Xylo-oligosaccharides inhibit enzymatic hydrolysis by influencing

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Biofuels and Biomass

Xylo-oligosaccharides inhibit enzymatic hydrolysis by influencing enzymatic activity of cellulase from Penicillium oxalicum Can Wang, Xianqin Lu, Jia Gao, Xuezhi Li, and Jian Zhao Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.8b01424 • Publication Date (Web): 02 Aug 2018 Downloaded from http://pubs.acs.org on August 9, 2018

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Energy & Fuels

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Xylo-oligosaccharides inhibit enzymatic hydrolysis by influencing

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enzymatic activity of cellulase from Penicillium oxalicum

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Can Wang1, Xianqin Lu1, Jia Gao1, Xuezhi Li1，Jian Zhao1,2*

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1

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Road, Qingdao 266237, P.R. China

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2

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Recycling of Argo-Waste in Cold Region, College of Life Science and Technology,

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Heilongjiang Bayi Agricultural University, Daqing, 163319, China

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*Corresponding author.

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State Key Laboratory of Microbial Technology, Shandong University, No. 72 Binhai

Heilongjiang Provincial Key Laboratory of Environmental Microbiology and

E-mail address: [email protected] (J. Zhao).

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ACS Paragon Plus Environment

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Abstract

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Xylo-oligosaccharides, as the important intermediates of hemicellulose degradation, are

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widely existed in hydrolysate from pre-treatment and enzymatic hydrolysis of

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lignocellulose. This study showed that xylo-oligosaccharides largely reduced the

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efficiency of cellulose hydrolysis, but this inhibition cannot be effectively relieved by

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increasing loading of cellulose substrate or cellulase. Xylo-oligosaccharides largely

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suppressed

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β-Glucosidase of cellulase complex, especially cellobiohydrolases, and adding

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cellobiohydrolases or suitable β-Glucosidase to hydrolysis system could increase

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cellulose conversion. Thin-layer chromatography assay indicated xylotetraose was

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quickly degraded in initial hydrolysis stage while xylotriose was still held a higher

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concentration until 24 h of hydrolysis, speculated that xylotriose was stubborn inhibitor

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on enzymatic hydrolysis. The molecular docking analysis showed that xylotriose

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preferred to combine in the tunnel of cellobiohydrolases, which was generally

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recognized as the substrate-binding site on cellobiohydrolases, thus reduced

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effectiveness of cellobiohydrolases.

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Keywords: Xylo-oligosaccharides, Enzymatic hydrolysis, Inhibition, Mechanism,

40

Molecular docking

enzymatic

activities

of

cellobiohydrolases,

endoglucanases

and

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1. Introduction

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Lignocellulosic biomass, as one of the most abundant and available resources in the

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world,1 is the promising raw material for producing biofuel.2 Lignocellulose is a kind of


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heterogeneous complex which mainly consists of cellulose, hemicellulose and lignin.3

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In plant cell walls, hemicellulose is considered as a complex network of polysaccharides

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embedded in the matrix of lignin,4, 5 and forms a physical barrier to block the cellulose

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degradation by cellulase.6 Currently, the pretreatment has become a necessary procedure

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before enzyme hydrolysis of lignocelluloses to enhance enzymatic digestibility of the

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substrates.5 However, the prehydrolysate is inevitably produced during pretreatment and

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it has been found as the strong inhibitor to enzymatic hydrolysis7,

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fermentation.8, 9 This inhibition could be reduced by separating the prehydrolysate and

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washing pretreated solid materials prior to enzymatic hydrolysis,9, 10 but the separation

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and washing process led to an amount of fermentable sugars in the prehydrolysate waste

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and cost increase. From the viewpoint of economy, if the prehydrolysate was directly

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used to produce ethanol, the higher concentration of ethanol could be obtained as well

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as the cost of the filtration and the washing step could also be expelled.9

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It has been reported that the strong cellulase inhibitors existing in the prehydrolysate

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mainly consisted of gluco-oligosaccharides (GOS) and xylo-oligosaccharides (XOS) by

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using the HPLC and mass-spectroscopy analyses.5 Besides, some GOS and XOS are

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also produced during enzymatic hydrolysis of pretreated lignocelluloses because the

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cellulose and hemicellulose can be greatly reserved in the pretreated lignocelluloses

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material after pretreatments under neutral and alkali conditions.5, 11, 12, 13 In which, the

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inhibition of GOS on cellulase was mainly due to its predominant hydrolysis product

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cellobiose14 and this inhibitory effect could be reduced by the supplementation of

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β-glucosidase (BGL) in vitro.15 Some literatures have reported the certain


8

and ethanol

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hemicellulose-derived sugars, including sparingly-soluble xylan and soluble XOS, were

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also strong inhibitors of cellulase and negatively influenced enzyme hydrolysis.3, 5, 6, 9,

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16-19

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xylose or xylan, which reflected on the decreased initial hydrolysis rate and reduced

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final glucose yield in the hydrolysis of Avicel (microcrystalline cellulose) by a

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commercial Trichoderma reesei cellulase.6 Furthermore, cellulase (mainly including

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endoglucanase (EG) and cellobiohydrolase (CBH)) and β-glucosidase (BGL)

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to be more sensitive to the product inhibition during the hydrolysis of lignocelluloses.16,

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20, 21

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Thermoascus aurantiacus decreased from 0.78 mg/mL to 0.59 mg/mL while the amount

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of glucose degraded by EGII reduced slightly.3 No significant difference in glucose

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yield from cellobiose hydrolysis by BGL (Novozyme 188) was observed when XOS

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existed.6 Moreover, adding β-xylosidase in vitro could not effectively improve the

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conversion of Avicel.17 Deeply understanding the effects and mechanism of XOS

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inhibition on enzyme and hydrolysis process are vital to optimize the cellulolytic

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enzyme system and improve the efficiency of lignocellulose conversion. Current studies

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about the negative influence of XOS mainly focused on the effect of addition of xylan

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and various XOS on enzymatic hydrolysis of Avicel and activity of pure cellulase.6, 19

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Because of the existence of hemicellulase in crude cellulase used in enzymatic

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hydrolysis, the content of XOS (DP 2-6) in hydrolysis system was changed during

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enzymatic hydrolysis of lignocellulose. However, so far, the changing of XOS with

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different degree of polymerization (DP) during enzymatic hydrolysis and its additional

Qing et al. reported that XOS had a great impact on enzymatic hydrolysis than

appear

When XOS were added in vitro, the amount of cellobiose released by CBHI of


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impact on hydrolysis efficiency are not reported. To look for the strategy to

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reduce/eliminate the negative effect of XOS on enzyme hydrolysis of cellulose, it is

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very important to understand how the XOS change during enzymatic hydrolysis and

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how the XOS affect enzymatic hydrolysis.

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The inhibition of XOS on cellulase activity had been researched in literatures. Baumann

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et al. found that the affinity of XOS bound to CBH I (TrCel7A) increased with DP of

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XOS and was seemly not affected by CBM through the study of isothermal titration

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calorimetry.18 By kinetic experiments analysis of CBHI from Thermoascus aurantiacus

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with XOS existed, Zhang et al. considered that the xylobiose and xylotriose presented

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competitive inhibition on cellulase. The reason was presumably that xylobiose and

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xylotriose bound into the active site of CBHI for their structural similarity toward

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cellobiose, and thus formed a steric hindrance to impede the combination of cellulose

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and enzyme. Moreover, the inhibition effect of xylobiose on CBHI was stronger than

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that of xylotriose.3 Besides, Moneni et al. revealed that xylotriose, xylotetraose and

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xylopentaose preferred to bind at the tunnel entrance of enzyme by determining the

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complex structure of Hypocrea jecorina (the anamorph of T. reesei 22) Cel7A with XOS

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using X-ray crystallography. As no xylobiose was detected in crystal structure of

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HjeCel7A co-crystallized or soaked with xylobiose, clear electron density of a

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cellobiose molecule was determined to bind in the catalytic domain of HjeCel7A.

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Momeni et al. inferred that the performance of xylobiose inhibition on HjeCel7A was

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due to the contamination with small amounts of cellobiose. And xylobiose had no

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chance for competing the binding sites with cellobiose,16 which was contradicted with


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the conclusion drawn by Zhang et al.3 The inhibition of HjeCel7A by various XOS and

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birchwood xylan with p-nitrophenyl lactoside as substrate also showed that there was a

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mixed type of inhibition rather than a simple pure competitive inhibition.16 Therefore,

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the inhibition mechanism of XOS on cellulase is still uncertain and more information is

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needed to be clarified with the variant laws of different XOS components during

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enzymatic hydrolysis.

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Penicillium oxalicumis is widely considered as the potential strain for bioenergy

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application23 due to its diversity of cellulolytic enzyme system and highly efficiency of

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enzymatic hydrolysis.24, 25 The present study attempted to investigate the effect of XOS

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on enzymatic hydrolysis with the cellulase of P. oxalicum and on the different

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components (mainly CBHI, EGI and BGLI) of the cellulase system. Feasibility of

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weakening XOS inhibition on enzymatic hydrolysis was explored by adding specific

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cellulase components originated from P. oxalicum. Changing of XOS components

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during enzymatic hydrolysis and a possible hypothesis of inhibitory mechanism of

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XOS on cellulase was proposed by using molecular docking.

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2. Materials and Methods

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2.1. Materials

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Avicel was purchased from Sigma–Aldrich (St. Louis, MO, USA). Cellulase, which

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from P. oxalicum, used in the enzyme hydrolysis was provided by Sino Biotechnology

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Co., Ltd (Gansu, China).26 Its different enzymes activities were as following: FPA 150

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FPU/g, pNPCase activity 42.82 IU/g, CMCase activity 538.20 IU/g, pNPGase activity

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47.22 IU/g, xylanase activity 9785.35 IU/g and pNPXase 20.25 IU/g, which stand for


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the activities of filter paper, CBH, EG, BGL, xylanase and β-xylosidase, respectively.

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XOS were provided by Longlive Bio-Technology Group Limited Company (Shandong,

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China). The standards of xylose (X1), xylobiose (X2), xylotriose (X3) and xylotetraose

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(X4) were purchased from Miragen (Shanghai, China). Pure cellobiohydrolases I

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(CBHI), endoglucanases I (EGI) and β-Glucosidase I (BGLI) were obtained by liquor

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fermentation with genetically modified P. oxalicum strains

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and purification using His Trap™ FF crude column and column superdex 100 (GE

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Healthcare, Sweden) according to the literature.27

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2.2. Enzymatic hydrolysis

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Enzyme hydrolysis was performed in 50 mmol/L acetate buffer (pH 4.8) in 50 mL

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erlenmeyer flask at 48± 0.3 oC for 72 h in a rotary shaker (QB-228). Avicel, cellulase

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and XOS were added to certain concentration depended on specific experiments

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conditions. For experiments of pure cellulase addition, different amounts of pure

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enzymes were firstly added into the enzyme complex, then used for enzymatic

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hydrolysis, in which the ratio of activities of original CBH or BG to the activities of

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additional CBH or BG were 5:1, 2:1 and 1:1, respectively. During enzymatic hydrolysis,

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the samples taken at specific time were centrifuged at 13,000 rpm for 10 min and the

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supernatant was boiled for 10 min, then stored at 4 oC for subsequent glucose

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determination and further experiments.

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2.3. Inhibition of pure cellulase

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XOS and different pure cellulase component (CBHI, EGI and BGLI) was mixed on the

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ice and then the mixture was used to measure different enzyme activities with


31

stored in our laboratory

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p-nitrophenyl-β-D-cellobioside (pNPC); sodium carboxymethy cellulose (CMC–Na);

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p-nitrophenyl-β-D-glucopyranoside (pNPG) as substrates, respectively. The controls

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were the mixture of equivalent buffer and pure cellulase, but without XOS. All

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experiments were performed in triplicate.

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2.4. Enzyme kinetic parameters

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Michaelis-Menten

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Lineweaver-Burk plot, and inhibition constants Ki were derived by Dixon plot.28

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2.5. TLC assay

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The silica gel 60 F254 plate (Millipore, Germany) used for TLC assay was purchased

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from Dingguo Corp. (Beijing, China).29 Aliquots of the samples (1 µL) were dotted on

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1.0 cm from the edge of the plate. The sugar mixtures were separated by the developing

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agent (containing butanol, isopropanol, acetic acid and water with a proportion of

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7:5:2:4)

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diphenylamine–aniline–phosphoric acid (DPA) reagent, after which, the plate was

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reacted at 110 oC for 10 min in the drying oven. In order to remove the disturbance of

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GOS, the samples analyzed by TLC assay were firstly treated by BGLI for 1 h and then

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boiled for 10min to terminate the reaction. Each sample was performed in triplicate in

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the same silicone plate. The photograph of chromogenic silica gel plate was detected

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and the gray value of each band on the plate was also given by Quantity One software.

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The relative abundance was the ratio of gray value of each band and that of the whole

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lane.30

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2.6. Molecular docking

for

kinetic

three

parameters

times,

(Km

and

and

Vmax)

then


were

detected

calculated

by

by

spaying

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Autodock was a kind of automated programs used for prediction of interaction between

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biomacromolecule and its ligand.31 This software was used here to analyze the binding

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sites and interactive amino acids of XOS and catalytic domain (CD) of CBH I from P.

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Oxalicum. The molecular structure of ligand was drawn by ChemDraw and then opened

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by Chem3D with the principle of energy minimization. And the 3D structure of CD of

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CBHI was predicted on SWISS-MODEL (https://swissmodel.expasy.org/) by homology

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modeling. Then the molecular docking was carried out by Autoduck and the graph of

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protein-ligand combination was finally generated.

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2.7. Analysis methods

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2.7.1 Sugar content determination

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The sugar components of XOS used in experiments including xylose, xylobiose,

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xylotriose, xylotetraose, xylotetraose and xylopentaose, were determined by HPLC

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(Shimadzu, Kyoto, Japan) with a refractive index detector (Shimadzu) on an Aminex

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HPX-42A column (Bio-Rad, Hercules, CA,USA) by using the ultrapure water (UPW)

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as the mobile phase according to the method described by Du et al.26 The amount of

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glucose in hydrolysis liquor during enzymatic hydrolysis was measured by SBA-40C

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biological sensor analyzer (BISAS, Shandong, China).24 Cellulose conversion was

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calculated by using formula (1). The apparent inhibition degree (AID) of XOS on

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enzymatic hydrolysis was calculated according toformula (2).21

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Cellulose conversion (%) =

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(1)

Glucose concentrat ion (g/L) * reaction volume (L) * 0.9 * 100 cellulose content in substrate (g)


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Conversion with inhibitor at specific time ）* 100 (2) conversion without inhibitor at specific time

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Apparent inhibition degree (%) =（1 -

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2.7.2 Analysis of enzyme activity

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The enzyme activities of Filter Paperase (FPA), pNPCase, CMCase, pNPGase, xylanase

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and pNPXase were determined according to the literatures.24, 32 To be specific, filter

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paper activity (FPA) was determined in 50 mmol/L NaAc-HAc buffer of pH 4.8 at 50℃

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for 60 min and expressed in filter paper units (FPU). The activity measurements of CBH,

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EG, BGL, xylanase and β-xylosidase were conducted under the same condition above

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for 30 min with 1% of pNPC (with D-Glucono-δ-lactone as inhibitor), CMC-Na, pNPG,

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xylan (Sigma-Aldrich, St. Louis, MO, USA) and pNPX as substrates, respectively. One

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unit (IU) of enzyme activity was defined as the quantity of enzyme that produced 1µmol

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of reducing sugar per minute.

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3. Results and discussion

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3.1 Inhibition of xylo-oligosaccharides on enzymatic hydrolysis with cellulase from

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Penicillium oxalicum

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3.1.1 Inhibition of xylo-oligosaccharides on enzymatic hydrolysis

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Xylo-oligosaccharides (XOS) are the important intermediates of hemicellulose

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degradation and widely exist in the prehydrolysates of pretreatment of lignocellulosic

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materials.5, 6, 12 Since XOS are a mixture of oligoses with different DP, the components

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of the XOS preparation used in this study was firstly analyzed by HPLC and the result

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was exhibited on Table 1. It showed that the XOS preparation was composed of XOS

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with different DP of 1 to 6. In which, xylobiose, xylotriose and xyotetraose were the


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main components which hold a proportion up to 70% (weight %) of the XOS

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preparation, others were xylose, xylopentaose and xylohexaose. In order to investigate

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the effect of the XOS on enzymatic hydrolysis, various amounts of the XOS were added

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to enzymatic hydrolysis reaction system that contained Avicel of 2% (w/v) and cellulase

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of 5FPU/g substrate.

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As shown in Fig. 1a, it was obviously that cellulose conversion continued rising up but

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appeared a negative correlation with the XOS concentration during enzymatic

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hydrolysis. Even slight addition of XOS (1 mg/mL) could obviously reduce the

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conversion of cellulose by 15% at initial hydrolysis time (1 h) compared to the control

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(without XOS addition). When the XOS concentration reached 10 mg/mL, the cellulose

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conversion was decreased by 78% and 26% at the hydrolysis time of 1 h and 72 h

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respectively compared to the control. This strong impact on cellulosic hydrolysis after

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XOS addition was also reported in other literatures.3, 5, 6, 18, 19, 33

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A parameter called the apparent inhibition degree (AID) which represents the extent of

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inhibition of XOS on enzymatic hydrolysis at a certain point of time21 was calculated

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and shown on Fig. 1b. It was found that the AID of all the samples exhibited the same

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change rules along with the hydrolysis time, which mainly manifested as that AID

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decreased rapidly at first 24 h and then slowed down at 72 h of hydrolysis. According to

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the previous report, higher DP of XOS had the stronger affinity to enzyme.18 Thus, the

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decrease of AID with hydrolysis time increasing maybe due to the degradation of XOS

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during enzymatic hydrolysis as there was xylanase and β-xylosidase activities existed in

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the cellulase powder. After 24 h of hydrolysis time, the changeable of AID seemed to


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stay stable and could not be eliminated as hydrolysis time going on. Based on the result,

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the hydrolysis time of 24 h was used in following study of enzymatic hydrolysis. It was

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also found that, the higher concentration of XOS companied with the higher AID value,

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which indicated that the inhibitory effect of XOS on enzymatic hydrolysis became

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stronger at higher XOS concentration. In conclusion, XOS were exactly a strong

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inhibitor for enzymatic hydrolysis of cellulose with cellulase, especially in the initial

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stage of enzymatic hydrolysis. Similar phenomenon was also found by Qing et al. that,

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when XOS were at a higher concentration of 12.5 mg/mL, the XOS could dramatically

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decrease the hydrolysis rates of Avicel by 82% in initial 1 h of enzymatic hydrolysis,

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and reduce the final hydrolysis yield by 38% at 72 h of enzymatic hydrolysis compared

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to no XOS addition.6

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3.1.2 Effects of substrate concentration and cellulase dosage during enzymatic

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hydrolysis on XOS inhibition

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Montella et al. reported that increasing cellulose substrate concentration and cellulase

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dosage might relieve the inhibitory effect of pretreatment products.34 In this study,

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different concentrations of cellulose and cellulase were used in enzymatic hydrolysis of

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Avicel to investigate their influences on the inhibition of XOS during enzymatic

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hydrolysis. Fig. 2a and 2b showed that whether XOS were existed or not, increasing

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cellulose substrate concentration could both improve the yield of glucose, but drop

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down the conversion of cellulose. However, when XOS were added to reaction system,

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the glucose yield and conversions of cellulose to glucose were both decreased under

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every concentration of cellulose substrate when compared to the control (no XOS


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addition) (Fig. 2a and 2b). Fig 2c showed that when XOS dosage was 10 mg/mL, the

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AID value at hydrolysis time of 1 h were decreased from 84% to 21% when substrate

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concentration increased from 0.5% to 10% (w/v), then the AID value dropped down

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quickly along with the enzyme hydrolysis kept on-going except 10% of substrate

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concentration, meant that the predominance (low inhibition on enzymatic hydrolysis)

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caused by high cellulose concentration became no longer significant. Fig. 2c also

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showed that, at 24 h of hydrolysis time, the higher substrate concentration resulted in

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higher AID when substrate concentration was below 2%. It may be due to the larger

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amounts of glucose and cellobiose produced under condition of higher substrate

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concentration, as a result, led to the stronger production inhibition. Moreover, the AID

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values almost had no obvious change from 12 h to 24 h of hydrolysis when substrate

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concentration was up to 5%, which may due to the combined effect of degradation of

277

Avicel and XOS. Fig 2a showed that the glucose consistency was increased with time

278

prolonging. However, a reverse phenomenon was found when cellulose concentration

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reached 10%, in which, the AID value kept growing with hydrolysis time increasing, in

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detail, from 21% at the hydrolysis time of 1 h to 50% at the hydrolysis time of 24 h.

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This result may be due to the feedback inhibition caused by the accumulation of large

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amounts of hydrolysis products at high cellulose substrate concentration which covered

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the effect of XOS. Therefore, it could be inferred that increasing cellulose substrate

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concentration could reduce the inhibition caused by XOS at initial stage of enzymatic

285

hydrolysis, but this effect would soon became inconspicuous as the hydrolysis time

286

increasing.


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Similar change rules were also found by investigating the effect of cellulase dosage on

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remission of XOS inhibition. As shown in Fig. 3a and 3b, glucose yield and cellulose

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conversion continued to grow when cellulase concentration increasing during enzymatic

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hydrolysis. It could also be found that the glucose yield and conversions of cellulose to

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glucose were both decreased under all the cellulase dosage when XOS added (Fig. 3a

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and 3b). As for the inhibitory effect, slight reduction of AID from 90% to 63% was

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observed by raising cellulase dosage from 1 to 15 FPU/g at initial hydrolysis time (1 h)

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under the condition of 10 mg/mL XOS (Fig. 3c), but then this superiority caused by

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raising cellulase loading also could not last for a long time. When hydrolysis time

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reached 24 h, there was no obvious difference on AID value under different

297

concentrations of cellulase. Consequently, it could be concluded that the effect of XOS

298

inhibition could not be relieved efficiently by increasing whether cellulose substrate

299

concentration or cellulase dosage during enzymatic hydrolysis.

300

3.2 Changes of xylo-oligosaccharides during enzymatic hydrolysis

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To clarify the variation rules of different components of XOS during enzymatic

302

hydrolysis, we tried to use TLC (thin-layer chromatography) method for analyzing the

303

concentration of different sugars of samples taken from enzymatic hydrolysis at

304

different hydrolysis time and conditions. For demonstrating the feasibility of this

305

method, the mixed solutions of XOS containing xylose (X1), xylobiose (X2) xylotriose

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(X3) and xylotetraose (X4) with different concentration of 5, 4, 3, 2, 1, 0.5 mg/mL

307

(from left to right) respectively, were firstly analyzed by TLC. Fig. 4a showed that

308

different sugar components in the mixed XOS solution could be completely separated


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309

by TLC. And Fig. 4b showed that there was obvious linear correlation between each

310

XOS concentration and the gray values detected by Quantity One software, with the

311

coefficients (R2) of around 0.99. It indicated that the method of determining gray value

312

was feasible for analyzing the amounts of different sugars in hydrolysis samples, as

313

previously reported.30, 35

314

The products patterns and the change of relative abundance of each XOS component in

315

different samples during enzymatic hydrolysis were shown in Fig. 5. It could be found

316

that all the sugar components of XOS in the samples held the similar changes during

317

enzymatic hydrolysis under different hydrolysis conditions. Specifically, under the

318

condition of 2% Avicel supplied with 5 FPU/g of cellulase, the relative content of X1

319

(xylose) was continually increased from 10% to 35% (Fig. 5e) while X3 (xylotriose)

320

was gradually hydrolyzed from 31% to 20% (Fig. 5g) during enzymatic hydrolysis of

321

24 h. Moreover, X2 (xylobiose) was quickly accumulated from 36% to 48% in the early

322

stages of hydrolysis (4 h), after that, its proportions stopped increasing and then slowly

323

reduced to 45% in the last 20 h (Fig. 5f). X4 (xylotetraose) was almost completely

324

degraded within the initial 4 h. During the hydrolysis time of 24 h, the relative

325

proportions of X1, X2, X3 and X4 in total sugar was changed from 10%, 36%, 31% and

326

23% (at the hydrolysis time of 0 h) to 35%, 45%, 20% and 0% (at the hydrolysis time of

327

24 h), respectively. Increasing Avicel loadings from 2% to 5% or adding 0.15 IU/g of

328

CBHI to hydrolysis system had slight effect on the variation laws of XOS components

329

during enzymatic hydrolysis. For example, compared to the control (2% Avicel loading

330

with 5 FPU/g of celluase), X1 was just increased by 3.8% and 5.0% while X3 was


Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

331

decreased by 2.9% and 11% after hydrolysis of 24 h under the conditions of 5% Avicel

332

loading and addition of 0.15 IU/g of CBHI, respectively. Although the rates of

333

accumulation and consumption of X2 became slight rapid in initial stage of enzymatic

334

hydrolysis by increasing Avicel and CBHI loadings, its final content was close to that at

335

the hydrolysis of 24 h. Fig. 5h showed that the varying patterns of X4 did not seem to

336

be effected by increasing cellulose substrate concentration or cellulase dosage as well as

337

adding extra CBHI. By increasing cellulase dosage from 5 FPU/g to 10 FPU/g, the

338

relative content of X1 was raised from 35% to 42% while X2 and X3 were reduced

339

from 45% and 20% to 43% and 14% respectively after 24 h of hydrolysis. However, X4

340

was decreased by 85% within the initial hydrolysis of 1 h. It meant that XOS with high

341

DP was more efficiently hydrolyzed and XOS with low DP was rapidly accumulated by

342

increasing cellulase supplement.

343

However, comparing the change laws of XOS components with that of AID during

344

enzymatic hydrolysis (Fig. 5 and Fig. 1b), it could be inferred that X1 and X2 could not

345

be the strong inhibitor for the enzymatic hydrolysis. As X1 content continually

346

enhanced during enzymatic hydrolysis, and the X2 concentration always held a high

347

level and almost unchanged within the range of 4 h to 24 h of hydrolysis, while AID

348

values continually decreased (inhibition degree lower) with hydrolysis time increase.

349

Momeni et al. also pointed out that X2 was not a stronger inhibitor as it showed no

350

advantage when competing the binding sites with cellobiose.16 The accumulation of X2

351

may be caused by the deficiency of β-xylosidase in P. oxalicum cellulase complex.

352

When comparison the change trends of X3 and X4 in XOS (Fig. 5g and 5h) with that of


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353

AID (Fig. 2c, 3c and Table 3), however, it could be assumed that the decrease in

354

contents of the XOS with high DP such as X3 and X4 contributed to the decrease of

355

AID value during enzymatic hydrolysis, especially X3, should be a strong inhibitor on

356

enzymatic hydrolysis as X4 (or XOS with higher DP) was rapidly depolymerized by

357

xylanase in cellulase complex, thus its adverse effect on enzymatic hydrolysis could be

358

usually disappeared within a few hours (for example, 4 h in Fig. 5h) in the beginning

359

stage of enzymatic hydrolysis. Increasing cellulose substrate concentration in reaction

360

system helped to the combination of cellulase to substrate, thus enhanced XOS with

361

high DP degradation to some extent in initial stage of hydrolysis, but resulted in the

362

accumulation of the XOS capable of strong inhibition such as X3, so the AID decreased

363

in initial hydrolysis stage then increased with the enzymatic hydrolysis time extending

364

(Fig. 2c and 3c). Increasing cellulase concentration or adding extra CBHI in reaction

365

system may accelerate the degradation process of the strong inhibitor (XOS with high

366

DP) or partly compensate for the loss of enzymatic activity due to XOS inhibition, as a

367

result, the AID could be alleviated (Fig. 2c, 3c and Table 3).

368

3.3 Effect of xylo-oligosaccharides on enzymatic activity of different components of

369

cellulase from Penicillium oxalicum

370

3.3.1 Effect on enzymatic activities and possible mechanism

371

The main components of cellulase are three groups of enzymes including

372

cellobiohydrolase (CBH), endoglucanase (EG) and β-Glucosidase (BGL).20 In this study,

373

the pure enzymes of CBHI, EGI and BGLI were used to investigate the effect of XOS

374

on their enzymatic activity. It was found that XOS strongly inhibited the activity of all


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Page 18 of 42

375

the pure enzymes, especially CBHI (Fig. 6a). For example, compared to the control

376

(without XOS addition), the activities of CBHI, EGI and BGLI were decreased by 61%,

377

23% and 40% respectively when XOS concentration reached 5 mg/mL. Previous studies

378

also reported that CBH was more susceptible to XOS

379

CBH activity was stronger than that on EG activity.5 Qing et al. reported that there was

380

no obvious difference in BGL activity by evaluating the glucose yield from cellobiose

381

hydrolysis no matter XOS existed or not.6 Fig. 6c showed that, however, the BGLI

382

activity was inhibited by XOS and dropped sharply along with the XOS concentration

383

increase in the range of 0~ 20 mg/mL, which may be owing to the difference in BGL

384

properties and substrate species. CBHI and BGLI almost lost all activities when XOS

385

concentration reached to 100 mg/mL. When the XOS concentration was below 10

386

mg/mL, in general, the decrease on enzyme activities resulted by XOS was CBHI >

387

BGLI > EGI. Because of the limitation of hemicellulose content in lignocellulose, the

388

XOS concentration could hardly reach the value (10 mg/mL) in reaction system during

389

enzymatic hydrolysis,34 thus the inhibition of XOS on enzymatic hydrolysis of

390

lignocellulosic may mainly due to the loss of CBH activity.

391

Enzymatic kinetic parameters (Table 2) represented that the both Michaelis–Menten

392

constants Km and Vmax were affected by adding XOS. Specifically, with XOS addition,

393

the values of Km were increased while the values of Vmax were reduced compared to no

394

XOS addition, which indicated that the XOS inhibition on CBHI or BGLI was a mixed

395

type of inhibition between competitive inhibition and non-competitive inhibition. The

396

original data of kinetic parameters were shown in Supporting Information.

2, 18, 19


and inhibition of XOS on

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Energy & Fuels

397

It was known by the above results that the activity of CBHI was strongly inhibited by

398

XOS, even at a very low consistency of XOS, and xylotriose was a strong inhibitor on

399

enzymatic hydrolysis. Zhang & Viikari observed that no obvious binding of XOS on

400

Avicel or wheat straw by incubating them for 2 h at room temperature and pH 5.0,35

401

indicated the inhibition caused by XOS on enzymatic hydrolysis of cellulose substrate

402

was not due to the effect of physical obstacle. In order to clarify the inhibition

403

mechanism of XOS on CBHI or enzymatic hydrolysis at a molecular level, the binding

404

mode of protein and ligand molecules was predicted by molecular docking. Xylotriose,

405

which was inferred as the most stubborn inhibitor according to above study, was chosen

406

to complete the docking into the catalytic domain (CD) of CBHI. The catalytic sites of

407

the CD of CBHI was located on a tunnel formed by β-pleated sheet and a loop.36 The

408

colorful spheres with color from blue to red (stand for binding energy from low to high),

409

as shown in Fig. 7a, represented the mass center of ten docking conformation based on

410

the principle of energy minimization. All the colorful balls were located in the tunnel of

411

CBH, predicted that the xylotriose had the preference to be bound at the tunnel of CD of

412

the CBH. The tunnel position in catalytic domain of CBH was generally considered as

413

the substrate-binding site represented previously by crystal structure of CBH of GH7,37

414

and also played an important role in efficient degradation of crystalline cellulose.38 In

415

the processive mechanism of enzyme action, the CBH was absorbed on the surface of

416

cellulose, disrupted the compact structure of the solid material, then guided the

417

single-carbohydrate chain threaded through the tunnel-liked cleft and finally released

418

the disaccharides at catalytic domain.37,

39-41

Thus, the nonproductive binding of


Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

419

xylotriose to the tunnel of CBHI would unavoidable hinder the combination of substrate

420

and CBH enzyme, which could be responsible for the loss of hydrolysis effectiveness.

421

Fig. 7b showed the optimal binding site of xylotriose on the CD of CBHI and the amino

422

acids which had interaction effect with the ligand. These amino acids mainly belonged

423

to hydrophobic amino acids (Trp and Tyr) and acidic amino acids (Asp and Glu). In

424

CBHI of T. reesei, four tryptophan (Trp) residues was distributed along the tunnel and

425

provided the hydrophobic interactions to participate in substrate bindingat the tunnel

426

entrance, tunnel center and around thecatalytic sites,42, 46 and the absence of them would

427

seriously impact the ligand binding and enzyme processivity.42, 44 Asp and Glu residues,

428

as the nucleophile in the tunnel, formed the enzyme-glycosyl intermediate and could

429

stable the interaction of enzyme and substrate.45 In addition, the Tyr residue preferred to

430

contact with its neighbouring residuein van der Waals and formed a link between

431

glucosyl residues, which had a role in promoting the processive actionof the enzyme.2, 46

432

In the catalytic domain of CBH I from P. oxalicum, Trp398, Glu241, Trp407 and

433

Asp238 which had interacted with xylotriose (Fig. 7b) were also found to participate in

434

the formation of glycosyl binding units of CBHI according to homology modeling.

435

Once the xylotriose molecule was nonproductively combined with the CBH and firstly

436

entered into the tunnel in catalytic domain of CBH, that is to say, the tunnel was blocked

437

by the xylotriose molecule, the cellulose chain could not be guided into the tunnel and

438

not to mention then catalyzed at the active sites of this CBH enzyme molecule, therefore,

439

influenced the effectiveness of the enzyme and enzymatic hydrolysis. This gave an

440

explanation about why xylotriose inhibited CBH activity and enzymatic hydrolysis of


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Energy & Fuels

441

cellulose.

442

3.3.2 Effect of adding pure cellulase components on XOS inhibition

443

As XOS were continually generated during lignocellulose degradation,9, 26 it was not a

444

proper method to remove the XOS inhibition by raising the substrate concentration. The

445

above results also showed that increasing substrate concentration in enzymatic

446

hydrolysis stage cannot effectively decrease the AID value. It had been reported that

447

adding extra cellulase could overcome the restriction directly caused by the combination

448

of inhibitor and enzyme.47 Since CBHI and BGLI were more susceptible to the

449

inhibition of XOS (Fig. 6a and 6c), pure cellulase components, CBHI and BGLI, were

450

supplied to Avicel hydrolysis with XOS to investigate the effect of adding pure cellulase

451

on XOS inhibition during enzymatic hydrolysis. It was found that, compared to the

452

control (5 FPU/g of cellulase), the glucose yield and cellulose conversion were

453

increased by adding CBH to hydrolysis system with and without XOS; and the increase

454

in glucose yield and cellulose conversion became more obvious with the increase in

455

pure CBH enzyme addition. When CBH of 0.15 IU/g substrate was added into the

456

hydrolysis system without XOS, the glucose yield and cellulose conversion were

457

increased by 50.6% and 50.5% compared to the control (5 FPU/g of cellulase), and just

458

a little lower than that at the cellulase dosage of 10 FPU/g. In the hydrolysis system

459

with XOS, adding CBHI could also significantly improve the cellulose conversion. For

460

instance, compared to the control (5 FPU/g of cellulase), only 0.075 IU/g of CBH

461

addition was able to increase conversion of cellulose from 8.3% to 12.4% at hydrolysis

462

time of 24 h, which was similar to the conversion of cellulose at cellulase dosage of 10


Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

463

FPU/g (13.0%). It was also found that the AID values decreased after adding extra CBH,

464

illustrated that addition of pure CBHI could reduce the inhibition caused by XOS in

465

hydrolysis of cellulose.

466

As also shown in Table 3, efficiency of enzymatic hydrolysis could also be enhanced by

467

adding BGLI in the hydrolysis system with and without XOS. For example, in the

468

hydrolysis system without and with XOS, supplying 0.08 IU/g of BGL could improve

469

the conversion of cellulose by 62.1% (from 11.1% to 18.0%) and 65.7% (from 8.3% to

470

13.8%) respectively at the cellulase dosage of 5 FPU/g at hydrolysis time of 24 h. In the

471

hydrolysis system with XOS existed, adding 0.16 IU/g of BGLI into the 5 FPU/g of

472

cellulase led to the cellulose conversion increase around 10.3% compared with the

473

cellulose conversion of 13.0% at 10 FPU/g of cellulase (from 13.0% to 14.3%) at the

474

hydrolysis time of 24 h. Adding lower amount of BGLI (0.032 IU of BGLI) could

475

decrease the AID value. However, the AID values cannot be continually reduced with

476

the amount of BGLI increased. It may be caused by the feedback inhibition of

477

production.

478

In general, the cellulose conversion was obviously improved via adding CBHI or BGLI

479

into hydrolysis system. In which, addition of CBHI could not only improve the glucose

480

yield and conversion of cellulose but also reduce AID value in enzymatic hydrolysis

481

with and without XOS. That is to say, decrease of XOS inhibition on enzymatic

482

hydrolysis by adding extra CBHI inferred that CBHI may be one of the key factors

483

which affected XOS inhibition during hydrolysis process.

484


Page 22 of 42

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Energy & Fuels

485

4. Conclusion

486

Xylo-oligosaccharides (XOS) had the strong inhibitory effect on enzymatic hydrolysis

487

and enzymatic activities of different components of cellulase complex, especially CBHI.

488

This inhibition effect of XOS could not be efficiently relieved by increasing cellulose or

489

cellulase loadings but can be reduced by adding CBHI and suitable amount BGLI

490

during enzymatic hydrolysis. Comprehensive analysis of the changes of AID and

491

different XOS components inferred that X3 was a stubborn inhibitor for enzymatic

492

hydrolysis. The binding mode of xylotriose to CBH showed that XOS could hamper the

493

combination of cellulose and CBH enzyme and result in the ineffectiveness of

494

enzymatic hydrolysis.

495 496

Abbreviations

497

XOS: xylo-oligosaccharides; AID: apparent inhibition degree; BGL: β-Glucosidase;

498

CBH: cellobiohydrolases; EG: endoglucanases; pNPC: p-nitrophenyl-β-D-cellobioside;

499

CMC–Na: sodium carboxymethy cellulose; pNPG: p-nitrophenyl-β-D-glucopyranoside;

500

pNPX: p-nitrophenyl-β-D-xylopyranoside; DP: degree of polymerization; FPU: filter

501

paper units; TLC: Thin-layer chromatography; CD: catalysis domain; HPLC: high

502

performanceliquidchromatographic; X1: xylose; X2: xylobiose; X3: xylotriose; X4:

503

xylotetraose.

504 505

Acknowledgments

506

This study was supported by the National Natural Science Foundation of China (Grant


Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

507

No. 21376141), and Heilongjiang Provincial Key Laboratory of Environmental

508

Microbiology and Recycling of Argo-Waste in Cold Region, Heilongjiang Bayi

509

Agricultural University, China (No.201715 ).

510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546

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(32) Tan, L.; Sun, W.; Li, X.; Zhao, J.; Qu, Y.; Choo, Y. M.; Loh, S. K., Bisulfite pretreatment changes the structure and properties of oil palm empty fruit bunch to improve enzymatic hydrolysis and bioethanol production. Biotechnology journal 2015, 10, (6), 915-25. (33) Kim, T. H.; Lee, Y. Y.; Sunwoo, C.; Kim, J. S., Pretreatment of corn stover by low-liquid ammonia recycle percolation process. Applied Biochemistry and Biotechnology 2006, 133, (1), 41-57. (34) Montella, S.; Balan, V.; da Costa Sousa, L.; Gunawan, C.; Giacobbe, S.; Pepe, O.; Faraco, V., Saccharification of newspaper waste after ammonia fiber expansion or extractive ammonia. AMB Express 2016, 6, (1), 18. (35) 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), 910-7. (36) Divne, C.; Stahlberg, J.; Reinikainen, T.; Ruohonen, L.; Pettersson, G.; Knowles, J.; Teeri, T.; Jones, T., The three-dimensional crystal structure of the catalytic core of cellobiohydrolase I from Trichoderma reesei. Science 1994, 265, (5171), 524-528. (37) Horn, S. J.; Sikorski, P.; Cederkvist, J. B.; Vaaje-Kolstad, G.; Sorlie, M.; Synstad, B.; Vriend, G.; Varum, K. M.; Eijsink, V. G., Costs and benefits of processivity in enzymatic degradation of recalcitrant polysaccharides. Proc Natl Acad Sci U S A 2006, 103, (48), 18089-94. (38) Borisova, A. S.; Eneyskaya, E. V.; Bobrov, K. S.; Jana, S.; Logachev, A.; Polev, D. E.; Lapidus, A. L.; Ibatullin, F. M.; Saleem, U.; Sandgren, M.; Payne, C. M.; Kulminskaya, A. A.; Stahlberg, J., Sequencing, biochemical characterization, crystal structure and molecular dynamics of cellobiohydrolase Cel7A from Geotrichum candidum 3C. The FEBS journal 2015, 282, (23), 4515-37. (39) Kari, J.; Kont, R.; Borch, K.; Buskov, S.; Olsen, J. P.; Cruyz-Bagger, N.; Väljamäe, P.; Westh, P., Anomeric Selectivity and Product Profile of a Processive Cellulase. Biochemistry 2017, 56, (1), 167-178. (40) Colussi, F.; Sorensen, T. H.; Alasepp, K.; Kari, J.; Cruys-Bagger, N.; Windahl, M. S.; Olsen, J. P.; Borch, K.; Westh, P., Probing substrate interactions in the active tunnel of a catalytically deficient cellobiohydrolase (Cel7). J Biol Chem 2015, 290, (4), 2444-54. (41) Sørensen, T. H.; Windahl, M. S.; McBrayer, B.; Kari, J.; Olsen, J. P.; Borch, K.; Westh, P., Loop variants of the thermophile Rasamsonia emersonii Cel7A with improved activity against cellulose. Biotechnology and bioengineering 2017, 114, (1), 53-62. (42) Taylor, C. B.; Payne, C. M.; Himmel, M. E.; Crowley, M. F.; McCabe, C.; Beckham, G. T., Binding site dynamics and aromatic-carbohydrate interactions in processive and non-processive family 7 glycoside hydrolases. The journal of physical chemistry. B 2013, 117, (17), 4924-33. (43) Nakamura, A.; Tsukada, T.; Auer, S.; Furuta, T.; Wada, M.; Koivula, A.; Igarashi, K.; Samejima, M., The tryptophan residue at the active site tunnel entrance of Trichoderma reesei cellobiohydrolase Cel7A is important for initiation of degradation of crystalline cellulose. J Biol Chem 2013, 288, (19), 13503-10. (44) Igarashi, K.; Koivula, A.; Wada, M.; Kimura, S.; Penttila, M.; Samejima, M., High-speed atomic force microscopy visualizes processive movement of Trichoderma reesei cellobiohydrolase I on crystalline cellulose. Journal of Biological Chemistry 2009. (45) Ghattyvenkatakrishna, P. K.; Alekozai, E. M.; Beckham, G. T.; Schulz, R.; Crowley, M. F.; Uberbacher, E. C.; Cheng, X., Initial recognition of a cellodextrin chain in the cellulose-binding tunnel may affect cellobiohydrolase directional specificity. Biophysical journal 2013, 104, (4), 904-12. (46) von Ossowski, I.; Ståhlberg, J.; Koivula, A.; Piens, K.; Becker, D.; Boer, H.; Harle, R.; Harris, M.; Divne, C.; Mahdi, S.; Zhao, Y.; Driguez, H.; Claeyssens, M.; Sinnott, M. L.; Teeri, T. T., Engineering the Exo-loop of


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Trichoderma reesei Cellobiohydrolase, Cel7A. A comparison with Phanerochaete chrysosporium Cel7D. Journal of Molecular Biology 2003, 333, (4), 817-829. (47) Kumar, L.; Arantes, V.; Chandra R.; Saddler J., The lignin present in steam pretreated softwood binds enzymes and limits cellulose accessibility. Bioresource technology 2012, 103, (1), 201-208.


Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

637 638

Figure captions

639

Fig.1. Effect of XOS concentration on cellulose conversion (a) and the apparent

640

inhibition degree (b) during 72 h of enzymatic hydrolysis. In which, cellulose and

641

cellulase loading was 2% (W/V) and 5 FPU/g substrate, respectively.

642

Fig.2. Effects of cellulose substrate concentration on glucose yield (a), cellulose

643

conversion (b), and the apparent inhibition degree (c) during 24 h of enzymatic

644

hydrolysis. In which, XOS concentration was 10 mg/mL, and cellulase dosage was 5

645

FPU/g substrate.

646

Fig.3. Effects of celulase dosage on glucose yield (a), cellulose conversion (b), and the

647

apparent inhibition degree (c) during 24 h of enzymatic hydrolysis. In which, XOS

648

concentration was 10 mg/mL, and cellulose substrate concentration was 2% (W/V).

649

Fig.4. Quantity determination of XOS standards by TLC assay. (a) TLC profile of

650

different concentration (mg/mL) of mixed XOS standards that consisted with xylose

651

(X1), xylobiose (X2), xylotriose (X3) and xylotetraose (X4), in which, the ratio of X1,

652

X2 , X3 and X4 was 1:1:1:1. (b) Correlation graph of concentrations of different

653

components of the mixed XOS standards and gray value corresponding to its band

654

assayed by TLC.

655

Fig.5. Hydrolysis products patterns (a, b, c, d) and changes in relative abundance of

656

bands of XOS with different DP (d, e, f, g) during enzymatic hydrolysis of Avicel under

657

different conditions by TLC. In which, (a) 2% Avicel + cellulase of 5 FPU/g as the

658

control, (b) 5% Avicel + cellulase of 5 FPU/g, (c) 2% Avicel + cellulase of 10FPU/g and

659

(d) 2% Avicel + cellulase of 5FPU/g + 0.15 IU CBHI. The XOS addition was 10 mg/mL.


Page 28 of 42

Page 29 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

660

And the band between xylose and xylobiose was glucose. (e), (f), (g) and (h) described

661

the change of xylose, xylobiose, xylotriose and xylotetraose, respectively.

662

Fig.6. Effects of XOS concentration on the activity of CBHI (a), EGI (b) and BGLI (c)

663

with pNPC, CMC and pNPG as substrate, respectively. The concentration of substrates

664

were 1%.

665

Fig.7. The pattern of molecular docking of P. oxalicum CBHI with xylotriose. (a)

666

Overall structure of combination between CBHI and xylotriose. The backbone of CD of

667

CBHI was shown as gray ribbon. The multi-colored spheres represented the mass center

668

of each docking conformation, and its color from blue to red signified the energy of

669

interaction from low to high. (b) The amino acids which had interacted with xylotriose

670

in the CD of CBHI. The best-ranked docking pose of xylotriose was shown as stick

671

model in purple.


Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

672

Tables

673

Table 1 Concentration of sugar components in XOSa

Name

674 675 676

Relative Concentration percentage (%，， (mg/mL) on total XOS)

xylose 1.10± 0.05 8.47b xylobiose 3.11± 0.65 23.96 xylotriose 3.66± 0.40 28.19 xylotetraose 2.28± 0.13 17.57% xylopentaose 1.50± 0.05 11.56 xylohexaose 1.33± 0.08 10.25 a XOS concentration was 10 mg/mL. b Represented by mass %.

677 678 679 680 681 682 683 684 685 686 687 688 689 690


Page 30 of 42

Page 31 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

691

Table 2 Enzymatic kinetic parameters of XOS inhibition on cellulase KM (mg/mL)a

692 693

Vmax (× ×10-6 M/ min)a

without XOS

with XOS

CBH

1.59

2.56±0.79

12.16

5.09±0.77

1.94±0.92

BGL

0.85

4.03±0.81

8.99

5.78±0.30

1.76±0.21

a b

without XOS

Ki (mg/mL)b

Calculated by Lineweaver-Burkplot. Derived by Dixon plot.

694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710


with XOS

Energy & Fuels 1 2 3 711 Table 3 Effects of different pure enzyme addition on XOSa inhibition 4 5 BGLd (IU/g ) CBHd (IU/g ) 10 FPU/g 6 Controlc Enzyme dosage cellulase 7 0.029 0.075 0.15 0.032 0.08 8 without 9 2.7±0.0 3.2±0.0 3.7±0.0 3.5±0.1 4.0±0.0 2.5± 0.0 3.9± 0.0 Glucose XOS b 10 yield 11 2.3±0.0 2.8±0.0 3.3±0.0 2.8±0.0 3.1±0.0 12 (mg/mL) with XOS 1.9± 0.1 2.9± 0.1 13 14 Cellulose without 11.1± 0.1 17.6± 0.1 12.1±0.0 14.3±0.2 16.7±0.2 15.8±0.4 18.0±0.2 XOS 15 conversion 16 (%) with XOS 8.3± 0.5 13.0± 0.4 10.4±0.0 12.4±0.0 14.8±0.2 12.7±0.0 13.8±0.0 17 18 19 Apparent inhibition 20 25.3 26.2 14.5 12.9 11.8 19.4 23.7 21 degree (%) 22 23 a 712 XOS concentration was 10 mg/mL 24 b 25 713 Glucose yield and cellulose conversion was determined at 24 h of hydrolysis time. 26 714 Enzymatic hydrolysis was performed at 48± 0.3 oC with 2% substrate loading 27 c 715 Control meant 5 FPU/g cellulase addition 28 d 716 These enzyme supplements were based on the control (5 FPU/g cellulase ) 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 ACS Paragon Plus Environment 60

Page 32 of 42

0.16 4.3±0.1 3.2±0.1 19.3±0.3 14.3±0.4

26.1

Page 33 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

717

Figures

718

Fig.1. Effect of XOS concentration on cellulose conversion (a) and the apparent

719

inhibition degree (b) during 72 h of enzymatic hydrolysis. In which, cellulose and

720

cellulase loading was 2% (W/V) and 5 FPU/g substrate, respectively.

721


Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

722

Fig.2. Effects of cellulose substrate concentration on glucose yield (a), cellulose

723

conversion (b), and the apparent inhibition degree (c) during 24 h of enzymatic

724

hydrolysis. In which, XOS concentration was 10 mg/mL, and cellulase dosage was 5

725

FPU/g substrate. 726


Page 34 of 42

Page 35 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

727

Fig.3. Effects of celulase dosage on glucose yield (a), cellulose conversion (b), and the

728

apparent inhibition degree (c) during 24 h of enzymatic hydrolysis. In which, XOS

729

concentration was 10 mg/mL, and cellulose substrate concentration was 2% (W/V).

730 731


Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

732

Fig.4. Quantity determination of XOS standards by TLC assay. (a) TLC profile of

733

different concentration (mg/mL) of mixed XOS standards that consisted with xylose

734

(X1), xylobiose (X2), xylotriose (X3) and xylotetraose (X4), in which, the ratio of X1,

735

X2 , X3 and X4 was 1:1:1:1. (b) Correlation graph of concentrations of different

736

components of the mixed XOS standards and gray value corresponding to its band

737

assayed by TLC.

738 739


Page 36 of 42

Page 37 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

740

Fig.5. Hydrolysis products patterns (a, b, c, d) and changes in relative abundance of

741

bands of XOS with different DP (d, e, f, g) during enzymatic hydrolysis of Avicel under

742

different conditions by TLC. In which, (a) 2% Avicel + cellulase of 5 FPU/g as the

743

control, (b) 5% Avicel + cellulase of 5 FPU/g, (c) 2% Avicel + cellulase of 10FPU/g and

744

(d) 2% Avicel + cellulase of 5FPU/g + 0.15 IU CBHI. The XOS addition was 10 mg/mL.

745

And the band between xylose and xylobiose was glucose. (e), (f), (g) and (h) described

746

the change of xylose, xylobiose, xylotriose and xylotetraose, respectively.

747


Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

748 749


Page 38 of 42

Page 39 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

750

Fig.6. Effects of XOS concentration on the activity of CBHI (a), EGI (b) and BGLI (c)

751

with pNPC, CMC and pNPG as substrate, respectively. The concentration of substrates

752

were 1%.

753


Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

754

Fig.7. The pattern of molecular docking of P. oxalicum CBHI with xylotriose. (a)

755

Overall structure of combination between CBHI and xylotriose. The backbone of CD of

756

CBHI was shown as gray ribbon. The multi-colored spheres represented the mass center

757

of each docking conformation, and its color from blue to red signified the energy of

758

interaction from low to high. (b) The amino acids which had interacted with xylotriose

759

in the CD of CBHI. The best-ranked docking pose of xylotriose was shown as stick

760

model in purple.

761 762


Page 40 of 42

Page 41 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

763

Supporting Information

764

Original data of kinetic parameters of enzyme

765

Additional file 1

766

Fig. S1 Measurement of kinetics parameter of CBHI (a) and BGLI (b) by

767

Lineweaver-Burk plot with pNPC, pNPG as substrates. In which, I 0, I 1, and I 2 stood

768

for inhibitor (XOS) concentration as 0, 10, 5 mg/mL, respectively. The regression

769

equations and variances (R2) corresponded to the trend lines were displayed on the

770

figures.

771 772


Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

773

Additional file 2

774

Fig. S2 Measurement of inhibitory constants of CBHI (a) and BGLI (b) by Dixon plot

775

with pNPC, pNPG as substrates. In which, S1, S2, and S3 stood for substrates (pNPC,

776

pNPG) concentration as 2, 5, 7 mg/mL, respectively. The regression equations and

777

variances (R2) corresponded to the trend lines were displayed on the figures.

778


Page 42 of 42

Xylo-oligosaccharides inhibit enzymatic hydrolysis by influencing

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