Extracellular Enzyme Composition and Functional Characteristics of

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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

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Journal of Agricultural and Food Chemistry

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Extracellular Enzyme Composition and Functional Characteristics of Aspergillus niger

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

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

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1

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

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2

College of Marine Science, Shandong University, Weihai, China

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*Corresponding Author: Professor Lushan Wang

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Address:

State Key Laboratory of Microbial Technology

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Shandong University

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27 Shandanan Road

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Jinan

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250100

13

China

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Tel: +86-531-88366202

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Fax: +86-531-88565610

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E-mail: [email protected]

ACS Paragon Plus Environment


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

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

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technology to analyze composition and functional characteristics of extracellular enzymes

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

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

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

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

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

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

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

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

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

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

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glycoside hydrolases; protease


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INTRODUCTION

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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hemicellulose-degrading enzymes and proteases6. Previous transcriptome studies revealed



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

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

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

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

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extracellular enzymes is needed8. Proteomes can provide comprehensive information

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

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

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

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enabling more efficient enzyme production10.

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

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

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

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

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

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

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

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

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

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MATERIALS AND METHODS

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

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

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stored in a sealed container at 4°C.


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

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

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

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

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

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

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

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

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

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

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Determination of concentration extracellular reducing sugar and activity of proteins.

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

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

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

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

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

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

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

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

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concentration had little effect on the measurement of protein concentration.

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

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



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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Sodium

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Electrophoresis was performed on a miniaturized vertical gel system using a

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

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

dodecyl

sulfate

polyacrylamide

gel


electrophoresis

(SDS-PAGE).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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



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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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sample.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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50°C for 24 h.

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

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

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

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



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determine the content of released soluble carbohydrates.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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RESULTS

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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induced by other substrates (Figure S2).

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Dynamic changes in oligosaccharides released during hydrolytic processing. To

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

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

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

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

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

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

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



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

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

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

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polysaccharides into large quantities of monosaccharides and oligosaccharides within 24 h

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

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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,

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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

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

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

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

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

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Analysis of the chemical composition and enzymatic hydrolysates of various

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

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

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

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

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

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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


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

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

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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

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

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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

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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



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.

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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


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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



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


Page 20 of 38

Page 21 of 38


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



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


Page 22 of 38

Page 23 of 38


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).



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.


Page 24 of 38

Page 25 of 38


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



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


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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.



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.


Page 28 of 38

Page 29 of 38


Figure 1



Figure 2


Page 30 of 38

Page 31 of 38


Figure 3



Figure 4


Page 32 of 38

Page 33 of 38


Figure 5



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


GH10

19±9.9

46.5±4.9

53.7±11.0

159.7±20.2

136±24.3

110.3±16.7

A0A100ITA6


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


GH11

A0A117E086


GH43

11.3±2.9

2.3±0.6

9.7±2.3

A0A117E0N6


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


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


GH51

13±1.4

12±1.4

1.7±0.6

4±0

6±3.5

2.7±0.6

A0A100IHT3


GH43

3±1.4

4.5±3.5

4±1.7

8±1.7

7.3±2.9

4.7±0.6

A0A100III9


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


4±1.7

Page 35 of 38


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


GH7

20±14.1

18±5.7

8±1.4

31.33±2.9

17±3.5

76.7±21.4

A0A100IHS6


GH6

4±1.7

9±0

4±1.7

14.3±2.9

11.3±1.2

22.3±8.1

A0A100ISK1


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


GH5

24±9.9

78±11.3

16±1.7

50.3±1.2

46.7±2.3

105.3±30.6

A0A100ILF3


GH5

37±1.4

57±4.2

57.3±16.7

54±6.9

49±22.5

32.3±15.0

A0A117DXN2


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


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


4.5±0.7

3±0

4±1.4

2±0

8±0

4±1.7


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


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


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


S10

12.7±5.5

25.3±7.6

28.3±8.1

18±3.6

16±3.5

20.7±5.8

A0A100ID38


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


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


S10

9±4.2

6.3±2.5

10.3±2.9

12.7±2.3

9.7±2.3

13.7±4.0


Page 37 of 38


Oligopeptidase

A0A100IQC7


S10

7±4.2

A0A100ITF4


S10

11±9.9

A0A117E0U5


S28

A0A100IU70


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


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


Graphic for Table of Contents


Page 38 of 38

Extracellular Enzyme Composition and Functional Characteristics of

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