Utility of Thermostable Xylanases of Mycothermus thermophilus in

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Utility of Thermostable Xylanases of Mycothermus thermophilus in Generating Prebiotic Xylooligosaccharides Rui Ma, Yingguo Bai, Huoqing Huang, Huiying Luo, Sanfeng Chen, Yunliu Fan, Lei Cai, and Bin Yao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05183 • Publication Date (Web): 09 Jan 2017 Downloaded from http://pubs.acs.org on January 10, 2017

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Journal of Agricultural and Food Chemistry 1

1

Utility of Thermostable Xylanases of Mycothermus thermophilus in Generating

2

Prebiotic Xylooligosaccharides

3 4

Rui Ma a,b,c, Yingguo Bai a, Huoqing Huang a, Huiying Luo a, Sanfeng Chen c, Yunliu

5

Fan b, Lei Cai d, Bin Yao a,*

6 7

a

8

Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081,

9

China

Key Laboratory of Feed Biotechnology of the Ministry of Agriculture, Feed

10

b

11

100081, China

12

c

13

China Agricultural University, Beijing 100094, China

14

d

15

Sciences, Beijing 100101, China

Biotechnology Institute, Chinese Academy of Agricultural Sciences, Beijing

State Key Laboratory for Agrobiotechnology, College of Biological Sciences,

State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of

16 17 18 19

* Corresponding author at: Key Laboratory of Feed Biotechnology of the

20

Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural

21

Sciences, Beijing 100081, China.

22

E-mail addresses: [email protected] (B. Yao).

23

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

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ABSTRACT

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Xylooligosaccharides as emerging prebiotics are able to promote the growth of

26

probiotic bacteria. In the present study, four neutral, thermostable xylanases

27

(MtXyn11A, MtXyn11At, MtXyn11B and MtXyn11C) from compost fungus

28

Mycothermus thermophilus CGMCC3.18119 were overexpressed in Pichia pastoris

29

GS115 and used to produce xylooligosaccharides from beechwood xylan. The

30

enzymes showed similar enzymatic properties (maximal activities at pH 6.0−6.5 and

31

65 °C) but varied in catalytic efficiency and cleaving actions. MtXyn11A,

32

MtXyn11At, and MtXyn11C mainly produced xylobiose (59−62%), xylose

33

(16−20%), and xylotriose (16−19%), while MtXyn11B released xylobiose (51%),

34

xylotriose (32%), and xylose (12%) as the main products. When using the xylan

35

hydrolysates of different xylanases as the carbon source, four probiotic Lactobacillus

36

strains L. brevis 1.2028, L. rhamnosus GG, L. casei BL23, and L. plantarum WCSF1

37

were confirmed to utilize the xylooligosaccharides efficiently (83.8−98.2%), with L.

38

brevis 1.2028 as the greatest.

39

KEYWORDS:

40

prebiotic, probiotic Lactobacillus

xylanase,

Mycothermus

thermophilus,

41


xylooligosaccharides,

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INTRODUCTION

43

Prebiotics are non-digestible oligosaccharides with low degree of polymerization

44

(2−20) that are able to selectively stimulate the growth and/or activity of beneficial

45

bacteria and improve the health of hosts.1 These oligomers play roles in the

46

prevention of diarrhea and constipation, improvement of immune system and

47

maintenance of host metabolic homeostasis.2,3 Prebiotics are various in origin and

48

chemical properties. According to the common criteria,4 prebiotics are classified into

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established ones (i.e., inulin, fructooligosaccharides, galactooligosaccharides,

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lactulose and polydextrose) and emerging ones (i.e., isomaltooligosaccharides,

51

xylooligosaccharides and lactitol). Of them, the xylooligosaccharides demonstrate

52

exceptional benefits including preferred substrates of beneficial bacteria,

53

antimicrobial and antioxidant activities and preventive effects on cancer, thus having

54

great application potential as functional foods.5

55

The occurrence of xylooligosaccharides in nature is low in amount, but can be

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produced by the hydrolysis of hemicellulose, in which xylan is the most abundant

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component. Xylan is composed of a main chain of β-1,4-linked xylopyranose

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residues and side chains of a variety of substituents attached by glycosidic or ester

59

linkages.6 To produce xylooligosaccharides from xylan-rich biomass, there are three

60

methods, i.e., chemical methods, autohydrolysis and enzymatic hydrolysis. In

61

comparison to the chemical methods that cause equipment corrosion and produce

62

excess xylose and other toxic by-products and slow-conversion and low-yield and

63

high-energy-dense autohydrolysis, enzymatic hydrolysis is much desirable for its



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specificity, high efficiency, and non-production of undesirable byproducts.7 In the

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recent decade, production of xylooligosaccharides from lignocellulosic materials has

66

been focused on those endo-xylanases (EC 3.2.1.8) that cleave heteroglygan into

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short xylooligosaccharides.8 Most endo-xylanases are classified into glycoside

68

hydrolase (GH; http://www.cazy.org) families 10 and 11, and minorities belong to

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families 5, 7, 8, 30 and 43. GH11 endo-xylanases are specific for xylan substrates

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and release short oligomers of 2−4 as the main products,9,10 thus representing an

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excellent candidate for the production of xylooligosaccharides.

72

Various microorganisms including bacteria, yeasts and fungi are found to

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naturally secret xylanases. However, thermotolerant or thermophilic fungi are of

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more industrial and biotechnological interest because they can produce thermostable

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xylanases with high yield, great thermo-adaptability and -stability, higher mass

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transfer rate, lower substrate viscosity, and less contamination risk.11,12 GH11

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xylananses from filamentous fungi and yeast13,14 have shown potentials for the

78

production of xylooligosaccharides. These enzymes varied in the catalytic efficiency

79

towards various substrates and released xylobiose to xylohexaose as the main

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hydrolysis products and trace amounts of xylose.

81

In the present study, four xylanases (MtXyn11A, MtXyn11At, MtXyn11B and

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MtXyn11C) of GH11 with and without a carbohydrate-binding domain (CBM) were

83

identified in the thermophilic compost fungus Mycothermus thermophilus

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CGMCC3.18119 (also known as Scytalidium thermophilum).15 The enzymes were

85

heterologously produced in Pichia pastoris, and their capacities of utilizing the


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model soluble xylan, originating from hardwood (beech), to obtain a range of

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xylooligosaccharides were assessed. Moreover, the utilization of beechwood xylan

88

hydrolysates by four probiotic Lactobacillus strains was also determined.

89 90

MATERIALS AND METHODS

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Strains, Vectors and Materials. M. thermophilus CGMCC3.18119 from the

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China General Microbiological Culture Collection Center (Beijing, China) was the

93

donor strain. To induce the production of xylanase, strain CGMCC3.18119 was

94

cultured at 45 °C for 3 days in the medium containing 5.0 g/L (NH4)2SO4, 1.0 g/L

95

KH2PO4, 0.5 g/L MgSO4⋅7H2O, 0.2 g/L CaCl2, 10.0 mg/L FeSO4⋅7H2O, 30.0 g/L

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wheat bran, 30.0 g/L soybean meal, and 30 g/L corncob. For gene cloning and

97

expression, the Escherichia coli Trans1-T1 competent cells and pEASY-T3 vector

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from TransGen (Beijing, China), and P. pastoris GS115 competent cells and pPIC9

99

vector from Invitrogen (Carlsbad, CA) were purchased. The SV Total RNA Isolation

100

System (Promega, Madison, WI) and ReverTra Ace-α-TM kit (TOYOBO, Osaka,,

101

Japan) were used to extract RNA and synthesize cDNA. The media for heterologous

102

gene expression in P. pastoris were prepared according to the manual of the Pichia

103

Expression kit (Invitrogen). The LA Taq DNA polymerase and DNA purification kit

104

were purchased from TaKaRa (Tsu, Japan). Restriction endonucleases (EcoRI, NotI

105

and BglII), T4 DNA ligase and endo-β-N-acetylglucosaminidase H (Endo H) were

106

purchased from New England Biolabs (Ipswich, MA). The substrate xylan from

107

beechwood X4252 and birchwood X0502 (Sigma-Aldrich, St. Louis, MO)



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with >90% xylose and soluble arabinoxylan P-WAXYL with 95% purity

109

(arabinose:xylose = 38:62) and insoluble arabinoxylan P-WAXYI with 80% purity

110

(arabinose:xylose = 36:51) from wheat (Megazymes, Wicklow, Ireland) were used

111

for substrate specificity activity assay and xylan hydrolysis experiment.

112

Xylooligosaccharides xylose to xylohexaose (Megazymes) were used as the

113

standards. All other chemicals were of analytical grade and commercially available.

114

Sequence Analysis and Gene Cloning. The genome sequence of M.

115

thermophilus

116

http://www.fungalgenomics.ca/wiki/Fungal_Genomes. Three xylanase genes of

117

GH11

118

KX867530−KX867532) were identified. Their nucleotide and deduced protein

119

sequences were analyzed by using the BLASTx and BLASTp programs

120

(http://www.ncbi.nlm.nih.gov/BLAST/), Vector NTI 10.0 (Invitrogen), SignalP 4.1

121

(http://www.cbs.dtu.dk/services/SignalP/),

122

(http://www.clustal.org/). The potential N-glycosylation sites were predicted by

123

NetNGlyc serve (http://www.cbs.dtu.dk/services/ NetNGlyc/).

(Mtxyn11A,

is

available

Mtxyn11B

and

on

Mtxyn11C;

the

GenBank

and

website

accession

ClustalW

nos:

1.6

124

Three-day-old mycelia were collected and immediately ground to a fine powder

125

in liquid nitrogen. Total RNA was extracted, cDNA was synthesized, and the cDNA

126

fragments coding for mature proteins were amplified with specific primer sets (Table

127

S1) containing restriction sites with an annealing temperature of 60 °C. A variant of

128

Mtxyn11A without the CBM1-coding sequence (Mtxyn11At) was also amplified. The

129

specific PCR products were ligated into the pEasy-T3 vector for sequencing.


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Expression and Purification of Recombinant Proteins. The correct PCR

131

products were digested with EcoRI and NotI and ligated into the corresponding sites

132

of pPIC9 vector. The recombinant plasmids pPIC9-Mtxyn11A, pPIC9-Mtxyn11At,

133

pPIC9-Mtxyn11B and pPIC9-Mtxyn11C were linearized using BglII and transformed

134

into P. pastoris GS115 competent cells by electroporation according to the

135

manufacturer’s instructions (Invitrogen). Gene transformation and expression was

136

conducted as described previously.16 The positive transformants with highest

137

xylanase activity were selected for further fermentation in 1 L conical flasks.

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The induced cultures were collected by centrifugation at 12,000 × g for 10 min at

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4 °C and concentration through a Vivaflow 200 membrane of 10-kDa molecular

140

weight cut-off (Vivascience, Göttingen, Germany). The crude enzymes were loaded

141

onto the HiTrapTM Desalting column and HiTrapTM Q Sepharose XL 5 mL FPLC

142

column (GE Healthcare, Uppsala, Sweden) that were both equilibrated with 20 mM

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Tris-HCl (pH 8.0). Proteins were eluted using a linear gradient of NaCl (0–1.0 M) at

144

a flow rate of 3.0 mL/min. The fractions with xylanase activity were combined, and

145

checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

146

The protein concentration was measured by a Protein Assay Kit (Bio-Rad, Hercules,

147

CA). To remove N-glycosylation, the recombinant proteins were deglycosylated by

148

Endo H at 37 °C for 2 h. The deglycosylated enzyme was also checked by

149

SDS-PAGE. Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight

150

(TOF)-Mass Spectrometry (MS) was used to identify each recombinant protein at the

151

Institute of Apiculture Research, Chinese Academy of Agricultural Sciences (Beijing,



Page 8 of 34 8

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China).

153

Biochemical Characterization. Xylanase activity was assayed by using the

154

3,5-dinitrosalicylic acid (DNS) method.17 Standard assay mixture was composed of

155

900 µL of McIlvaine buffer (200 mM Na2HPO4, 100 mM citric acid, pH 6.5)

156

containing 1% (w/v) beechwood xylan and 100 µL of appropriately diluted enzyme

157

(approximately 10−20 µg). The amount of enzyme releasing 1 µmol of reducing

158

sugar per min at given assay conditions (pH 6.0 or 6.5 and 65 °C for 10 min) was

159

defined as one unit (U) of xylanase activity. All reactions were performed in

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triplicate and corrected for background hydrolysis using a reference of identical

161

composition to the reaction mixture with thermo-inactivated enzyme.

162

The pH properties of MtXyn11A, MtXyn11At, MtXyn11B and MtXyn11C were

163

determined in the following solutions, McIlvaine buffer for pH 3.0–8.0, 100 mM

164

Tris-HCl for pH 8.0–9.0, and 100 mM glycine-NaOH for pH 9.0–12.0. The

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pH-activity profiles were examined at 50 °C over the pH range of 3.0–9.0 for 10 min.

166

The pH stability of each enzyme was determined by measuring the residual enzyme

167

activity under optimal conditions (pH 6.0 or 6.5 and 65 °C for 10 min) after

168

pre-incubation of the enzymes in buffers of pH 3.0–12.0 at 37 °C for 1 h without the

169

substrate. The temperature optima were examined at each optimal pH by measuring

170

the enzyme activities over the temperature range of 30 to 80 °C. The thermostability

171

assay was carried out by incubating the enzymes at optimal pH and at 60 °C, 70 °C

172

or 80 °C without substrate for 5, 10, 20, 30, and 60 min, and then measuring the

173

residual enzyme activities under the optimal assay conditions.


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To investigate the effects of different metal ions and chemical reagents on the

175

enzymatic activities, the reaction systems containing 5 mM of each Na+, K+, Ag+,

176

Cu2+, Mn2+, Ca2+, Pb2+, Co2+, Zn2+, Mg2+, Fe3+, Cr3+, SDS, EDTA, and

177

β-mercaptoethanol was subject to enzyme activity assay under the standard conditions

178

and compared to the blank control without any additives.

179

The enzyme substrate specificities were tested by measuring the enzyme specific

180

activity against 1% (w/v) of polysaccharides (birchwood xylan, beechwood xylan,

181

soluble/insoluble wheat arabinoxylan) in McIlvaine buffer (pH 6.0 or 6.5),

182

respectively.

183

The kinetic parameters, Km and Vmax, of each enzyme were determined after 5-min

184

incubation at 65 °C and pH 6.0 or 6.5 by using 1–10 mg/mL beechwood xylan as the

185

substrate. The GraphPad Prism version 5.01 (La Jolla, CA) was used for data analysis

186

with the Michaelis-Menten model. Each experiment was repeated three times.

187

Hydrolysis of Beechwood Xylan. Hydrolysis reactions were done at 65 °C

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under stirring in a 1 mL system containing 10 U of each enzyme and 10 mg

189

beechwood xylan dissolved in McIlvaine buffer, pH 6.5. Samples were collected at

190

12 h, followed by centrifugation (12,000 × g, 4 °C, 10 min) through a 3-kDa Amicon

191

Ultra centrifugal filter (Millipore) to remove excess unreacted enzymes. The

192

xylooligosaccharide

193

anion-exchange chromatography (HPAEC, Dionex, Sunnyvale, CA; model 2500)

194

using a 250 mm × 3 mm CarboPac PA200 guard column and a mobile phase (0.5

195

mL/min) of constant 1 M NaOH. Xylooligosaccharide standards (1−16 µg/mL) were

hydrolysates

were

analyzed

with


high

performance


Page 10 of 34 10

196

used to identify the peaks in the chromatograms. Samples were appropriately diluted

197

(50 to 200 times) in Milli-Q water before analysis and compared with the standards.

198

Fermentation Experiment with Xylooligosaccharides as the Carbon Source.

199

The fermentation experiment was conducted as Falck et al.18 described with some

200

modifications. Four Lactobacillus strains L. brevis 1.2028, L. rhamnosus GG, L.

201

casei BL23, and L. plantarum WCSF1 were used to test the utilization of the

202

beechwood xylan hydrolysates. The bacteria were pre-cultivated aerobically in MRS

203

broth (pH 6.7) containing 10 mg/mL glucose as the carbon source at 37 °C overnight.

204

Cells were harvested by centrifugation at 3000 g and 4 °C for 15 min. After washing

205

twice with 0.9% sterile saline solution, 2% (v/v) inoculum of each bacterium was

206

grown at 37 °C with the agitation rate of 150 rpm in MRS broth (pH 6.7) containing

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glucose, beechwood xylan, or hydrolyzed beechwood xylan by MtXyn11A,

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MtXyn11At, MtXyn11B or MtXyn11C as the carbon source at a concentration of 5

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mg/mL. Fermentations were conducted in glass test tubes with a working volume of

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10 mL. Each fermentation experiment had triplicate tubes, and those without any

211

addition of Lactobacilli were treated as controls. Samples of the culture supernatants

212

were collected after 48 h incubation for the analysis of pH, cell growth and

213

utilization of carbon source. pH was measured by using the portable pH meter

214

(206-pH2; Testo, Sparta, NJ). The cell density of each 200 µL culture sample,

215

dispensed into the wells of a 96-well microplate, was measured at 620 nm using a

216

microplate reader (BioTek, Winnoski, VT). Significant difference was defined when

217

the growth yields of each strain on different substrates had a difference of more than


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20%. The xylooligosaccharide-utilizing ability of each Lactobacillus strain was

219

determined by measuring the amounts of residual oligosaccharide (RO) with

220

HPAEC-PAD. Glucose and xylooligosacchardies with the polymerization degree of

221

1 to 6 were used as standards.

222

RESULTS AND DISCUSSION

223

Sequence Analysis. Thermophilic M. thermophilus is a well-known mushroom

224

compost fungus that produces lignocellulose-degrading enzymes.19,20 Genome

225

sequence

226

xylanase-encoding genes of GH11 (Table 1), which share 76−100% sequence

227

identities to known proteins but low identities (36−48%) to each other. The mature

228

proteins are predicted to be extracellular and have an estimated molecular mass of

229

23.3−29.2 kDa and an alkaline pI value of 8.04−8.71. Deduced MtXyn11A,

230

MtXyn11B and MtXyn11C have putative N-glycosylation sites of two (N74 and

231

N239), one (N10), and two (N28 and N77), respectively, suggesting the possible

232

occurrence of N-glycosylation during heterologous expression in P. pastoris.21

233

Multi-modular MtXyn11A comprises of a catalytic domain, a Gly/Asn-rich linker,

234

and a carbohydrate-binding module of family 1 (CBM1), and other xylanases are

235

single-modular. These xylananse isozymes share a so-called β-sandwich structure

236

containing one α-helix and two β-sheets,22 with two catalytic residues of glutamate

237

(E99 and E190 for MtXyn11A and MtXyn11B and E96 and E188 for MtXyn11C).

analysis

of

M.

thermophilus

reveals

the

presence

of

three

238

Preparation of Recombinant Xylanases. CBM refers to the non-catalytic

239

polysaccharide-recognizing module of GHs with diverse ligand specificity.23 To date,



Page 12 of 34 12

240

79329 putative CBM sequences in 80 different species have been identified in the

241

CAZy database (http://www.cazy.org/Carbohydrate-Binding-Modules.html; Aug 31,

242

2016). CBM1 is found almost exclusively in fungi and contains approximately 40

243

residues (serine/threonine/asparagines/glycine-rich). Although its presence in GH11

244

xylanases has been demonstrated in many cases, 24−26 its functional role rather than

245

binding to insoluble and crystalline polysaccharides is unidentified yet. To verify the

246

role of CBM1, the cDNA fragments coding for the three full-length enzymes and

247

truncated MtXyn11A without the CBM1 (i.e. MtXyn11At) were successfully

248

expressed in P. pastoris. With methanol induction, the endo-xylanase activities of

249

MtXyn11A, MtXyn11At, MtXyn11B and MtXyn11C were as high as 377.2, 263.8,

250

136.3 and 203.8 U/mL in the culture supernatants, respectively. The results indicated

251

that the four genes encoded functional xylanases and successfully expressed in P.

252

pastoris. The xylanase yields are higher than or comparable to their close,

253

biochemically characterized homologs (32.2−154.5 U/mL).25,26 After purification by

254

a one-step anion-exchange chromatography and treatment with/without Endo H to

255

remove N-glycosylation, the purified MtXyn11A, MtXyn11At and MtXyn11C had

256

predicted molecular masses, while MtXyn11B still retained a higher band (see Fig.

257

1). MALDI-TOF-MS analysis identified a few specific sequences of MtXyn11B,

258

APFDFVPRDN,

259

NPLVEYYVIESYGTYNPGSQAQYKGTFYTDGDQYDIFVSTRYNQPSIDGTRTF

260

QQYWSIRK. The results altogether indicated that the purified enzymes are

261

recombinant MtXyn11A, MtXyn11At, MtXyn11B and MtXyn11C, and other

NTGNFVGGKGWNPGTGR,


and

Page 13 of 34


262

post-translation modification than N-glycosylation may occur on MtXyn11B.21

263

Biochemical Characterization of Recombinant Enzymes. Characterized

264

enzymes (trehalase, glucosidase and arabinofuranosidase) from M. thermophilus

265

demonstrated neutral and high-temperature-active properties.27−29 The four GH11

266

xylanases from M. thermophilus CGMCC3.18119 had similar characteristics. The

267

enzymes were active over the pH range from 3.0 to 10.0, with the pH optima at

268

5.5–6.5 (Fig. 2A). MtXyn11A and MtXyn11At with and without the CBM1 had

269

similar pH-activity profiles, retaining >60% maximal activity at pH 5.0–7.0. In

270

contrast, MtXyn11B and MtXyn11C showed greater pH adaptability to alkaline

271

conditions, exhibiting 76% and 77% activities at pH 8.0 and 73% and 49% activities

272

at pH 9.0, respectively. The four enzymes had similar temperature-activity profiles

273

(Fig. 2B) over the temperature range of 30 °C to 80 °C, showing the maximum

274

activities at 65 °C. For pH stability assay, all the enzymes remained highly active

275

(Fig. 2C) after incubation at pH 5.0–10.0, 37 °C for 1 h without substrate. After

276

incubation at 60 °C, pH 6.0 or 6.5 (optimal pH) for 1 h without substrate, MtXyn11A

277

and MtXyn11B remained >65% initial activities, while MtXyn11At with truncation

278

of the CBM1 and MtXyn11C almost completely lost the activities (Fig. 2D). These

279

enzymes were thermolabile at 70 °C and above, losing >90% activities within 5 min

280

(data not shown). In comparison to the four acidic and mesophilic GH11 xylanases

281

from the xylanolytic fungus Talaromyces versatilis30 and other biochemically

282

characterized homologs from thermphilic fungi22,31, the GH11 xylanases of M.

283

thermophilus

have

broad

pH

adaptability

and


stability

ranges

and


Page 14 of 34 14

284

high-temperature-active properties, but weaker thermostability at temperatures of >

285

70 °C than the counterparts from Chaetomium thermophilum and Nonomuraea

286

flexuosa. These characters facilitate the xylanases to degrade xylan substrates at

287

higher mass transfer rate and lower substrate viscosity, thus showing potentials for

288

application in a variety of industrial fields, especially the efficient production of

289

xylooligosaccharides.

290

The four M. thermophilus GH11 xylanases exhibited strong resistance to all tested

291

metal ions and chemical reagents except for SDS, retaining >78% activity (Table S2).

292

SDS as a strong enzymatic inhibitor reduced the xylanase activities by 5.7−54.1%. On

293

the other hand, the enzymatic activities were also enhanced by up to 145.7% in the

294

presence of Ca2+, Ni2+, Ag+ or β-mercaptoethanol.

295

The preferences of MtXyn11A, MtXyn11At, MtXyn11B and MtXyn11C for

296

different xylan substrates are shown in Table 2. By defining the enzymatic activity

297

towards beechwood xylan as 100%, the MtXyn11A and MtXyn11At exhibited the

298

highest activities towards birchwood xylan (132.5% and 122.1%), while MtXyn11B

299

and MtXyn11C were most active on soluble wheat arabinoxylan (110.8%) and

300

beechwood xylan, respectively. The presence of CBM1 contributed to the improved

301

activity on insoluble xylan substrate from wheat (23.2%), which was higher than that

302

of MtXyn11At, MtXyn11B and MtXyn11C (9.4%, 15.9% and 14.1%) without the

303

CBM. The low sequence similarities and big variance in the amino acid

304

compositions in the active pocket and substrate binding sites may account for the

305

different substrate preference.


Page 15 of 34


306

The kinetic values of these enzymes against beechwood xylan are shown in Table

307

2. Of the four enzymes, MtXyn11A showed the greatest maximum velocity (Vmax),

308

turnover rate (kcat), and catalytic efficiency (kcat/Km), while MtXyn11B exhibited the

309

highest substrate affinity (Km). Comparison of the enzymatic properties of MtXyn11A

310

and MtXyn11At revealed that the CBM1 in GH11 xylanase not only involves in

311

binding to insoluble xylan substrate, but also plays a role in enzymatic catalysis and

312

stability and resistance to metal ions and chemical reagents.

313

Analysis of Hydrolysis Products. The action modes of GH11 xylanases are

314

various. Those from Achaetomium sp. Xz-8 have xylobiose as the main products,25

315

while that from H. insolens mainly produces xylotriose.26 The four M. thermophilus

316

xylanases showed different cleavage modes on beechwood xylan (Table 3). After

317

incubation at 65 °C for 12 h, the total contents of xylose to xylotriose were

318

9.50−9.66 µg/mL in hydrolyzed beechwood xylan, indicating the almost complete

319

hydrolysis of beechwood xylan (10 µg/mL). However, the compositions of xylose,

320

xylobiose, and xylotriose were different. MtXyn11A, MtXyn11At, and MtXyn11C

321

mainly produced xylobiose (59−62%), xylose (16−20%), and xylotriose (16−19%),

322

while MtXyn11B released xylobiose (51%), xylotriose (32%), and xylose (12%) as

323

the main products.

324

Growth of Probiotic Bacteria on the Xylooligosaccharide Hydrolysates. The

325

present batch fermentation experiments showed that the four probiotic bacteria L.

326

brevis 1.2028, L. rhamnosus GG, L. casei BL23, and L. plantarum WCSF1 grew

327

well using xylooligosaccharide hydrolysates from beechwood xylan as the carbon



Page 16 of 34 16

328

source (Table 4). All strains showed an increase in bacterial population and a

329

decrease in pH. Under the same conditions, the strains achieved high growth on

330

glucose but no growth on nontreated polymeric xylan, which might be explained by

331

the fact that glucose is more readily utilized whereas beechwood xylan is not.

332

Carbohydrate utilization measured by the HPAEC-PAD after 48 h of fermentation

333

confirmed the results of bacterial growth, pH changes, and xylooligosaccharide

334

utilization. Moreover, the HPAEC-PAD chromatograms indicated that there are

335

differences among the fermentation patterns of Lactobacillus strains. For L. brevis

336

xylooligosaccharides were fermented at the highest level (up to 98.2%), whereas

337

other tested Lactobacillus strains showed relatively weak capacity of utilizing

338

xylooligosaccharides (84.0−94.8%), especially xylobiose and xylotriose (see Table

339

S3). The composition of xylooligosaccharides also played a role in the fermentation

340

of Lactobacillus strains, i.e. the xylan hydrolysates from MtXyn11A hydrolysis

341

showed great capacity to support the growth of probiotic Lactobacilli, while those of

342

MtXyn11B were more readily utilized by all tested strains (Table 4).

343

A few studies have been conducted to test the carbohydrate utilization of

344

probiotic Lactobacillus strains, including mono- and disaccharides, amino acids,

345

carboxylic acids, fatty acids, nucleosides, and dietary fibre carbohydrates.18,32,33

346

Dietary fibre carbohydrates such as glucan, xylan, mannan are unfavorable carbon

347

sources. Comparative genomics analysis indicates the extensive loss and acquisition

348

of genes for efficient carbon and nitrogen utilization during the coevolution of

349

Lactobacillus strains with their niches.34 And phenotype profiling demonstrates the


Page 17 of 34


350

correlation of Lactobacillus genotypes and carbohydrate utilization signatures.35

351

Although there is no model for the uptake of xylooligosaccharides in Lactobacillus,

352

genes coding for the xylosidase of GH3 and GH43 and arabinofuranosidase of GH51

353

instead of GH10 and GH11 xylanase are present in the Lactobacillus genomes.36

354

Moreover, the characterized xylosidases of GH43 are distinguished for the high

355

catalytic efficiency against xylobiose, xylotriose and xylotetraose.37 These studies

356

may explain why the tested Lactobacillus strains have no capacity of utilizing xylan

357

polymer but demonstrate high efficiency of xylooligosaccharide utilization.

358

Therefore, the comprehensive study of the enzymatic properties and action modes of

359

xylanase, composition of xylan hydrolysates, and xylooligosaccharide fermentability

360

by different strains will ensure a cost-effective and value-added process for the

361

production and application of prebiotic xylooligosaccharides.

362

In summary, four GH11 xylanase isozymes were identified in M. thermophilus

363

and successfully expressed in P. pastoris. These enzymes showed similar neutral,

364

thermostable properties and great hydrolysis capacities of xylan substrates. The

365

beechwood xylan hydrolysates of different enzymes varied in xylooligosaccharide

366

compositions, which further affected their utilization and Lactobacilli growth. This

367

study

368

xylooligosaccharides and probiotic Lactobacillus strains.

reveals

the

importance

of

synbiotic

combination

369 370

ASSOCIATED CONTENTS

371

Supporting Information


of

prebiotic


Page 18 of 34 18

372

Primers used in this study (Table S1); effects of metal ions and chemical reagents on

373

the enzymatic activities of the four GH11 xylanases of M. thermophilus

374

CGMCC3.18119 (Table S2); quantity of xylooligosaccharides in the MRS media

375

containing different carbon sources at the beginning (time 0) and after 48 h growth

376

of the four Lactobacillus strains (Table S3).

377 378

AUTHOR INFORMATION

379

Corresponding Author

380

*E-mail: [email protected] (B. Yao). Phone: +86 10 82106065

381

Notes

382

The authors declare no conflict of interest.

383 384

ACKNOWLEDGEMENTS

385

We thank Dr. Zhigang Zhou of the Feed Research Institute, Chinese Academy of

386

Agricultural Sciences for providing the Lactobacillus strains. This study was

387

supported by the National High-Tech Research and Development Program of China

388

(863 Program, 2013AA102803), the Special Fund for Agro-Scientific Research in

389

the Public Interest of China (201403047), the National Science Foundation for

390

Distinguished Young Scholars of China (31225026) and the China Modern

391

Agriculture Research System (CARS-42).

392 393

ABBREVIATIONS USED


Page 19 of 34


394

CBM, carbohydrate-binding module; DNS, 3,5-dinitrosalicylic acid; endo H,

395

endo-β-N-acetylglucosaminidase H; GH, glycoside hydrolase; HPAEC, high

396

performance anion-exchange chromatography; MALDI-TOF-MS, matrix-assisted

397

laser

398

oligosaccharide; SDS-PAGE, dodecyl sulfate-polyacrylamide gel electrophoresis.

desorption/ionization-time-of-flight-mass

spectrometry;

RO,

residual

399 400

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401

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Page 25 of 34


524

FIGURE LEGENDS

525 526

Figure 1. SDS-PAGE analysis of the purified recombinant xylanases from M.

527

thermophilus CGMCC3.18119 with or without N-deglycosylation with Endo-H.

528

Lanes M, the molecular markers; 1 and 2, the purified MtXyn11A and MtXyn11At; 3

529

and 4, the deglycosylated, purified MtXyn11B and MtXyn11C.

530 531

Figure 2. Enzymatic properties of the four xylanases from M. thermophilus

532

CGMCC3.18119. (A) pH-activity profiles. (B) Temperature-activity profiles. (C)

533

pH-stability profiles at 37 °C for 60 min. (D) Thermostability profiles at 60 °C for 60

534

min.



Page 26 of 34

26

Table 1. Gene Information of the GH11 Xylanases of M. thermophilus CGMCC3.18119 DNA length

cDNA length

Genes

Signal peptide

Mature protein

Introns (bp)

(bp)

Mtxyn11A

1003

885

Mtxyn11B

815

Mtxyn11C

752

MW

Protein sequence identity

(kDa)

(%)

pI (aa)

(aa)

2

1−19

275

8.71

29.2

95

684

2

1−19

208

8.04

23.9

100

684

1

1−19

208

8.53

23.3

76


Page 27 of 34


Table 2. Substrate Specificity and Kinetic Values of the Four GH11 Xylanases of M. thermophilus CGMCC3.18119a Parameters

MtXyn11A

MtXyn11At

MtXyn11B

MtXyn11C

Beechwood xylan (U/mg)

2332 ± 761

1764 ± 802

1686 ± 162

2129 ± 661

Birchwood xylan (U/mg)

3089 ± 931

2154 ± 212

1720 ± 143

1284 ± 154

2563 ± 461

1826 ± 502

1868 ± 122

2019 ± 221

541 ± 141

166 ± 92

268 ± 23

300 ± 84

Vmax (U/mg)

2270 ± 1541 1255 ± 262

1557 ± 113

1618 ± 134

Km (mg/mL)

2.01 ± 0.521 3.23 ± 0.112

0.87 ± 0.063

1.53 ± 0.094

kcat (/s)

1105

538

222

240

kcat/Km (mL/s⋅mg)

549.8

166.6

255.2

156.9

Substrate specificity

Soluble

wheat

arabinoxylan

(U/mg) Insoluble wheat arabinoxylan (U/mg) Kinetics

a

Substrate specificity was determined under the given assay conditions (pH 6.0 or 6.5

and 65 °C for 10 min) by using 10 mg/mL beechwood xylan as the substrate, while kinetic parameters were determined at pH 6.0 or 6.5 and 65 °C for 5 min by using 1–10 mg/mL beechwood xylan as the substrate. Data are shown as mean ± SD (n = 3). Different superscripts of the same row indicate significant differences at P < 0.05.



Page 28 of 34 28

Table 3. Xylooligosaccharides Present in the Reaction Mixtures of Beechwood Xylan (10 mg/mL) Treated with Different GH11 Xylanases of M. thermophilus CGMCC3.18119a Sugars (mg/mL)

MtXyn11A

MtXyn11At MtXyn11B

MtXyn11C

Xylose

1.98 ± 0.101 1.63 ± 0.042 1.20 ± 0.063 1.92 ± 0.071

Xylobiose

6.05 ± 0.241 6.15 ± 0.191 5.10 ± 0.112 5.89 ± 0.101

Xylotriose

1.59 ± 0.071 1.86 ± 0.102 3.20 ± 0.053 1.84 ± 0.082

Other xylooligosaccharides 0.38 ± 0.021 0.37 ± 0.011 0.50 ± 0.042 0.34 ± 0.001 a

Data are show as means ± SD (n = 3). Different superscripts of the same row

indicate significant differences at P < 0.05.


Page 29 of 34


29

Table 4. Relative Growth of the Four Lactobacillus Strains and the pH and Oligosaccharides (w/v) Present in the MRS Media Containing Different Carbon Sources after 48 h Incubation L. brevis 1.2028

L. casei BL23

L. plantarum WCSF1

L. rhamnosus GG

Carbon source a Growth b

pH

RO (mg/mL) c

Growth

pH

RO (mg/mL)

Growth

pH

RO (mg/mL)

Growth

pH

RO (mg/mL)

1.62 ± 0.051

5.50

01

1.61 ± 0.061

4.81

0.05 ± 0.011

1.85 ± 0.011

4.91

01

2.00 ± 0.011

4.87

0.11 ± 0.031

−

6.36

−

−

6.67

−

−

6.18

−

−

6.49

−

BX-MtXyn11A

1.48 ± 0.061

5.63

0.12 ± 0.022

1.48 ± 0.031

5.64

0.80 ± 0.062

1.80 ± 0.051

5.75

0.26 ± 0.032

1.93 ± 0.021

5.67

0.65 ± 0.052

BX-MtXyn11At

1.40 ± 0.011

5.68

0.09 ± 0.012

1.35 ± 0.101

5.52

0.80 ± 0.112

1.40 ± 0.022

5.66

0.79 ± 0.083

1.58 ± 0.032

5.65

0.62 ± 0.032

BX-MtXyn11B

1.38 ± 0.011

5.62

0.09 ± 0.002

1.32 ± 0.051

5.60

0.58 ± 0.043

1.32 ± 0.012

5.69

0.34 ± 0.042

1.46 ± 0.082

5.69

0.59 ± 0.022

BX-MtXyn11C

1.45 ± 0.061

5.68

0.12 ± 0.012

1.21 ± 0.002

5.66

0.77 ± 0.092

1.68 ± 0.041

5.70

0.66 ± 0.073

1.51 ± 0.032

5.71

0.72 ± 0.062

Glucose BX

a

The carbon sources are glucose, beechwood xylan (BX), and beechwood xylan hydrolysates treated by MtXyn11A (BX-MtXyn11A),

MtXyn11At (BX-MtXyn11At), MtXyn11B (BX-MtXyn11B) and MtXyn11C (BX-MtXyn11C) at the starting concentration of 5 mg/mL. b

Growth is defined by the cell density measured at 620 nm. Data are show as means ± SD (n = 3). “−” means no growth, and different

superscripts mean significant difference of more then 20%. c

RO, the amounts of residual oligosaccharides (glucose or xylooligosaccharides) identified by HPAEC-PAD. Data are show as means ± SD (n =



Page 30 of 34

30

3), and different superscripts mean significant difference at P < 0.05.


Page 31 of 34


kDa 135 100 75 65 55 45

M

4

3

2

1

35 25

Endo-H

15 10 Figure 1.



Page 32 of 34 32

B 1600

MtXyn11A MtXyn11A MtXyn11At MtXyn11At MtXyn11B MtXyn11B MtXyn11C MtXyn11C

Specific activity (U/mg)

1400 1200 1000 800 600 400

2400 2000

Specific activity (U/mg)

A

1600 1200 800 400

200

0

0 3

4

5

6

7

8

9

30

10

40

50

C

70

80

D 120

120

100

100 Relative activity (%)

Relative activity (%)

60

Temperature (°C)

pH

80 60 40 20

80 60 40 20

0

0 3

4

5

6

7

8

9

10

11

12

0

10

pH

20

30 Time (min)

Figure 2


40

50

60

Page 33 of 34


For TOC only MtXyn11A, MtXyn11At, MtXyn11B & MtXyn11C from M. thermophilus CGMCC3.18119 Enzyme characterization MtXyn11A MtXyn11A MtXyn11At MtXyn11At MtXyn11B MtXyn11B MtXyn11C MtXyn11C

2000 2000 1600 1600 1200 1200

800

800

400

400

0

44

55

66

77

88

99

10 10

pH pH

0 30 30

40 40

D

120

120

50 60 70 50 60 70 Temperature (°C) Temperature (°C)

60

60

40

40

20

20

0 3

3

44

55

66

7

7

88 pH pH

99

10 10

12 11 12

11

100

100

80

80

60

60

40

40

20

20

0

0

Xylobiose Xylobiose

Xylotriose Xylotriose

Others Others

MtXyn11A MtXyn11A MtXyn11At MtXyn11At MtXyn11B MtXyn11B MtXyn11C MtXyn11C

Xylooligosaccharide utilization Residual sugars (mg mL−1)

Relativeactivity activity (%) (%) Relative

80

80

Xylose Xylose

1.0

120

100

7 7.0 6 6.0 5 5.0 4 4.0 3 3.0 2 2.0 1 1.0 0 0.0

80 80

120

100

0

2400 2400

Amounts (mg mL−1)

1600 1400 1400 1200 1200 1000 1000 800 800 600 600 400 400 200 200 00 3 3

C Relative activity (%) Relative activity (%)

Hydrolysis products analysis

B 1600

−1 Specific activity Specific activity (U(U /mg) mg )

−1 Specific activity (U/m g) mg ) Specific activity (U

A

00

10 10

20 20

30 40 30 40 Time (min) Time (min)

50 50

6060

1.0

MtXyn11A MtXyn11A

MtXyn11At MtXyn11At

MtXyn11B MtXyn11B

MtXyn11C MtXyn11C

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0.0

0.0


brevis L. L.brevis

L. casei L. plantarum L. plantarum L.L. rhamnosus L. casei rhamnosus


254x190mm (96 x 96 DPI)


Page 34 of 34

Utility of Thermostable Xylanases of Mycothermus thermophilus in

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