate the reaction mechanism in more detail, degradation of

494

labeled chitin oligosaccharides (GlcNAc)3-pNP and (GlcNAc)2-pNP was

495

followed in time by HPAEC (Figure S3 B, Supporting Information).

496

Hydrolysis of (GlcNAc)3-pNP yielded predominantly GlcNAc-pNP and

497

(GlcNAc)2 (about 90% end product) and low amounts of (GlcNAc)3 (about

498

10% end product). In time the released (GlcNAc)3 was degraded further

499

to (GlcNAc)2 and GlcNAc. Products detected from the hydrolysis of

500

(GlcNAc)2-pNP

501

(GlcNAc)3 (Figure S4 B, Supporting Information). The substrate used in

502

this experiment did not contain (GlcNAc)3 (Figure S4 C, Supporting

503

Information).

504

transglycosylation reaction catalyzed by chitinase Chi1 and it is in

505

agreement with results reported for chitinase MBP-CfcA from Aspergillus

506

niger.36

were

(GlcNAc)2,

Production

of

GlcNAc-pNP

(GlcNAc)3

can

and

be

small

amounts

explained

by

of

the

22


Page 23 of 52


507

DISCUSSION

508

Analysis of the amino acid sequence of chitinase Chi1 confirmed that

509

chitinase Chi1 is a real glycoside hydrolase from GH 18 family, which

510

contains characteristic for this family D-X-E motif. The 3D modelled

511

structure revealed that chitinase Chi1 has (β/α)8 barrel fold (TIM) which is

512

another characteristic for chitinases from GH 18.

513

Chitinase Chi1 was purified to homogeneity as confirmed by MALDI-TOF-

514

MS and HPLC-MS/MS analysis of the proteolytic digests of the purified

515

enzyme. The increase in specific activity after each purification step was

516

not very extensive, and this might be due to the additive activity of the

517

accompanying N-acetylglucosaminidase that was detected in the crude

518

enzyme extract and which was still present in the fractions obtained after

519

the first purification step using anion exchange chromatography. The

520

ability of N-acetylglucosaminidase to act on the amorphous parts of chitin

521

was reported for β-N-acetylhexosaminidase (LeHex20A) by Konno and co-

522

workers.37

523

chromatography enabled a clear separation between chitinase Chi1 and

524

the remaining N-acetylglucosaminidase, resulting in the isolation of a pure

525

enzyme.

526

Molecular weight of chitinase Chi1 measured with MALDI-TOF-MS was 43

527

kDa, which was different from a molecular weight calculated from the

528

deduced protein sequence of 43.8 kDa. This difference shows that full-

529

length chitinase Chi1 undergoes post-translational proteolytic modification

530

in the host M. thermophila C1. Detection of peptides from C-terminus end

The

second

step

of

purification

on

size

exclusion

23



Page 24 of 52

531

of chitinase Chi1 with HPLC-MS/MS indicates that proteolytic removal

532

takes place from N-terminus end of chitinase Chi1. According to the

533

calculated molecular weight, approximately 9 amino acids might be

534

removed. Proteolytic processing has been previously described for

535

chitinases from other microorganisms including chitinase ChiC from

536

Serratia marcescens38, chitinases from Streptomyces olivaceoviridis39 and

537

Janthinobacterium

538

exochitinase rMtChit obtained by heterologous expression of the same

539

gene sequence obtained from M. thermophila BJA in Pichia pastoris. This

540

latter protein has a molecular weight of 48 kDa. The difference in

541

molecular weight between chitinase Chi1 and rMtChit can be explained by

542

the fact that different cloning approaches and different hosts were used

543

for enzyme production, i.e. chitinase Chi1 was expressed homologously in

544

M. thermophila while exochitinase rMtChit was expressed heterologously

545

in the yeast P. pastoris. Furthermore, chitinase Chi1 and exochitinase

546

rMtChit differ clearly in the extent of glycosylation. Staining with PAS

547

confirmed that chitinase Chi1 was not or hardly glycosylated because no

548

magenta

549

concentration of 1 mg mL-1. In contrast, exochitinase rMtChit gave an

550

intense magenta color, indicating glycosylation, as reported by Dua et

551

al.25

552

Chitinase Chi1 was found to have a pH optimum at pH 6.0 and a pI of

553

3.98, while the calculated pI from the amino acid sequence was 4.95. This

554

difference between the theoretical pI predicted from the primary structure

color

lividum40.

formation

Recently,

was

Dua

detected

for

et

al.25

chitinase

reported

Chi1

the

at

a

24


Page 25 of 52


555

and the experimentally determined pI is common, since the pI of proteins

556

is affected by several factors, including the solvent accessibility of amino

557

acids. Some charged amino acids could be shielded by the folded structure

558

of the enzyme and may not be exposed to the solvent, changing therefore

559

the observed pI.

560

The highest activity of chitinase Chi1 was detected at 55 °C which is in

561

agreement with other thermophilic chitinases (Table 2). Chitinase Chi1

562

showed excellent thermostability at 50 °C (>168 h, 90% activity) and at

563

55 °C (t½= 48 h). Reported thermostable fungal chitinases have also

564

thermostability up to 50 °C, but all are less stable in time than chitinase

565

Chi1 (Table 2). For example, the exochitinase rMtChit retained only 70%

566

of its activity when exposed to 45 °C for 5 h and showed a t½= 113

567

minutes at 65 °C. During incubation at 50 °C (1 h) chitinase from T.

568

lanuginosus retained 71% of its activity whereas other chitinase from T.

569

lanuginosus was able to preserve about 70% of the enzyme activity after

570

6 h at 50 °C. In contrast, another chitinase from T. lanuginosus SY2 was

571

100 % active for 1h when incubating at 50 °C. Therefore, it can be

572

concluded that chitinase Chi1 can be classified as thermostable chitinase

573

which shows excellent thermostability among other chitinases from

574

thermophilic fungi.

575

Besides activity on chitin, chitinase Chi1 showed also activity on the

576

deacetylated chitin derivative - chitosan. Solubilized chitosan was more

577

efficiently degraded by the enzyme than swollen chitin. This is in

578

agreement with previous studies reporting that chitinases from GH 18 are 25



Page 26 of 52

579

able to cleave the glycosidic linkage of not only GlcNAc-GlcNAc but also

580

GlcNAc-GlcN present in chitosan as long as a GlcNAc residue is bound at

581

the -1 subsite.42 Furthermore, higher activity on chitosan than on chitin

582

indicates that substrate accessibility is an important parameter influencing

583

chitinase activity as it was also observed for bacterial chitinase from

584

Ralstonia sp.41

585

The activity of chitinase Chi1 was strongly affected by DDA, Mw and

586

presence of side groups (i.e. aldehyde, carboxyl) at the chitosan chain. In

587

general, chitosan with a lower DDA (77 DDA) was degraded more

588

efficiently than chitosan with high DDA (94 DDA). This result confirmed,

589

that chitinase Chi1 is a real GH 18 enzyme, which is dependent on the

590

acetyl group of GlcNAc for catalysis. Thus, a decrease in the number of

591

GlcNAc moieties present in the chitosan chain will result in less productive

592

binding sites of chitinase Chi1. Chitosan with low Mw (100 kDa) was

593

degraded more efficiently than chitosan with high Mw (3,000 kDa) and the

594

same DDA. This result denotes, that chitosan with higher Mw, which also

595

shows higher viscosity, is less accessible for the enzyme than the chitosan

596

with a low Mw. Fully deacetylated modified chitosans with pending

597

aliphatic side groups - such as glycol chitosan and hydroxypropyl-chitosan,

598

were not degraded at all by the enzyme, showing that chitinase Chi1 is a

599

real chitinase, which is not able to cleave GlcN-GlcN bonds. Although

600

steric hindrance due to side chains cannot be excluded, the results clearly

601

suggest that the chitinase Chi1 activity depends on the presence and the

602

number of acetyl groups. It was shown for enzymes from GH 18, that the 26


Page 27 of 52


603

carbonyl oxygen from GlcNAc moiety act as a nucleophile during catalytic

604

reaction of GH 18 enzymes. Similar activity on chitosan was reported for

605

chitinase from Streptomyces griseus.42

606

Chitinase Chi1 released mainly (GlcNAc)2 from chitin. Release of dimers

607

was reported for other chitinases from GH 18 family.7,43 In contrast,

608

exochitinase rMtChit released only monomers from chitin.25 Although the

609

amino acid sequence of chitinase Chi1 and exochitinase rMtChit should be

610

the same, differences in expression host and glycosylation influence the

611

activity and mode of action of chitinase Chi1 and exochitinase rMtChit.

612

Next to (GlcNAc)2, chitinase Chi1 released small amount of GlcNAc and

613

(GlcNAc)3, with a ratio of (GlcNAc)2/GlcNAc equal to 11. This ratio is

614

commonly used for a rough assessment of enzyme processivity.44,45 Thus,

615

chitinase Chi1 may be considered as a processive chitinase. A decrease in

616

the rate of chitin hydrolysis, which was observed after 30 min reaction is

617

most likely due to the fact that chitinase Chi1 enriched the recalcitrant

618

regions of substrate. It was previously stated, that the activity of

619

processive enzymes tends to decrease as the substrate is consumed and

620

when the enzymes reach regions that hinder processive binding.45

621

The release of (GlcNAc)2 in higher molar amount than other chitin

622

oligosaccharides from chitin was observed for processive and non-

623

processive enzymes degrading

624

processive chitinases ChiA, ChiB and non-processive ChiC from S.

625

marcescens.43 Therefore, results obtained for chitinase Chi1 with natural

626

substrates indicate its processivity, but this conclusion is not indisputable.

recalcitrant polysaccharides, like

for

27



Page 28 of 52

627

Important feature of processive exo-acting chitinases is so-called α + β

628

insertion domain that forms one ‘wall’ of the substrate binding cleft, which

629

were found in ChiA and ChiB.46,9 This domain was not found in chitinase

630

Chi1. However, the active site of chitinase Chi1 is aligned with 60 amino

631

acid residues, which may be important for interactions with the substrate

632

and promote processivity of the enzyme. Aromatic residues in the active

633

site of processive enzymes were shown to interact with the substrate

634

during the processive mode of action.45

635

Processivity has been studied for other chitinases and also for cellulases.47

636

It was shown, that processive enzymes slide with their active site on the

637

single-polymer chain and stay closely associated with the substrate

638

between

639

degradation of chitin the enzymes release mainly dimers because the

640

successive sugar units in the polymer are rotated by 180° and sliding of

641

such polymers through the enzyme’s active site will result in the

642

productive binding only after every second sugar moiety. Rotation of the

643

sugar units is particularly important for chitinases from family GH 18,

644

since these enzymes require a correctly positioned N-acetyl group in

645

their -1 subsite.43

646

It may be concluded, that chitinase Chi1 is an endo-chitinase with high

647

degree of processivity. It was stated that both endo- and exo-mechanisms

648

can be combined with processive action46 and that the most important

649

difference between chitinases may be related to the ability of the enzymes

subsequent

hydrolytic

reactions.

During

the

processive

28


Page 29 of 52


650

to act in processive or non-processive way, rather than to their binding

651

preferences (endo- or exo-manner).9

652

Chitinase Chi1 was able to release a broad spectrum of chitin- and chito-

653

oligosaccharides (DP2-DP12) from chitosan with 90% DDA. The chito-

654

oligosaccharide composition of the reaction mixture changed over time,

655

indicating

656

oligosaccharides might be simultaneously catalyzed by chitinase Chi1, as

657

it was shown in the experiment with chitin oligosaccharides (GlcNAc)2-6.

658

The composition of chito-oligosaccharides and the accumulation of a

659

(GlcNAc, GlcN) dimer at longer reaction time may indicate the ability of

660

chitinase Chi1 to cleave glycosidic linkages between GlcN and GlcNAc

661

moieties in the chitosan chain, as it was reported for the chitinase G from

662

Streptomyces coelicolor A3(2) from bacterial family GH19 chitinase48 and

663

other bacterial chitinases49. Release of longer chito-oligosaccharides may

664

indicate, that similarly to chitin degradation, chitinase Chi1 can degrade

665

chitosan in a processive way. Chitinases ChiA and ChiB from S.

666

marcescens50 were shown to degrade chitosan in a processive way. In

667

case of chitosan, processive enzymes stay attached to the substrate after

668

productive (with a correctly positioned N-acetyl group in the sugar unit) or

669

non-productive (with lack of a correctly positioned N-acetyl group in the

670

sugar unit) initial binding to the substrate. Binding of the substrate will be

671

followed by sliding of the substrate through the active site cleft by two

672

sugar units at the time, until a new productive complex will be formed and

673

an enzymatic reaction occurs.43

that

release

and

further

degradation

of

some

chito-

29



Page 30 of 52

674

Chitinase Chi1 degraded chitin oligosaccharides with DP ≥ 3. The absence

675

of activity on (GlcNAc)2 ruled out the possibility that chitinase Chi1 is an

676

N-acetylglucosaminidase. All initial released chitin oligosaccharides with

677

DP ≥ 3 were subject to further hydrolysis that yielded (GlcNAc)2 and

678

GlcNAc as final products. Chitinase Chi1 showed increasing activities with

679

increasing DP of chitin oligosaccharides. These data indicate that chitinase

680

Chi1

681

oligosaccharides with DP≥4 is more efficient than with DP 3, resulting in

682

about 10 times faster conversion for longer-chain chitin oligosaccharides

683

than for shorter ones. Additionally, experiments with pNP-(GlcNAc)3 and

684

pNP-(GlcNAc)2 oligosaccharides bound to chitinase Chi1 at subsites -2 to

685

+2 in the active site during hydrolysis.

686

Overall, we showed here that homologous expressed chitinase Chi1

687

releases mainly dimers from chitin and might use a processive mechanism.

688

Depolymerization of chitosan resulted in the production of a wide range of

689

chito-oligosaccharides. Chitin and chito-oligosaccharides are an emerging

690

class of bioactive ingredients with potential biomedical, cosmetic and

691

pharmaceutic

692

technologies for the production of chitin and chito-oligosaccharides reveals

693

new perspectives for the application of biocatalysts. With its remarkable

694

thermostability and activity in a wide range of pH, chitinase Chi1 is a

695

promising biocatalyst for bioconversion of chitin waste sources and

696

production of chitin and chito-oligosaccharides from both chitin and

697

chitosan at industrial scale.

has

a

multi-subsite

applications.

binding

The

cleft

need

for

and

positioning

green

and

of

chitin

biocompatible

30


Page 31 of 52


698

FUNDING SOURCES

699

This research received funding from the Netherlands Organisation for

700

Scientific Research (NWO) in the framework of the TASC Technology Area

701

BIOMASS.

702

SUPPORTING INFORMATION AVAILABLE: DESCRIPTION

703

-

with HPLC-ESI-MS/MS analysis (Figure S1).

704 705

Protein oligomers detected after proteolytic digestion of chitinase Chi1

-

Spectra from matrix assisted laser-desorption time-of-flight mass

706

spectrometry

707

released by chitinase Chi1 from chitosan 90 DDA/100 and their

708

molecular mass (Figure S2, Table S1).

709

-

(MALDI-TOF-MS)

obtained

for

chito-oligosaccharides

Specific activity of chitinase Chi1 for GlcNAc-pNP, (GlcNAc)2-pNP and

710

(GlcNAc)3-pNP (A) and mode of action of chitinase Chi1 on (GlcNAc)3-

711

pNP and (GlcNAc)2-pNP (B). (Figure S3).

712

-

High Performance Anion Exchange (HPAEC) elution profiles of

713

standards of chito-oligosaccharides (GlcNAc)1-3 and GlcNAc-pNP (A)

714

and reaction products released after 60 min incubation of (GlcNAc)2-

715

pNP with purifed chitinase Chi1 (B) (Figure S4).

716

ABBREVIATIONS

717

DDA, degree of deacetylation; DP, polymerization degree; GlcNAc, N-

718

acetyl-D-glucosamine; (GlcNAc)2, diacetyl-chitobiose; (GlcNAc)3, triacetyl-

719

chitotriose; (GlcNAc)4, tetraacetyl-chitotetraose; (GlcNAc)5, pentaacetyl-

720

chitopentaose;

(GlcNAc)6,

hexaacetyl-chitohexaose;

GlcNAc-pNP,

4-

31



(GlcNAc)2-pNP,

Page 32 of 52

721

nitrophenyl–N-acetylglucosamine;

722

diacetyl-β-D-chitobioside;

723

triacetylchitotriose; LPMO, lytic polysaccharide monooxygenase; GH 18,

724

glycoside hydrolase family 18; GH 20, glycoside hydrolase family 20; GH 3,

725

glycoside hydrolase family 3; Mw, molecular weight; SDS-PAGE, sodium

726

dodecyl sulfate-polyacrylamide gel electrophoresis; pI, isolectric point;

727

PAHBAH,

728

assisted laser-desorption time-of-flight mass spectrometry; HPAEC, High

729

Performance Anion-Exchange Chromatography;

730

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731

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732

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Thermophilic Fungus, Thermomyces lanuginosus in Saccharomyces

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22.

Visser, H.; Joosten, V.; Punt, P.J.; Gusakov, A.; Olson, P.T.; Joosten,

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efficient library construction and high throughput screening in the

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measurements in a sensitive, fast, reproducible assay. J. Biomol.

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Screen. 2010, 15, 213–217.

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azure

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846

Camarillo, R. Colloidal chitin stained with Remazol Brilliant Blue R®, a

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useful substrate to select chitinolytic microorganisms and to evaluate

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chitinases. J. Microbiol. Methods 2004, 56, 213–219.

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the head of bacteriophage T4. Nature 1970, 227, 680–685.

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Characterization of β-N-acetylhexosaminidase (LeHex20A), a member

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of glycoside hydrolase family 20, from Lentinula edodes (shiitake

864

mushroom). AMB Express. 2012, 2, 29. 37



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Bahk J.D.; Cho M.J. Cloning of the 52-kDa chitinase gene from Serratia

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marcescens KCTC2172 and its proteolytic cleavage into an active 35-

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kDa enzyme. FEMS Microbiol Lett. 1998, 160, 151–158.

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Streptomyces

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multiplicity. J. Bacteriol. 1992, 174, 3450–3454.

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sequencing of a gene encoding the 69-kDa extracellular chitinase of

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Janthinobacterium lividum. FEMS Microbiol Lett. 1995, 131, 279-288.

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41.

Sutrisno, A.; Ueda, M.; Abe, Y.; Nakazawa, M.; Miyatake, K. A

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chitinase with high activity toward partially N-acetylated chitosan from

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a new, moderately thermophilic, chitin-degrading bacterium, Ralstonia

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sp. A-471. Appl. Microbiol. Biotechnol. 2003, 63, 398–406.

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42.

Ohtakara,

A.;

Matsunaga,

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Action

Pattern

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Streptomyces griseus Chitinase on Partially N-Acetylated Chitosan.

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Agric. Biol. Chem. 1990, 54, 3191–3199.

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43.

Horn, S.J.; Sørbotten, A.; Synstad, B.; Sikorski, P.; Sørlie, M.; Vårum,

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K.M.; Eijsink, V.G.H. Endo/exo mechanism and processivity of family

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18 chitinases produced by Serratia marcescens. FEBS J. 2006, 273,

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491–503.

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44.

Teeri, T.T.; Koivula, A.; Linder, M.; Wohlfahrt, G.; Divne, C.; Jonest,

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T.A. Trichoderma reesei cellobiohydrolases: why

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crystalline cellulose? Biochem Soc Trans. 1998, 26, 173–178. 38


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889


45.

Hamre, A.G.; Jana, S.; Reppert, N.K.; Payne, C.M.; Sørlie, M.

890

Processivity, Substrate Positioning, and Binding: The Role of Polar

891

Residues in a Family 18 Glycoside Hydrolase. Biochemistry. 2015, 54,

892

7292–7306.

893

46.

Zees, A.C.; Pyrpassopoulos, S.; Vorgias, C.E. Insights into the role of

894

the (α+β) insertion in the TIM-barrel catalytic domain, regarding the

895

stability and the enzymatic activity of Chitinase A from Serratia

896

marcescens. BBA-Proteins Proteom. 2009, 1794, 23-32.

897

47.

Wilson, D.B. Processive and nonprocessive cellulases for biofuel

898

production-lessons from bacterial genomes and structural analysis.

899

Appl. Microbiol. Biotechnol. 2012, 93, 497–502.

900

48.

Heggset, E.B.; Hoell, I.A.; Kristoffersen, M.; Eijsink, V.G.H.; Vårum,

901

K.M. Degradation of Chitosans with Chitinase G from Streptomyces

902

coelicolor A3(2): Production of Chito-oligosaccharides and Insight into

903

Subsite Specificities. Biomacromolecules 2009, 10, 892–899.

904

49.

W.-J.;

Park,

R.-D.

Bioproduction

of

Chitooligosaccharides:

Present and Perspectives. Mar. Drugs 2014, 12, 5328–5356.

905 906

Jung,

50.

Horn, S.J.; Sørlie, M.; Vaaje-Kolstad, G.; Norberg, A.L.; Synstad, B.;

907

Vårum, K.M.; Eijsink, V.G.H. Comparative studies of chitinases A, B

908

and C from Serratia marcescens. Biocatal. Biotransformation 2006, 24,

909

39–53.

910

FIGURE CAPTIONS

911

Figure 1. Sequence analysis of chitinase Chi1 from Myceliophthora

912

thermophila C1. A. Multiple sequence alignment of active site of chitinase 39



Page 40 of 52

913

Chi1 from Myceliophthora thermophila C1 with active site of Chitinase A

914

from Serratia marcescens (1NH6_A), Chitinase B from Arthrobacter

915

TAD20 (1KFW_A), Janthinobacterium lividum (AAA83223), and chitinase

916

from Clostridium paraputrificum (BAD12045). Conserved residues are

917

colored in yellow and marked with (#) sign. Conserved D-X-E motif is

918

shown in the green box. Chitinase Chi1 shares 10 amino acids with

919

conserved hydrolases family 18 (GH 18) domain. Analysis was performed

920

with Conserved Domain Search and Conserved Domain Database. B.

921

Secondary structure of chitinase Chi1 predicted with Phyre2. C. 3D

922

modelling of chitinase Chi1 was performed with Phyre2 software with

923

chitinase from Clonostachys rosea belonging to GH 18 family (PDB entry

924

3G6MA) used as template.

925

Figure 2. Purification of chitinase Chi1 from Myceliophthora thermophila

926

C1 by ion exchange chromatography on DEAE-FF Sepharose (A) and size

927

exclusion chromatography on Superdex 75 (B). Proteins (blue line) were

928

detected at 280 nm. Chitinase activity(green line) was measured with

929

chitin azure at pH 6.0, 50 °C. Activity of N-acetylglucosaminidase (red

930

line)was assayed with GlcNAc-pNP at pH 4.0, 50 °C.

931

Figure 3. Analysis of purified chitinase Chi1 from Myceliophthora

932

thermophila C1. A. molecular weight of chitinase Chi1 determined by SDS-

933

PAGE under reducing denaturing conditions. Lanes: M, standard protein

934

molecular weight markers; 1, crude enzyme preparation of chitinase Chi1;

935

2, purified chitinase Chi1. B. Protein staining with periodic acid (PAS).

sp.

40


Page 41 of 52


936

Lanes: M, standard protein molecular weight markers; 1, purified chitinase

937

Chi1; 2, yeast invertase; 3, bovine serum albumin. C. Isoelectric focusing

938

(IEF). Lanes: M, pl marker; 1, purified chitinase Chi1. D. Molecular weight

939

of chitinase Chi1 determined by MALDI-TOF-MS.

940

Figure 4. Enzyme characteristics of chitinase Chi1: A. Enzyme activities at

941

various pH (pH 3.0–9.0) were measured at 50 °C in 0.1 M citrate-

942

phosphate-borate buffer; B. Enzyme activities at various temperatures

943

(30–80 °C) were measured at pH 6.0 in 0.1 M citrate-phosphate-borate

944

buffer; C. Thermostability was measured by incubating chitinase Chi1 at

945

various temperatures (40–60 °C), and the residual activities were assayed

946

at 50 °C. Reactions were performed with 1.6 µM purified chitinase Chi1

947

and 1% (w/v) swollen chitin in 0.1 M citrate-phosphate-borate buffer for

948

30 min. The error bars represent the range of duplicate experiments.

949

Figure 5. Specific activity of chitinase Chi1 on different types of chitosans.

950

Chitosans were used in a concentration of 0.1% (w/v) with 40.7 nM of

951

purified chitinase Chi1 in 1 mL 0.05 M sodium phosphate buffer pH 6.0.

952

The reaction mixtures were incubated at 50 °C, for 15 min. The reducing

953

sugars produced in the supernatant were measured using the p-

954

hydroxybenzoic acid hydrazide (PAHBAH) assay. The results represent the

955

average of duplicate experiments.

956

Figure 6. Release of chitin oligosaccharides during swollen chitin

957

hydrolysis by chitinase Chi1. Swollen chitin (0.45% (w/v)) was incubated

958

with purified chitinase Chi1 (100 nM) at 50 °C in 1 mL 0.05 M sodium 41



Page 42 of 52

959

phosphate buffer pH 6.0. Aliquots were taken at different time intervals

960

and the hydrolysis products were analyzed by High-Performance Anion-

961

Exchange Chromatography (HPAEC). Detected products were GlcNAc

962

(cross), (GlcNAc)2 (triangle) and (GlcNAc)3 (square). Soft lines are only

963

drawn as visual aids. The error bars represent the range of duplicate

964

experiments.

965

Figure 7. Hydrolysis of chitin oligosaccharides (GlcNAc)3 (A), (GlcNAc)4 (B),

966

(GlcNAc)5 (C), and (GlcNAc)6 (D) by chitinase Chi1. Chitin oligosaccharides

967

(2 mM) in 0.5 mL 0.05 M sodium phosphate buffer pH 6.0 were incubated

968

with purified chitinase Chi1 (25 nM). Aliquots were taken at different time

969

intervals and the hydrolysis products were analyzed by High-Performance

970

Anion-Exchange Chromatography (HPAEC). Detected products were

971

GlcNAc (star), (GlcNAc)2 (triangle), (GlcNAc)3 (square), (GlcNAc)4 (circle),

972

(GlcNAc)5 (cross), (GlcNAc)6 (diamond). Experimental work performed in

973

duplicates and the standard deviation was less than 5 %.

974 975

42


Page 43 of 52


A

B

C

Figure 1. Figure in color

43



Page 44 of 52

B

A 2500

1200

35

Fraction II

12

1500

20 15

1000

10 500 5 0

0 140

160

180

200

220

240

260

1000

10

800

8

600

6

400

4

200

2

0

chitinase activity (U mL-1)

25

chitinase activity (U mL-1)

Absorbance at 280 nm (mAU) NAG activity (U mL-1)

30 2000

Absorbance at 280 nm (mAU) NAG activity (U mL-1)

Fraction I

0 40

50

60

70

80

mL

mL


44


Page 45 of 52


A

B M

1

2

C M

1

2

D

3


45



A

B 100 Relative activity (%)

100 Relative activity (%)

Page 46 of 52

80 60 40 20

80 60 40 20 0

0 2

4

6

8

10

25

35

45

55

65

75

Temperature [°C]

pH

C 140

Relative activity (%)

120 100 80 40 C

60

50 C 40

55 C

20

60 C

0 0

20

40

60

80

100

120

140

160

180

Time (h)


46


Page 47 of 52


Specific activity [µmol min-1 mg protein-1]

12 10 8 6 4 2 0


47



Page 48 of 52

0.25

0.2

mM

0.15

0.1

0.05

0 0

100

200

300

400

time (min)


48


Page 49 of 52


A

B

C

D


49



Page 50 of 52

Table 1. Characterization of chitinase-containing fractions obtained during Organism

Molecular

pH

Temp.

Temp.

Thermostability

weight

optimum

optimum

range (°C)

(as % activity retained activity)

(kDa)

Reference

(°C)

purification of chitinase Chi1 from Myceliophthora thermophila C1.

Purification step

Crude extract

Volume

Total activity

Total protein

Specific activity (U

(mL)

(U)

(mg)

mg-1)a

50

373

142

2.6

10

126

51

2.5

2.5

23

6.6

3.5

Anion exchange chromatography Fraction I

Size exclusion chromatography Fraction II

a

Specific activity was assayed with 5 % (w/v) colloidal chitin azure at 50 °C and pH

7.0, and was calculated per mg protein.

50


Page 51 of 52


Aspergillus fumigatus YJ-407

46

5

50-60

45 to 60

100% activity after 1 h at 45 °C;

14

≈70% activity after 1 h at 55 °C; ≈20% activity after 1 h at 60 °C;

47

Chaetomium thermophilum

5.5

60

40 to 70


15

≈90% activity after 1 h at 60 °C;

51

Gliocladium catenulatum

Rhizopus oryzae

5.0-6.0

60

20 to 70

40% activity after 20 min at 50 °C;

16

5.5-6.0

60

50 to 70

100% activity after 30 min at 50 °C;

17

≈40% activity after 30 min at 60 °C;

Myceliophthora thermophila BJA

48

5.0

55

30-70

70% activity after 5h at 45°C;

25

≈50% activity after 113 min at 65; ≈50% activity after 48 min 75 °C; 48

Thermoascus aurantiacus vs.

8.0

50

40 to 60


15


levisporus 44.1

Thermomyces langinousus

5.0

50

30 to 60

50% activity at 50 °C after 630 min;

18

56% activity at 60 °C after 30 min 36.6

Thermomyces langinosus

4.0

40

30 to 60

50% activity at 40 °C after 577.5 min;

18

71% activity at 50 °C after 60 min

48

Thermomyces langinosus SY2

55

55

50 to 60


19


Thermomyces langinosus

42

60

60

30 to 70

Trichoderma viride

28

3.5

55 to 60


20


22

≈10% activity after 3 h at 60 °C; Myceliophthora thermophila C1

43

6.0

55

30-70

≈90% activity after >168 h at 50 °C;

This work

50% activity after 48 h at 55 °C 10% activity after 10 min at 60 °C

Table 2 Properties of chitinases from thermophilic fungi.

GlcNAc

Chi1

GlcN

chitinase

Thermostability 50 °C active>168 h 55 °C t ½ = 48 h

51

Chi1

ACS Paragon Plus Environment Chitin Chi1


Page 52 of 52

Figure For Table of Contents Only

52


Chitinase Chi1 from Myceliophthora thermophila C1, a Thermostable

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