Extracellular Enzyme Composition and Functional Characteristics of

Jan 15, 2018 - ... Characteristics of Aspergillus niger An-76 Induced by Food Processing Byproducts and ... In this study, we utilized integrated func...
0 downloads 0 Views 2MB Size
Subscriber access provided by READING UNIV

Article

Extracellular Enzyme Composition and Functional Characteristics of Aspergillus niger An-76 Induced by Food Processing Byproducts and Based on Integrated Functional-omics Lin Liu, Weili Gong, Xiaomeng Sun, Guanjun Chen, and Lushan Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05164 • Publication Date (Web): 15 Jan 2018 Downloaded from http://pubs.acs.org on January 15, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

Journal of Agricultural and Food Chemistry

1

Extracellular Enzyme Composition and Functional Characteristics of Aspergillus niger

2

An-76 Induced by Food Processing Byproducts and Based on Integrated Functional-omics

3

Lin Liu1,2, Weili Gong1, Xiaomeng Sun1, Guanjun Chen1,2, Lushan Wang1,*

4

1

State Key Laboratory of Microbial Technology, Shandong University, Jinan, China

5

2

College of Marine Science, Shandong University, Weihai, China

6

*Corresponding Author: Professor Lushan Wang

7 8

Address:

State Key Laboratory of Microbial Technology

9

Shandong University

10

27 Shandanan Road

11

Jinan

12

250100

13

China

14

Tel: +86-531-88366202

15

Fax: +86-531-88565610

16

E-mail: [email protected]

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

17

ABSTRACT: Byproducts of food processing can be utilized for the production of

18

high-value-added enzyme cocktails. In this study, we utilized integrated functional-omics

19

technology to analyze composition and functional characteristics of extracellular enzymes

20

produced by Aspergillus niger grown on food processing byproducts. The results showed

21

that oligosaccharides constituted by arabinose, xylose, and glucose in wheat bran were able

22

to efficiently induce the production of extracellular enzymes of A. niger. Compared with

23

other substrates, wheat bran was more effective at inducing the secretion of β-glucosidases

24

from GH1 and GH3 families, as well as >50% of proteases from A1-family aspartic

25

proteases. Compared with proteins induced by single wheat bran or soybean dregs, the

26

protein yield induced by their mixture was doubled, and the time required to reach peak

27

enzyme activity was shortened by 25%. This study provided a technical platform for the

28

complex formulation of various substrates and functional analysis of extracellular enzymes.

29 30 31 32 33 34 35

KEY WORDS: Aspergillus niger; food processing byproducts; functional-omics;

36

glycoside hydrolases; protease

ACS Paragon Plus Environment

Page 2 of 38

Page 3 of 38

Journal of Agricultural and Food Chemistry

37

INTRODUCTION

38

Annual production of soybeans, wheat, and corn is estimated at 300, 700, and 800 million

39

tons, respectively, and represent the main food sources for humans1. Deep processing of

40

grain produces a large number of byproducts, such as soybean hulls (SHs), soybean dregs

41

(SDs), wheat bran (WB), and corn bran (CB), which account for ~8% to ~10% of the grain

42

weight2. These byproducts are generally used as low-value products, such as animal feed3.

43

These materials are also important biomass resources, representing good raw materials for

44

the production of high-value commercial enzymes, dietary fiber, and food additives.

45

The composition of plant-cell walls exhibits significant differences in polysaccharides

46

and proteins4. In monocotyledonous plants, such as corn and wheat, additional genes

47

encoding xylan and β-1,3;1,4-D-glucan synthase are detected, whereas genes encoding

48

pectin, mannan, and xyloglucan synthase are more abundant in dicotyledonous plants, such

49

as soybean4. As early as 1990, WB, CB, SHs, and SDs were utilized as raw materials to

50

produce crude enzymes, with the enzyme activity improved by optimizing culture

51

temperature, pH, time, and moisture during fermentation. However, the different chemical

52

compositions and structures of raw materials results in significant differences in enzyme

53

species and activities5. Additionally, the composition, abundance, and dynamic changes in

54

various enzyme components have not been quantitatively analyzed in detail, and the effects

55

of different raw materials on enzyme systems have not been systematically studied.

56

Aspergillus niger has become one of the main microbes capable of producing enzymes

57

on an industrial scale, as its genome contains a high number of genes encoding

58

hemicellulose-degrading enzymes and proteases6. Previous transcriptome studies revealed

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

59

that the enzyme-secretion order and species of A. niger were dynamically regulated by

60

different substrates7. However, genome and transcriptome analyses were unable to

61

determine the dynamic changes in functional proteins, proteomics can make up for this

62

deficiency. Therefore, comprehensive analysis of the type, abundance, and activity of

63

extracellular enzymes is needed8. Proteomes can provide comprehensive information

64

associated with extracellular enzymes, including species, concentration, modification, and

65

subcellular location9. Systematic studies of proteomes aid in the understanding and

66

evaluation of enzyme characteristics, especially their specific induction conditions, thereby

67

enabling more efficient enzyme production10.

68

The species and functions of enzymes secreted by A. niger cultured on different food

69

processing byproducts has not been systematically researched, which limits their industrial

70

application. To elucidate the dynamic changes in functional proteins on complex substrates,

71

it is necessary to integrate proteomics and metabolomics using a variety of high-throughput

72

techniques11 to systematically analyze the types, functions, and interactions of biological

73

macromolecules, and well as their dynamic changes12. In this study, the composition and

74

function of enzymes secreted by A. niger An-76 were analyzed by integrated

75

functional-omics, and suitable substrates for the industrial production of efficient and stable

76

enzymes was revealed, promoting the development of green industrial biotechnology.

77

MATERIALS AND METHODS

78

Byproducts of food processing. SHs, WB, and CB were ground using a pulverizer, and all

79

milled materials were stored at room temperature. SDs without any pretreatment were

80

stored in a sealed container at 4°C.

ACS Paragon Plus Environment

Page 4 of 38

Page 5 of 38

Journal of Agricultural and Food Chemistry

81

Solid-state fermentation and sample preparation. SHs (or WB or CB) were mixed with

82

distilled water at a 1:2 ratio (w/v) to culture A. niger An-76 in solid-state fermentation. All

83

mixed media were prepared by mixing two materials at a 1:1 ratio (w/w), with the moisture

84

of the final medium at ~67%. A total of 30 g medium was added to a 300-mL Erlenmeyer

85

flask and sterilized at 115°C for 30 min. A total of 200 µL of spore (7 × 107/mL)

86

suspension of A. niger was inoculated into medium and cultivated at 30°C. The samples

87

were collected every 24 h, and after collection, 100 mL distilled water was immediately

88

added to medium. To extract the extracellular mixture, the Erlenmeyer flasks were shaken

89

at 200×g for 1 h at 4°C and then centrifuged at 8000×g for 20 min to obtain the supernatant.

90

The samples were stored at 4°C until further use.

91

Determination of concentration extracellular reducing sugar and activity of proteins.

92

Bovine serum albumin (0.1 mg/mL) was used to obtain the standard curve, and 100 µL of

93

protein sample and 1 mL Coomassie Brilliant Blue G-250 dye were reacted at room

94

temperature for 10 min. Each sample was tested in triplicate, and all mixtures were

95

measured at 595 nm using a microplate spectrophotometer (Tecan, Morrisville, NC, USA).

96

Previous experiments suggested that the presence of sugars may interfere with the

97

measurement of extracellular-protein concentration13. In order to examine this effect,

98

protein concentration were measured at gradient concentrations of different sugars (xylose,

99

glucose, and xylo-oligosaccharide). As shown in Figure S1, the sugars with low

100

concentration had little effect on the measurement of protein concentration.

101

Standard curves were prepared using 0.1 mg/mL xylose or glucose, and 1% xylan

102

(Sigma-Aldrich, St. Louis, MO, USA) (w/v) or 1% sodium carboxymethylcellulose (CMC,

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 6 of 38

103

Sangon Biotech, Shanghai, China) (w/v) in sodium hydrogen phosphate/citric acid buffer

104

(pH 5.0) was used as a substrate to measure xylanase or endoglucanase activities. Enzyme

105

(400 µL) and 600 µL of substrate were mixed and reacted at 50°C for 30 min. After the

106

reaction, 800 µL DNS was added to each sample, which was then boiled for 10 min. The

107

reaction system was comprised of a 10-mL volume, and products were determined by

108

ultraviolet spectrophotometer (Puyuan Instruments, Ltd., Shanghai, China) at 550 nm.

109

p-nitrophenyl-β-D-cellobioside (pNPC)14, p-nitrophenyl-β-D-glucopyranoside (pNPG) 15

110

were used to measure the activities of the exoglucanase and β-glucosidase, respectively.

111

And p-nitrophenol (pNP) was used to obtain standard curves. 50 µL of sample, 50 µL of 2

112

mM pNPC (or pNPG) (Sigma-Aldrich) and 100 µL of acetic acid/sodium acetate buffer

113

(pH5.0) were mixed and reacted at 50°C for 30 min. Then 50 µL of 1M Na2CO3 was added

114

to terminate the reaction. All mixtures were measured at 420 nm using a microplate

115

spectrophotometer. Filter paper assay were performed according to the protocol published

116

before16. Whatman No. 1 filter paper strip (50 mg) was submerged in the mixture of 1.5 mL

117

of sodium hydrogen phosphate/citric acid buffer (pH 5.0) and 500µL of crude enzymes, the

118

mixture was reacted at 50°C for 1 h. Then 3 mL DNS was added to each sample, which

119

was then boiled for 10 min. The reaction system was diluted with distilled water to 25-mL

120

volume, and products were determined by ultraviolet spectrophotometer at 550 nm.

121

Sodium

122

Electrophoresis was performed on a miniaturized vertical gel system using a

123

Mini-PROTEAN 3PowerPac basic power supply (Bio-Rad, Hercules, CA, USA). The 12%

124

SDS-PAGE involved 15 µL of sample/well. Electrophoresis was performed at 80 V for

dodecyl

sulfate

polyacrylamide

gel

ACS Paragon Plus Environment

electrophoresis

(SDS-PAGE).

Page 7 of 38

Journal of Agricultural and Food Chemistry

125

around 2 h and the gel was stained with Coomassie Brilliant Blue R250 (Sangon Biotech)

126

for 30 min before destaining (glacial acetic acid: absolute ethanol: distilled water at a

127

volumetric ratio of 1:1:8) and scanning (Canon, Tokyo, Japan).

128

Fluorescence-assisted carbohydrate electrophoresis (FACE). The detailed protocol for

129

FACE was described previously17. Samples (5 µL) were fluorescently labeled by mixing

130

with 5 µL of 0.2 M 7-amino-1, 3-naphthalenedisulfonic acid monopotassium salt

131

monohydrate (Sigma-Aldrich) dissolved in 15 % acetic acid and incubating for 1 h in the

132

dark, followed by addition of 5 µL of 1 M NaCNBH3 (Sangon Biotech) and incubation at

133

40°C for 12 h. Samples (15 µL) were added to each well. Electrophoresis was performed at

134

7 mA for around 2 h, and gels were scanned using the ChemiDoc MP system (Bio-Rad).

135

LC-MS/MS. Extracellular proteins secreted by A. niger for 72 h on different substrates

136

were ultrafiltered using a 3-kDa cut-off membrane. All the extracellular proteins collected

137

from each experimental condition were ultrafiltered using a 3-kDa cut-off membrane and

138

precipitated using 1% trichloroacetic acid and 0.1% dithiothreitol (DTT, Sigma–Aldrich)

139

dissolved in acetone, then the total of precipitated extracellular proteins were dried and

140

resuspended in equal volume of double-distilled water, and the concentration of redissolved

141

protein from each sample (>20µg/µL) was determined with Coomassie Brilliant Blue

142

staining method, then 10µL dissolved protein solution was mixed with 50 µL degeneration

143

buffer (0.5 M Tris–HCl, 2.75 mM ethylenediaminetetraacetic acid, 6 M guanidine-HCl [pH

144

8.1]; Sigma-Aldrich), and 30 µL of 1 M DTT was added and incubated at 37°C for 2 h. To

145

alkylate the samples, 50 µL of 1 M iodoacetamide (Sigma-Aldrich) was added, and the

146

solution was incubated in the dark for 25 min. The mixture was transferred to a Microcon

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

147

YM-10 centrifuge tube (3-kDa membrane; Sigma-Aldrich) and washed three times with

148

360 µL of 25 mM NH4HCO3 (Sigma-Aldrich) for 15 min at 14,000×g at 4°C. The washed

149

proteins were obtained by centrifugation at 1000×g for 3 min at 4°C; digested with trypsin

150

at a ratio of 1:50 (w/w, trypsin: centrifuged proteins); and incubated at 37°C overnight. The

151

peptides were desalted using ZipTip C18 columns (Millipore, Burlington, MA, USA), and

152

the desalted peptides were dissolved with 0.1% (v/v) trifluoroacetic acid and subjected to

153

LC-MS/MS analysis on a Prominence nano LC system (Shimadzu, Kyoto, Japan) coupled

154

to an LTQ-Orbitrap Velos Pro ETD mass spectrometer (Thermo Fisher Scientific, Waltham,

155

MA, USA). To separate the peptides, a custom-made silica column (75 µm × 15 cm)

156

packed with Reprosil-Pur 120 C18-AQ (Dr.Maish GmbH, Ammerbuch, Germany) was

157

used. Mobile phases were solvent A (2.0 % ACN in water [v/v] with 0.1 % [v/v] formic

158

acid) and solvent B (98 % ACN in water [v/v] with 0.1 % [v/v] formic acid), the procedure

159

of stepping gradient elution was set as: 2 % (v/v) solvent B (0.0–5.0 min), 2–15 % (v/v)

160

solvent B (5.0–25.0 min), 15–40 % solvent B (25.0–55.0 min), 40–98 % (v/v) solvent B

161

(55.0–60.0 min), 98 % solvent B (60.0–70.0 min), 98–2 % (v/v) solvent B (70.0– 75.0 min),

162

and 2 % (v/v) solvent B (75.0–90.0 min) at a flow of 300 nL/min. A nanospray ion source

163

with a voltage of 2000 V and a transfer-capillary temperature of 275°C were used to spray

164

peptides into the mass spectrometer. The system was run in a data-dependent acquisition

165

mode using Xcalibur 2.2.0 software (Thermo Fisher Scientific) to perform MS/MS

166

experiments. Fullscan MS spectra (from 400 to 1800 m/z) were detected in the Orbitrap

167

with a resolution of 60000 at 400 m/z. The ten most intense precursor ions greater than the

168

threshold of 5000 counts in the linear ion trap were selected for MS/MS fragmentation

ACS Paragon Plus Environment

Page 8 of 38

Page 9 of 38

Journal of Agricultural and Food Chemistry

169

analysis at a normalized collision energy of 35 %. Three replicates were performed for each

170

sample.

171

Database searches. All data searches used Proteome Discover software version 1.4

172

(Thermo Fisher Scientific) with the SEQUEST search engine. The reference databases of A.

173

niger An-76 was downloaded from Uniprot (http://www.uniprot.org). The settings for

174

MS/MS searches were as follows: 1) trypsin was used to digest the proteins, allowing two

175

missed cleavages; 2) precursor mass tolerance was set at 10 ppm, with a 0.8-Da fragment

176

mass tolerance; and 3) oxidation of methionine was chosen as the dynamic modification,

177

and carbamidomethylation of cysteine residues was selected as the fixed modification.

178

Only peptides with at least six amino acid residues showing 95% certainty (q ≤ 0.05) were

179

included in the results. At least two peptides (q < 0.05) were needed to be considered for

180

protein identification, and the false-discovery rate was set at 1%. The relative abundance of

181

proteins was characterized by peptide-spectrum matches (PSMs) 18, and protein abundance

182

in different cultivation was positively correlated with PSM according to previous studies19.

183

Extracellular protein digestion experiments. The proteins secreted by A. niger on WB or

184

WBSH at 72 h were inactivated at 95°C for 10 min. Then inactivated proteins were

185

digested by the proteins (secreted on WB or WBSH at 120 h) at a ratio of 1:1 (w/w) at

186

50°C for 24 h.

187

Enzyme hydrolysis of food-processing byproducts. The enzymes used in this assay were

188

collected at 72 h. Byproducts of food processing (3.6 g) were added into 60 mL of cell-free

189

enzyme solution, and the mixture was incubated at 50°C for 24 h. Samples were

190

centrifuged at 8000×g for 15 min to separate the hydrolysate, which was analyzed to

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

191

determine the content of released soluble carbohydrates.

192

Ion chromatography (IC). To detect the compositions of byproducts, all samples need to

193

be hydrolyzed into monosaccharides. Each substrate was hydrolyzed by 1% H2SO4 at

194

120°C for 2 h. Then, the hydrolysate was freeze-dried, and resolubilized in double-distilled

195

water. An ICS-2000 ion chromatograph (Dionex, Sunnyvale, CA, USA) was utilized for

196

chromatographic separations. This instrument included a model EG50 eluent generator, a

197

model AS50 autosampler, a model ED50 electrochemical detector operated in conductivity

198

mode, and a model GP50 gradient pump. The columns used were a Dionex Carbopac PA1

199

(2 × 250 mm) and a PA20 (3 × 150 mm). The elution system of the PA20 was 0.8% NaOH

200

and 99.2% H2O, and the elution system for the PA1 was 1.8% NaOH and 98.2%H2O. The

201

flow rate was 0.30 mL/min, and the column temperature was 30°C. Data were collected

202

using a Chromeleon 6.80 chromatogram workstation (Dionex). Sugar identification was

203

performed by comparison with reference sugars (D-galactose, L-arabinose, L-rhamnose,

204

D-glucose, D-xylose, and D-mannose). Calculation of the molar ratio of the

205

monosaccharides was performed based on the peak area of the monosaccharide.

206

RESULTS

207

Dynamic changes in extracellular reducing sugar, and protein concentration, and GHs.

208

As shown in Figure 1A, A. niger was able to grow on SD, SH, CB, WB, and their mixtures.

209

WB consisted of high concentrations of soluble sugars easily broken down by the enzymes,

210

leading to the highest concentration of extracellular reducing sugar (9 mg/mL), whereas the

211

reducing sugar in SH was the lowest (4.6 mg/mL) (Figure 1A). The concentration of

212

reducing sugars reached the maximum at 24 h using a mixture of WB with SH or SD as

ACS Paragon Plus Environment

Page 10 of 38

Page 11 of 38

Journal of Agricultural and Food Chemistry

213

carbon sources. High concentrations of reducing sugar in WB promoted the growth of A.

214

niger, with extracellular-protein content reaching a peak (0.5 mg/mL) at 120 h and 2- to

215

3-fold higher than that seen from the other three substrates (Figure 1B). Xylanases secreted

216

by A. niger were detected at 24 h on SH, WB, WBSH and WBSD (Figure 1C), earlier than

217

endoglucanases at 48 h, which was consistent with previous reports10. After 48 h, the

218

activities of xylanases produced from WB and its mixtures were higher than those induced

219

by SH, SD, and CB (Figure 1C). A significant difference was observed in the

220

endoglucanase activity of these substrates measured within 72 h, and endoglucanase

221

activity produced on WB and its mixtures after 72h was higher than that produced on SH,

222

SD, and CB as single carbon sources (Figure 1D). The activities of exoglucanses and

223

β-glucosidases were also measured. Obvious exoglucanase and β-glucosidase activities

224

were detected at 72 h and 48 h, respectively (Figure S2). The activities of exoglucanses,

225

β-glucosidases and FPase produced on WB and its mixtures were also higher than those

226

induced by other substrates (Figure S2).

227

Dynamic changes in oligosaccharides released during hydrolytic processing. To

228

characterize the dynamic changes in species and concentration of reducing sugars during

229

the growth process, FACE was used for detection and quantitative analysis20. Because the

230

reducing-sugar concentration of WB and its mixtures was 2- to 3-fold higher than that of

231

the other substrates (Figure 1A), the reducing sugar was diluted. As shown in Figure 2, the

232

types of soluble sugars released on different substrates were significantly different. The

233

main components of soluble sugars maintained in non-degraded SD (Figure 2A), WB,

234

WBSH and WBSD (Figure 2D-F) were oligosaccharides between X2 and X3, whereas

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

235

those of SH and CB were monosaccharides near X1 (Figure 2B, C).

236

A trace of soluble reducing sugar in raw materials was absorbed by A. niger to support

237

its growth and induce the release of large amounts of GHs capable of degrading

238

polysaccharides into large quantities of monosaccharides and oligosaccharides within 24 h

239

(Figure 1B). At 24 h, the oligosaccharides degraded from different substrates accumulated

240

rapidly (the band intensities increased significantly). When SD, SH, CB, or WB was used

241

as carbon source, respectively, the species and total concentrations of reducing sugars

242

reached the maximum at 48 h, which was consistent with the results shown in Figure 1A,

243

whereas the major reducing sugars were diverse of oligosaccharides and monosaccharides

244

in SD and SH at 48 h, and mainly monosaccharide (a single band near X1) in CB (Figure

245

2A–C). More than two types of oligosaccharides were produced from 24 h to 48 h in WB

246

(Figure 2D). When SD or SH was added to WB, the concentration of X3 to X5

247

oligosaccharides at 24 h is similar to that on WB (Figure 2E, F). Additionally, there were

248

more types of monosaccharides observed in WBSH as compared with WBSD (Figure 2F).

249

Analysis of the chemical composition and enzymatic hydrolysates of various

250

substrates. Analysis of chemical compositions associated with different food-processing

251

byproducts using IC showed that SH consisted of higher kinds of sugar units (Figure 3A),

252

which included large amounts of mannose, rhamnose and xylose (23% , 22%, and 23.9%,

253

respectively), and trace amounts of glucose and arabinose. There were differences in

254

composition between SH and SD obtained from soybeans, with different tissues reflecting

255

higher amounts of rhamnose (28%) and galactose (48%) in SD (Table S2) and suggesting

256

that SD contained large amounts of pectin. The chemical composition of WB is similar to

ACS Paragon Plus Environment

Page 12 of 38

Page 13 of 38

Journal of Agricultural and Food Chemistry

257

that of CB, mainly consisting of glucose, xylose, arabinose, and galactose; however, the

258

content of arabinose and glucose in CB was higher than that in WB (34% and 33%,

259

respectively), and the amount of xylose (29%, Table S2) was higher in WB. The diverse

260

structural units reflect the heterogeneity in the types and contents of polysaccharides from

261

various plant-cell walls, which can lead to differences in the types of enzymes produced by

262

specific induction 21.

263

The hydrolysates of various substrates degraded by the enzymes secreted by A. niger

264

(after a 72-h culture) differed significantly (Figure 3). As shown in Figure 3B, hydrolysates

265

in SD were comprised mainly of galactose, rhamnose, glucose, and lacked xylose. Xylose

266

and xylooligosaccharide (XOS) were reported as efficient inducers for triggering enzyme

267

production in A. niger22; therefore, the lower content of xylose and XOS in SD might affect

268

the induction of xylanases and other GHs. The content of xylose in SH was much higher

269

than that in SD (Figure 3B); therefore, SH more rapidly induced the expression of xylanase

270

genes than SD (Figure 1C). The soluble sugar produced by A. niger after degradation of CB

271

and WB for 24 h was comprised mainly of glucose, xylose, and arabinose, with the xylose

272

content in WB also higher than that in CB (Figure 3B).

273

Analysis of the hydrolysates of residues after degradation (Figure 3C) showed that SD

274

mainly contained glucose and galactose, indicating that the degree of hemicellulose and

275

pectin degradation was higher when the main residue was cellulose. The relative content of

276

glucose in SH residue increased from 7.9% to 30.1% (Table S2), suggesting that

277

hemicellulose had been significantly degraded. The types and contents of sugar units before

278

and after degradation of WB and CB showed no obvious changes. In monocots, arabinose

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

279

is the main side chain of xylan23. The proportion of arabinose in CB residue decreased by

280

~7%, indicating xylan degradation.

281

Due to the complex chemical composition of SH and SD, various structural units were

282

produced by degradation catalyzed by the induced enzymes (Figure 3B, D). Although the

283

structural units of WB and CB is similar, but the concentration of induced-enzyme and

284

enzymatic hydrolysates (Figure 3D) are significant different. Compared with CB, WB

285

could be degraded into more oligosaccharides above X3, as shown in Figure 3D, and more

286

extracellular proteins were produced by A. niger grown on WB(Figure 1B). Therefore,

287

soluble oligosaccharides in WB might be the ingredients capable of inducing A. niger to

288

produce extracellular enzymes efficiently and continuously. However, the induction

289

mechanism of the oligosaccharides in WB required further study.

290

SDS-PAGE analysis of dynamic changes in proteins. As shown in figure 4, SD, WB,

291

WBSH, and WBSD contained small amounts of soluble proteins with lower molecular

292

weights (50% of the total extracellular proteases, which was consistent

371

with the perference acid environment of A. niger and the optimal pH of extracellular GHs25.

372

This indicated that GHs and proteases exhibited co-adaption mechanisms related to the

373

growth environment. In addition to increases in aminopeptidase concentration, the types of

374

proteases induced by CB were similar to those on SD. Compared with the other substrates,

375

the amount of proteases induced by WB was higher, specifically 10-fold and 1-fold

376

increases in aminopeptidase and oligopeptidase concentrations, respectively. Additionally,

377

some cysteine proteases and metalloproteases were also produced by A. niger. Although the

378

content of aspartic protease in WB was less than that in SH, the total amount of protease

379

was still the highest (Figure 5D). In WBSH and WBSD, no metalloproteinase or cysteine

380

protease was detected. The species and concentrations of protease induced by WBSD were

381

similar to those induced by WB, indicating that WB plays a dominant role in the induced

382

expression of proteases.

383

Microorganisms degrade extracellular proteins into small peptides or amino acids

384

by secreting proteases to support their growth and protein synthesis. The induction of

385

proteases is significantly different from that of GHs (Table 3 and Figure 5D). The protein

386

content of SD was high, but not all extracellular proteases were induced. SH was able to

387

induce secretion of the A1-family aspartic protease (A0A100IK58) at 1- to 2-fold higher

388

levels than that induced by other substrates. The species and concentrations of proteases

ACS Paragon Plus Environment

Page 18 of 38

Page 19 of 38

Journal of Agricultural and Food Chemistry

389

secreted in CB were similar to those of SD. The protease species induced by WB were the

390

richest, and several proteases, including one type of cysteine protease (A0A117DUE3), two

391

types of metalloproteinases (A0A100INJ6 and A0A100IEJ2), and five types of

392

aminopeptidases (A0A117DXE7, A0A117E4Q1, A0A100IN20, A0A100IAD1, and

393

A0A100I9B5) were specifically induced by WB. Increases in these components enable

394

complete degradation, which provides additional nitrogen sources for growth.

395

Compared with WB, the concentrations of aspartic protease (A0A100IK58) induced by

396

WBSH or WBSD increased slightly, but metalloproteinases (A0A100INJ6 and

397

A0A100IEJ2) and cysteine protease (A0A117DUE3) were almost undetectable. The

398

advantage of WB and its combinations for inducing the secretion of various proteases

399

enables the production of efficient proteinase preparations.

400

DISCUSSION

401

The polysaccharide components of different types of cell walls were differentiated during

402

evolution26, especially the chemical compositions and structures of monocotyledonous and

403

dicotyledonous plants, which showed obvious differences27, thus, A. niger produced diverse

404

oligosaccharides during the degradation process (Figure 1 and 2) 28. The concentration of

405

oligosaccharides released from WB was higher than that from SD from 24 h to 48 h.

406

Additionally, these oligosaccharides were also detected in WBSH and WBSD (Figure 2),

407

suggesting the dominant inducing role of soluble sugars in WB. These oligosaccharides

408

were composed mainly of xylose, arabinose and glucose detected by IC (Figure 3B).

409

Xylose and XOS rapidly triggered the expression of transcription factor XlnR from A.

410

niger An-7629; therefore, soluble sugars of WB might contribute to the high expression of

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

411

extracellular GHs30. SH and SD were not efficient at inducing the secretion of enzymes

412

from A. niger, which might be related to the lower content of xylan in their structures.

413

Therefore, it is necessary to analyze the composition and dynamic changes of hydrolysates

414

during the substrate-degradation process before efficient utilization of byproducts, which

415

represents the foundation for further comprehensive utilization31.

416

Analysis of the A. niger genome revealed a preference for degradation of dicotyledonous

417

plants10, such as SH and SD. However, proteomics results indicated that WB is an efficient

418

substrate for inducing the secretion of extracellular GHs. Compared with the other three

419

substrates, WB was more preferable for A. niger32, and the quantity of extracellular-protein

420

secretion and activity (GHs) induced by WB was higher (Figure 1). Compared with

421

dicotyledonous plants, such as SH and SD, WB contributed to the higher expression levels

422

of xylan- and cellulose-degradation enzymes33, particularly xylanase (F5CI28) and CBH

423

(A0A117DZQ3). Additionally, β-glucosidases induced by WB were able to remove

424

cellobiose produced by CBH and relieve product inhibition of CBH, which further

425

improved the degradation efficiency of crystalline cellulose34.

426

The protease-induction mechanism was significantly different from that of GHs28.

427

Despite large amounts of soy proteins in SD, it was not an efficient substrate for inducing A.

428

niger protease production (Table 3). We hypothesized that fragmentation of soybean

429

proteins after processing resulted in the efficient degradation by limited numbers of

430

proteases31. WB induced numerous types of proteases, including aminopeptidase,

431

carboxypeptidase, oligopeptidase, aspartic protease, serine protease, cysteine protease, and

432

metalloproteinase. S8-family serine endopeptidase exhibits activity similar to that of

ACS Paragon Plus Environment

Page 20 of 38

Page 21 of 38

Journal of Agricultural and Food Chemistry

433

trypsin35 and works in conjunction with aspartate protease to provide additional

434

degradation sites for exopeptidases36. For endopeptidases, WB can specifically induce the

435

secretion of metalloproteases, which mainly degrade water-insoluble fibrous proteins, such

436

as gluten proteins in WB. Systematic analysis of these mechanisms can promote strategies

437

supporting the specific hydrolysis of proteins and improve the efficiency of enzymatic

438

hydrolysis, which is critical for the application of proteases.

439

The mixture of WB and SD significantly accelerated A. niger growth, and the

440

concentrations and species of extracellular proteins remained stable at 72 h. Moreover, the

441

combination of WB and SD resulted in efficient synthesis of GH7-family CBH

442

(A0A117DZQ3) and GH12-family endoglucanase (A0A117DY27). WB also enabled

443

synthesis of high concentrations of β-glucosidases (I1Z9C3 and A0A117E1I2) and

444

AA9-family LPMO, thereby improving cellulose-degradation efficiency. Furthermore, SD

445

possessed of largest amount of easily degradable proteins37 and lignocellulose are

446

necessary for efficient enzyme production38, which can provided continuous carbon sources

447

or amino acids39 for the synthesis of proteins. Therefore, when A. niger An-76 was utilized

448

as a strain for the production of lignocellulolytic enzymes, a combination of WB and SD

449

could shorten enzyme-production time and produce more efficient enzyme preparations.

450

In this study, an integrated functional technology was established to analyze the dynamic

451

changes in extracellular enzymes during the degradation process of complex substrates. We

452

analyzed the composition and abundance of enzymes secreted by A. niger in different

453

food-processing byproducts, finding that the effective combination of WB and SD

454

optimized the composition of the enzyme system and prolonged the shelf life of the

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

455

enzyme, which has direct significance toward increasing the added-value of agricultural

456

waste and optimizing industrial enzyme production.

457

AUTHOR INFORMATION

458

Corresponding author

459

*E-mail: [email protected]

460

Notes

461

The authors declare no competing financial interest.

462

ABBREVIATIONS USED

463

SH, soybean hulls; SD, soybean dreg; CB, corn bran; WB, wheat bran; WBSH, wheat bran

464

and

465

carboxymethylcellulose; FACE, Fluorescence-assisted carbohydrate electrophoresis;

466

SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis; DTT,

467

dithiothreitol; IC, Ion chromatography; XOS, xylooligosaccharide; LPMO, lytic

468

polysaccharide monooxygenases; CBH, cellobiohydrolase.

469

ASSOCIATED CONTENT

470

Supporting Information

471

Figure S1. The protein concentration after the addition of sugars. A: 10mg/mL BAS +

472

20mg/mL XOS; precipitated and dissolved protein +20mg/mL xylose. B: precipitated and

473

dissolved protein +20mg/mL glucose.

474

Figure S2. The pNPGase (A), pNPCase (B) and FPase (C) produced by A. niger An-76 on

475

different carbon sources.

476

Figure S3. The digestion experiment of extracellular proteins according to SDS–PAGE

477

analysis. A: The zymogram of inactivated proteins secreted on WB at 72 h hydrolyzed for

soybean

hulls; WBSD,

wheat bran and soybean dregs; CMC, sodium

ACS Paragon Plus Environment

Page 22 of 38

Page 23 of 38

Journal of Agricultural and Food Chemistry

478

24 h by enzymes produced on WB at 120 h. B: The zymogram of inactivated proteins

479

secreted on WBSH at 72 h digested for 24 h by enzymes produced on WBSH at 120 h.

480

Table S1. Pectin and (Galacto) mannan-degrading enzymes secreted by Aspergillus niger

481

An-76 on different substrates.

482

Table S2. The proportion of monosaccharide units detected by IC.

483

ACKNOWLEDGEMENTS

484

This work was supported by a grant from The National Natural Science Foundation

485

of China (31770054),and the National Key Research and Development Program of China

486

(2016YFD0800601).

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530

REFERENCES 1.

Prado, J. R.; Segers, G.; Voelker, T.; Carson, D.; Dobert, R.; Phillips, J.; Cook, K.; Cornejo, C.; Monken, J.;

Grapes, L., Genetically engineered crops: from idea to product. Annual review of plant biology 2014, 65. 2.

Elleuch, M.; Bedigian, D.; Roiseux, O.; Besbes, S.; Blecker, C.; Attia, H., Dietary fibre and fibre-rich

by-products of food processing: Characterisation, technological functionality and commercial applications: A review. Food chemistry 2011, 124 (2), 411-421. 3.

Gaurav, N.; Sivasankari, S.; Kiran, G. S.; Ninawe, A.; Selvin, J., Utilization of bioresources for sustainable

biofuels: A Review. Renewable and Sustainable Energy Reviews 2017, 73, 205-214. 4.

Calderan-Rodrigues, M. J.; Jamet, E.; Douché, T.; Bonassi, M. B. R.; Cataldi, T. R.; Fonseca, J. G.; San

Clemente, H.; Pont-Lezica, R.; Labate, C. A., Cell wall proteome of sugarcane stems: comparison of a destructive and a non-destructive extraction method showed differences in glycoside hydrolases and peroxidases. BMC plant biology 2016, 16 (1), 14. 5.

da Silva Menezes, B.; Rossi, D. M.; Ayub, M. A., Screening of filamentous fungi to produce xylanase and

xylooligosaccharides in submerged and solid-state cultivations on rice husk, soybean hull, and spent malt as substrates. World journal of microbiology & biotechnology 2017, 33 (3), 58. 6.

Pel, H. J.; de Winde, J. H.; Archer, D. B.; Dyer, P. S.; Hofmann, G.; Schaap, P. J.; Turner, G.; de Vries, R. P.;

Albang, R.; Albermann, K., Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88. Nature biotechnology 2007, 25 (2), 221. 7.

Adav, S. S.; Li, A. A.; Manavalan, A.; Punt, P.; Sze, S. K., Quantitative iTRAQ secretome analysis of

Aspergillus niger reveals novel hydrolytic enzymes. Journal of proteome research 2010, 9 (8), 3932-3940. 8.

Aebersold, R.; Mann, M., Mass-spectrometric exploration of proteome structure and function. Nature

2016, 537 (7620), 347-55. 9.

Hu, Q.; Noll, R. J.; Li, H.; Makarov, A.; Hardman, M.; Graham Cooks, R., The Orbitrap: a new mass

spectrometer. Journal of mass spectrometry 2005, 40 (4), 430-443. 10. Gong, W.; Zhang, H.; Liu, S.; Zhang, L.; Gao, P.; Chen, G.; Wang, L., Comparative Secretome Analysis of Aspergillus niger, Trichoderma reesei, and Penicillium oxalicum During Solid-State Fermentation. Appl Biochem Biotechnol 2015, 177 (6), 1252-71. 11. Van Emon, J. M., The omics revolution in agricultural research. Journal of agricultural and food chemistry 2015, 64 (1), 36-44. 12. Guerriero, G.; Sergeant, K.; Hausman, J.-F., Integrated-omics: a powerful approach to understanding the heterogeneous lignification of fibre crops. International journal of molecular sciences 2013, 14 (6), 10958-10978. 13. Banik, S. P.; Pal, S.; Ghorai, S.; Chowdhury, S.; Khowala, S., Interference of sugars in the Coomassie Blue G dye binding assay of proteins. Analytical biochemistry 2009, 386 (1), 113-115. 14. Lahjouji, K.; Storms, R.; Xiao, Z.; Joung, K.-B.; Zheng, Y.; Powlowski, J.; Tsang, A.; Varin, L., Biochemical and molecular characterization of a cellobiohydrolase from Trametes versicolor. Applied microbiology and biotechnology 2007, 75 (2), 337-346. 15. Parry, N. J.; Beever, D. E.; Emyr, O.; Vandenberghe, I.; Van Beeumen, J., Biochemical characterization and mechanism of action of a thermostable β-glucosidase purified from Thermoascus aurantiacus. Biochemical Journal 2001, 353 (1), 117-127. 16. Ghose, T., Measurement of cellulase activities. Pure and applied Chemistry 1987, 59 (2), 257-268. 17. Zhang, Q.; Zhang, X.; Wang, P.; Li, D.; Chen, G.; Gao, P.; Wang, L., Determination of the action modes of cellulases from hydrolytic profiles over a time course using fluorescence ‐ assisted carbohydrate electrophoresis. Electrophoresis 2015, 36 (6), 910-917.

ACS Paragon Plus Environment

Page 24 of 38

Page 25 of 38

Journal of Agricultural and Food Chemistry

531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574

18. Zhou, J.-Y.; Schepmoes, A. A.; Zhang, X.; Moore, R. J.; Monroe, M. E.; Lee, J. H.; Camp, D. G.; Smith, R. D.; Qian, W.-J., Improved LC− MS/MS spectral counKng staKsKcs by recovering low-scoring spectra matched to confidently identified peptide sequences. Journal of proteome research 2010, 9 (11), 5698-5704. 19. Liu, H.; Sadygov, R. G.; Yates, J. R., A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Analytical chemistry 2004, 76 (14), 4193-4201. 20. Gong, W.; Zhang, H.; Tian, L.; Liu, S.; Wu, X.; Li, F.; Wang, L., Determination of the modes of action and synergies of xylanases by analysis of xylooligosaccharide profiles over time using fluorescence-assisted carbohydrate electrophoresis. Electrophoresis 2016, 37 (12), 1640-50. 21. Sharma Ghimire, P.; Ouyang, H.; Wang, Q.; Luo, Y.; Shi, B.; Yang, J.; Lu, Y.; Jin, C., Insight into Enzymatic Degradation of Corn, Wheat, and Soybean Cell Wall Cellulose Using Quantitative Secretome Analysis of Aspergillus fumigatus. Journal of proteome research 2016, 15 (12), 4387-4402. 22. Delmas, S.; Pullan, S. T.; Gaddipati, S.; Kokolski, M.; Malla, S.; Blythe, M. J.; Ibbett, R.; Campbell, M.; Liddell, S.; Aboobaker, A., Uncovering the genome-wide transcriptional responses of the filamentous fungus Aspergillus niger to lignocellulose using RNA sequencing. PLoS genetics 2012, 8 (8), e1002875. 23. Scheller, H. V.; Ulvskov, P., Hemicelluloses. Annual review of plant biology 2010, 61. 24. Popper, Z. A.; Michel, G.; Hervé, C.; Domozych, D. S.; Willats, W. G.; Tuohy, M. G.; Kloareg, B.; Stengel, D. B., Evolution and diversity of plant cell walls: from algae to flowering plants. Annual review of plant biology 2011, 62, 567-590. 25. Rawat, R.; Srivastava, N.; Chadha, B. S.; Oberoi, H. S., Generating fermentable sugars from rice straw using functionally active cellulolytic enzymes from Aspergillus niger HO. Energy & Fuels 2014, 28 (8), 5067-5075. 26. Fangel, J. U.; Ulvskov, P.; Knox, J. P.; Mikkelsen, M. D.; Harholt, J.; Popper, Z. A.; Willats, W. G., Cell wall evolution and diversity. Frontiers in plant science 2012, 3. 27. Burton, R. A.; Gidley, M. J.; Fincher, G. B., Heterogeneity in the chemistry, structure and function of plant cell walls. Nature chemical biology 2010, 6 (10), 724-732. 28. de Castro, R. J. S.; Nishide, T. G.; Sato, H. H., Production and biochemical properties of proteases secreted by Aspergillus niger under solid state fermentation in response to different agroindustrial substrates. Biocatalysis and Agricultural Biotechnology 2014, 3 (4), 236-245. 29. Hasper, A. A.; Visser, J.; De Graaff, L. H., The Aspergillus niger transcriptional activator XlnR, which is involved in the degradation of the polysaccharides xylan and cellulose, also regulates d‐xylose reductase gene expression. Molecular microbiology 2000, 36 (1), 193-200. 30. Hasper, A. A.; Trindade, L. M.; van der Veen, D.; van Ooyen, A. J.; de Graaff, L. H., Functional analysis of the transcriptional activator XlnR from Aspergillus niger. Microbiology 2004, 150 (5), 1367-1375. 31. Li, B.; Qiao, M.; Lu, F., Composition, Nutrition, and Utilization of Okara (Soybean Residue). Food Reviews International 2012, 28 (3), 231-252. 32. Prückler, M.; Siebenhandl-Ehn, S.; Apprich, S.; Höltinger, S.; Haas, C.; Schmid, E.; Kneifel, W., Wheat bran-based biorefinery 1: Composition of wheat bran and strategies of functionalization. LWT-Food Science and Technology 2014, 56 (2), 211-221. 33. Xing, S.; Li, G.; Sun, X.; Ma, S.; Chen, G.; Wang, L.; Gao, P., Dynamic changes in xylanases and β-1, 4-endoglucanases secreted by Aspergillus niger An-76 in response to hydrolysates of lignocellulose polysaccharide. Applied biochemistry and biotechnology 2013, 171 (4), 832-846. 34. Guerriero, G.; Hausman, J. F.; Strauss, J.; Ertan, H.; Siddiqui, K. S., Lignocellulosic biomass: Biosynthesis, degradation, and industrial utilization. Engineering in Life Sciences 2016, 16 (1), 1-16. 35. Laskar, A.; Rodger, E. J.; Chatterjee, A.; Mandal, C., Modeling and structural analysis of evolutionarily

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

575 576 577 578 579 580 581 582 583

diverse S8 family serine proteases. Bioinformation 2011, 7 (5), 239. 36. Yin, L.-J.; Hsu, T.-H.; Jiang, S.-T., Characterization of acidic protease from Aspergillus niger BCRC 32720. Journal of agricultural and food chemistry 2013, 61 (3), 662-666. 37. Pleissner, D.; Venus, J., Utilization of protein-rich residues in biotechnological processes. Applied Microbiology and Biotechnology 2016, 100 (5), 2133-2140. 38. Turk, B., Targeting proteases: successes, failures and future prospects. Nature reviews. Drug discovery 2006, 5 (9), 785. 39. Dasuri, K.; Zhang, L.; Keller, J. N., Oxidative stress, neurodegeneration, and the balance of protein degradation and protein synthesis. Free Radical Biology and Medicine 2013, 62, 170-185.

584

ACS Paragon Plus Environment

Page 26 of 38

Page 27 of 38

Journal of Agricultural and Food Chemistry

585

Figure Captions

586

Figure 1.Time course of enzyme production during solid-state fermentation by Aspergillus

587

niger An-76 with different substrates. A: Reducing-sugar concentration. B: Protein

588

concentration. C: Xylanase activity. D: Endoglucanase activity. Bars represent standard

589

deviations of three replicates.

590

SD: soybean dregs; SH: soybean hulls; CB: corn bran; WB: wheat bran; WBSH:

591

wheat bran and soybean hull; WBSD: wheat bran and soybean dregs.

592 593

Figure 2. Extracellular reducing sugar from Aspergillus niger An-76 on different substrates

594

observed using FACE during 120-h solid-state fermentation. A – F: SD, SH, CB, WB,

595

WBSH, and WBSD. A and B samples were diluted 10-fold; C – F: Samples were diluted

596

15-fold. X1–X4: xylose, xylobiose, xylotriose, and xylotetraose.

597 598

Figure 3.Monosaccharide and oligosaccharide content detected using IC and FACE.

599

A: Substrates used in this study. B: Supernatant of substrate hydrolyzed by enzymes for 24

600

h. C: Residues after degradation by Aspergillus niger An-76 for 120 h. D: Supernatant of

601

substrates hydrolyzed for 24 h by enzymes produced by A. niger An-76. Abbreviations

602

before the line represent the enzymes induced by this substrate; abbreviations after the line

603

represent the substrate.

604 605

Figure 4. Extracellular proteins secreted by Aspergillus niger An-76 according to SDS–

606

PAGE analysis. A – F: SD, SH, CB, WB, WBSH, and WBSD.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

607 608

Figure 5. Classification of proteins identified in the secretomes of Aspergillus niger An-76

609

using LC-MS/MS. A: Correlation analysis of total extracellular protein PSM value and

610

corresponding concentration of different substrates. B: Correlation analysis of PSM value

611

and corresponding enzymatic activities of different substrates. C: PSM of CAZymes

612

secreted by A. niger An-76 on different substrates, respectively. D: The PSM of proteases

613

secreted by A. niger An-76 on different substrates, respectively.

ACS Paragon Plus Environment

Page 28 of 38

Page 29 of 38

Journal of Agricultural and Food Chemistry

Figure 1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Figure 2

ACS Paragon Plus Environment

Page 30 of 38

Page 31 of 38

Journal of Agricultural and Food Chemistry

Figure 3

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Figure 4

ACS Paragon Plus Environment

Page 32 of 38

Page 33 of 38

Journal of Agricultural and Food Chemistry

Figure 5

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 34 of 38

Table 1. Xylan-degrading enzymes secreted by Aspergillus niger An-76 on different substrates. Substrate

Accession number

Protein

Family

SD

SH

CB

Xylan-backbone

F5CI28

Endo-β-1,4-xylanase

GH11

76±12.7

177±25.4

272.7±27.0

283.7±20.4

283.7±29.1

291±13.9

A0A100I6F6

Endo-β-1,4-xylanase

GH10

19±9.9

46.5±4.9

53.7±11.0

159.7±20.2

136±24.3

110.3±16.7

A0A100ITA6

Endo-β-1,4-xylanase

GH11

45±12.1

43±13.9

50.67±8.1

44±5.2

5±0

1.7±1.2

Xylan-side chain

3.5±0.7

5.5±0.7

WB

WBSH

WBSD

A0A100I6U9

Endo-β-1,4-xylanase

GH11

A0A117E086

Endo-β-1,4-xylanase

GH43

11.3±2.9

2.3±0.6

9.7±2.3

A0A117E0N6

Endo-β-1,4-xylanase

GH11

5.7±2.9

6±0

2±0

A0A100I443

β-xylosidase

GH3

27±17.0

73±8.5

33.3±4.6

92.3±21.9

38.7±5.8

77.3±20.2

A0A117DZC8

α-L-arabinofuranosidase

GH54

145.5±13.4

188±11.3

231±31.2

107±34.6

129±46.8

146.3±39.3

A0A100I6G0

α-L-arabinofuranosidase

GH62

78.5±13.4

132.5±9.2

143.3±30.6

115±17.3

192.3±26.4

138.3±16.7

A0A100ITZ9

α-L-arabinofuranosidase

GH51

13±1.4

12±1.4

1.7±0.6

4±0

6±3.5

2.7±0.6

A0A100IHT3

α-L-arabinofuranosidase

GH43

3±1.4

4.5±3.5

4±1.7

8±1.7

7.3±2.9

4.7±0.6

A0A100III9

α-L-arabinofuranosidase

GH51

4±1.7

6.7±4.0

5.7±2.3

A0A100IK16

α-glucuronidase

GH67

W6GEY2

Feruloyl esterase

CE1

A0A100IPH2

Acetylxylan esterase

CE1

A0A100IKK3

Acetylxylan esterase

CE1

19.5±3.5 8.5±0.7

19.5±3.5

24.3±8.1

46.3±2.3

23.7±7.5

34.3±4.6

11±1.4

22.7±5.8

59.3±8.1

27. 3±8.1

24±3.5

10±0

5±0

12.3±2.3

9.67±1.2

21±5.2

8.5±2.1

4.3±2.9

ACS Paragon Plus Environment

4±1.7

Page 35 of 38

Journal of Agricultural and Food Chemistry

Table 2. Cellulose-degrading enzymes secreted by Aspergillus niger An-76 on different substrates. Substrate

Accession number

Protein

Family

SD

SH

CB

WB

WBSH

WBSD

Cellulose

A0A117DZQ3

Exo-β-1,4-glucanase

GH7

44±18.4

152.5±6.4

52.7±23.1

211±1.7

96±24.3

292.3±16.2

A0A100I8W6

Exo-β-1,4-glucanase

GH7

20±14.1

18±5.7

8±1.4

31.33±2.9

17±3.5

76.7±21.4

A0A100IHS6

Exo-β-1,4-glucanase

GH6

4±1.7

9±0

4±1.7

14.3±2.9

11.3±1.2

22.3±8.1

A0A100ISK1

Exo-β-1,4-glucanase

GH6

2±0

4.7±2.3

12.7±1.2

5.3±1.2

16±1.7

A0A100IKG6

Endo-β-1,4-glucanase

GH12

45±1.4

91.3±11.6

131±12.1

95.7±16.2

82.7±12.7

Cellubiose

Crystalline cellulose

27.5±2.1

A0A117DY27

Endo-β-1,4-glucanase

GH5

24±9.9

78±11.3

16±1.7

50.3±1.2

46.7±2.3

105.3±30.6

A0A100ILF3

Endo-β-1,4-glucanase

GH5

37±1.4

57±4.2

57.3±16.7

54±6.9

49±22.5

32.3±15.0

A0A117DXN2

Endo-β-1,4-glucanase

GH5

29.5±0.7

32.5±6.4

15.7±0.6

8.3±0.6

11±1.7

19.3±8.1

A0A100INM5

Endo-β-1,4-glucanase

GH12

3±1.4

4±1.7

2.33±1.2

3.67±0.6

I1Z9C3

β-glucosidase

GH3

28±11.3

48±2.8

49.3±9.8

90.3±16.7

39.7±4.0

72.3±16.7

A0A117E2F4

β-glucosidase

GH3

17±12.7

33±7.1

22.3±4.6

32.7±4.0

22±3.5

42.3±6.4

A0A100I7V3

β-glucosidase

GH31

11.7±5.8

36.3±9.8

9.3±6.4

34±12.1

A0A117E1I2

β-glucosidase

GH1

31±1.7

28.3±8.1

40.3±11.6

A0A124BXC2

β-glucosidase

GH3

27.7±8.1

6±3.5

16±3.5

4.7±2.3

17.7±4.0

3.7±2.3

A0A100IJJ3

β-glucosidase

GH3

27.3±2.3

A0A117DXA6

β-glucosidase

GH1

1.7±0.6

A0A100IG02

β-glucosidase

GH3

8.3±1.2

A0A100IQ43

LPMO

AA9

7.3±2.9

2.3±0.6

A0A117E071

LPMO

AA9

3.3±0.6

7.7±2.3

A0A100IFW8

LPMO

AA9

2±1.7

5.3±1.2

A0A100IKJ9

LPMO

AA9

4.3±1.2

2.7±0.6

ACS Paragon Plus Environment

4.5±0.7

3±0

4±1.4

2±0

8±0

4±1.7

Journal of Agricultural and Food Chemistry

Page 36 of 38

Table 3. Proteases secreted by Aspergillus niger An-76 on different substrates. Types Asparitic endopeptidase

Accession number

Enzyme activity

Family

SD

SH

CB

WB

WBSH

WBSD

A0A100IK58

Asparitic protease

A1

143.3±14.3

290±62.9

142±6.9

134±1.7

192±3.5

163.7±5.8

A0A117E4L6

Asparitic protease

A1

14.3±5.0

20.7±8.7

8±1.7

5.3±0.6

18±3.5

16. 7±4.0

A0A100IER6

Asparitic protease

A1

3±0

1.3±0.6

A0A117E3G6

Asparitic protease

A1

2.5±0.7

A0A100IKE2

Aspartyl protease

A4

2.5±0.7

A0A100IA04

Aspartyl protease

A1

2.5±0.7

A0A117DXA4

Serine endopeptidase

S8

8±6.1

19±8.5

16.7±2.3

7.7±4.0

8±1.7

8±3.5

A0A100II79

Serine endopeptidase

S8

9.7±2.9

16±1.7

25±8.7

7±0

10±1.7

12.3±2.3

4.7±1.2

4.7±0.6

4±0

Endopeptidase Serine endopeptidase

A0A100IE06

Serine endopeptidase

S8

11.7±2.3

Cysteine endopeptidase

A0A117DUE3

Cysteine endopeptidase

C1

6.3±2.9

Metallopeptidase

A0A100INJ6

Metallopeptidase

M1

7.3±2.9

A0A100IEJ2

Metallopeptidase

M3

30±5.2

A0A117DXE7

Lysine aminopeptidase

M1

50.7±1.2

57.7±12.7

63±10.9

A0A117DVZ2

Aminopeptidase

M28

18.7±4.6

15.3±2.9

15.7±2.3

A0A117E4Q1

Aspartyl aminopeptidase

M18

36.7±9.2

A0A100IN20

Aspartyl aminopeptidase

M18

26.3±0.6

3.7±0.6

15.3±2.9

A0A100IAD1

Aminopeptidase

S9

19.3±4.6

10±5.2

7±3.5

A0A100I9B5

Aminopeptidase

S9

3.7±0.6

3±0

1.7±0.6

Aminopeptidase

Exopeptidase Carboxypeptidase

2.5±2.1

8.3±3.2

14±1.7

15.3±8.1

A0A100I4P6

Serine carboxypeptidase

S10

23.3±8.7

36±12.8

44.3±6.4

20.3±1.2

17±3.5

17.7±4.0

A0A100INL4

Serine carboxypeptidase

S10

12.7±5.5

25.3±7.6

28.3±8.1

18±3.6

16±3.5

20.7±5.8

A0A100ID38

Serine carboxypeptidase

S10

7.3±3.5

22.3±4.0

31.3±6.4

8.7±2.3

6±1.7

8.3±0.6

A0A100IPA5

Serine carboxypeptidase

S28

7±5.3

20.7±6.7

12.7±0.6

5.7±0.6

6.3±1.2

7.3±1.2

A0A100ILU3

Serine carboxypeptidase

S10

9±4.2

6.3±2.5

10.3±2.9

12.7±2.3

9.7±2.3

13.7±4.0

ACS Paragon Plus Environment

Page 37 of 38

Journal of Agricultural and Food Chemistry

Oligopeptidase

A0A100IQC7

Serine carboxypeptidase

S10

7±4.2

A0A100ITF4

Serine carboxypeptidase

S10

11±9.9

A0A117E0U5

Serine carboxypeptidase

S28

A0A100IU70

Serine carboxypeptidase

S10

24.3±1.2

6.3±1.2

2.7±0.6

A0A100IRN1

Carboxypeptidase

M20

21.3±0.6

13.7±7.5

19.3±8.1

A0A100IPJ6

Depetidyl-peptidase

S9

9±1.7

3.3±1.2

24±1.7

A0A100IAX5

Depetidyl-peptidase

29±1.7

5±0

32.7±7.5

A0A117DWA5

Depetidyl-peptidase

M19

14.7±2.3

3.3±1.2

8.3±2.9

A0A100IML6

Depetidyl-peptidase

S9

13.3±2.9

2±0

5±0

A0A124BXM8

Tripeptidyl-peptidase

S8

10.7±4.7

21±3.5

3.7±0.6

33.7±7.5

37.7±11.0

23±3.5

A0A117E271

Tripeptidyl-peptidase

S8

6.3±1.2

20±5.6

19±6.9

9±1.7

15.3±6.4

12.3±2.9

5±2.8

30.3±7.0

ACS Paragon Plus Environment

4.3±3.1

19.7±4.2

4±0

4±0

15.7±5.8

4.3±2.9

2.67±0.6

2.7±0.6

2±1.7

5±0

2±0

2±0

5±1.7

Journal of Agricultural and Food Chemistry

Graphic for Table of Contents

ACS Paragon Plus Environment

Page 38 of 38