A ChiE1 from Coprinopsis cinerea is characterized as a processive

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A ChiE1 from Coprinopsis cinerea is characterized as a processive exochitinase and revealed to have a significant synergistic action with endochitinase ChiIII on chitin degradation Jiangsheng Zhou, Lingling Chen, Liqin Kang, Zhonghua Liu, Yang Bai, Yao Yang, and Sheng Yuan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04261 • Publication Date (Web): 08 Nov 2018 Downloaded from http://pubs.acs.org on November 16, 2018

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Journal of Agricultural and Food Chemistry

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A ChiE1 from Coprinopsis cinerea is characterized as a processive exochitinase and revealed

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to have a significant synergistic action with endochitinase ChiIII on chitin degradation

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Running Title: Chitinase from Coprinopsis cinerea

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Jiangsheng Zhou1*, Lingling Chen1*, Liqin Kang1, Zhonghua Liu1, Yang Bai1, Yao Yang2,

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Sheng Yuan1**

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1Jiangsu

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Engineering and Technology Research Center for Industrialization of Microbial Resources,

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College of Life Science, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210023, PR

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China.

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PR China.

2

Key Laboratory for Microbes and Microbial Functional Genomics, Jiangsu

Ginling College, Nanjing Normal University, 122 Ninghai Road, Nanjing, 210097,

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*

15

**

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Sheng Yuan

17

College of Life Science

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Nanjing Normal University

19

1 Wenyuan Rd

20

Xianlin University Park

21

Nanjing, 210023

22

PR China

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Tel: 86-25-85891067 (O)

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Fax: 86-25-85891067 (O)

Co-first author.

Corresponding author: [email protected]

1

ACS Paragon Plus Environment


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Abstract

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Fruiting bodies that exhibit strong autolysis of Coprinopsis cinerea are a good resource for the

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chitinolytic system. In this study, a new chitinase ChiE1 from C. cinerea was cloned,

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heterologously expressed and characterized. Biochemical analysis demonstrated that ChiE1 is an

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exochitinase with a processive mode of action. Although ChiE1 contains only a single catalytic

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domain without a binding domain, it can bind to and degrade insoluble chitin powder and colloidal

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chitin. The combination of ChiE1 and C. cinerea endochitinase ChiIII could increase the amount

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of reducing sugar released from chitin powder by approximately 120% compared to using ChiE1

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and ChiIII alone. The synergistic action of ChiE1 and ChiIII on degradation of chitin powder is

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higher than all previously reported synergism of chitinases. The recombinant chitinase ChiE1

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expressed in Pichia pastoris may be used as a synergistic chitinase for a reconstituted

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chitinolytic system for agricultural, biological, and environmental applications.

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Key words: Coprinopsis cinerea; chitinase; exochitinase; processivity; synergistic action

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INTRODUCTION

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Chitin is composed of β-1,4-linked N-acetyl-D-glucosamine and it is one of the most abundant

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biopolymers on earth. Not only is it one of main structural components of the fungal cell walls, but

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is also widely distributed in the exoskeletons of insects and crustaceans.1,2 Chitin from crustaceans

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is abundantly available and can be converted by chitinases into chito-oligosaccharides, which are

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reported to possess diverse biological activities including antitumor, antioxidant, antimicrobial

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action and other bioactivities.3

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Chitinases (EC 3.2.1.14) are hydrolytic enzymes which degrade chitin polymers by cleaving the

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1,4-β-glycosidic linkage. According to the classification of the carbohydrate active enzymes

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(www.cazy.org), fungal chitinases are included exclusively in glycoside hydrolases (GH) family

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18.4 Depending on the mode of action, fungal chitinases are divided to endochitinases which

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cleave the chitin polymer at random positions and exochitinases which degrade the chitin chain

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from the non-reducing end or the reducing end.5 According to the cleavage patterns, chitinases are

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classified as processive and non-processive chitinasess.1 The processivity of family 18 chitinases

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can be determined by analysis of the size distributions of enzyme-digested oligomers products of

54

one kind of water-soluble chitosan (fraction of N-acetylated units (FA) = 0.65).6,7 An N-acetyl

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group in the sugar residue bound to the -1 subsite is essential for catalysis by GH18 chitinases. If

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the glucosamine residue bound in the -1 subsite lacks an N-acetyl group, binding of chitosan

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would be non-productive, processive enzymes would remain loosely association with the substrate

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and proceed forward along the chitosan chain at every two sugar units untill the sugar with an

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N-acetyl group bound in the -1 subsite for formation of a productive complex. Thus, every

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chitooligosaccharide come from the same initial enzyme-substrate complex, except for the very

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first, would be even-numbered in length in the early stages of degradation. Non-processive

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chitinases dissociate from the substrate when the enzyme and substrate form a non-productively

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bound complex, resulting in a more random distribution of odd- and even-numbered products.

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According this measurement, exochitinases ChiA and ChiB from bacterium Serratia marcescens

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were suggested to be processive enzymes and endochitinase ChiC was a non-processive enzyme.6

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It should be noted that endo- and exo- activity each may or may not be accompanied by

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processivity.8 Prior to the family classification of the carbohydrate active enzymes, plant 3



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chitinases were divided into five classes, Class I , Class II, Class III, Class IV, and Class V, based

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on the amino acid sequence homology. Classes I, II, and IV chitinases are included in GH family

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19, while classes III and V chitinases belong to GH family 18.9 Therefore, GH 18 chitinases were

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also subdivided into classes III and V. The class III chitinases were also termed fungal/plant

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chitinases and class V were called fungal/bacterial chitinases due to their predominant occurrence

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in selected organisms. Class V chitinases have deep, tunnel-shaped substrate binding clefts and are

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exo-chitinases (corresponding to processive enzymes), and class III chitinases have shallow, open

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substrate binding clefts and show endo-acting activities (corresponding to non-processive

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enzymes).1 Fungal chitinases from sequenced fungal genomes can be divided phylogenectically

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into three different groups, Group A, Group B and Group C. Group A and C chitinases belong to

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class V chitinases, and group B belongs to class III chitinases.10 Group A chitinases have a

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catalytic domain without CBMs and possess a molecular mass of 40-50 kDa. Group B chitinases

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have a molecular mass of 30-90 kDa, contain frequently CBMs. Group C chitinases possess a

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molecular mass of 140-170 kDa, and contain CBM and distinctive LysM-motifs.1

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Autolysis of the fruiting body of C. cinerea shows a remarkable feature - the complete

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disintegration and liquefaction of the mature pileus in order to disperse basidiospores.11,12

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Disintegration and liquefaction of the mature pileus were considered to result from the hydrolysis

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of cell walls by a series of glycoside hydrolases, such as chitinases and glucanases.11,13-15 We

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previously reported that a class III endochitinase, ChiIII, from C. cinerea could degrade insoluble

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chitin powder.16 However, a class V exochitinase, called ChiB1,15 and a so-called endochitinase,

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ChiEn1,17 from C. cinerea could not degrade insoluble chitin power, in contrast to the

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exochitinases ChiA and ChiB, and endochitinase ChiC from the bacterium Serratia marcescens.6

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To be an efficient chitinolytic system for degradation of insoluble chitin, C. cinerea should

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contain other exochitinases for synergistic action with endochitinase ChiIII on insoluble chitin

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substrates. The phylogenetic relationship analysis of predicted eight chitinases in the genome of C.

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cinerea showed that a putative chitinase ChiE1 has the most homology to the exochitinse ChiB1

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from C. cinerea and two bacterial class V exochitinases, ChiA and ChiB from Serratia

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marcescens, forming a subgroup in the phylogenetic tree.16 And ChiE1 has been shown to be

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expressed during maturation of pilei.15 Therefore, it is necessary to heterologously expressed 4


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recombinant chitinase ChiE1 from C. cinerea to determine its enzyme features. This study

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characterized ChiE1 as an exochitinase with a processivity. ChiE1 can efficiently degrade

99

insoluble chitin powder and colloidal chitin, and exhibited a significant synergistic action with the

100

endochitinase ChiIII, implying a potential application value for degradation of chitin biomass..

101 102

MATERIALS AND METHODS

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Chemicals. Chitin powder from shrimp shells, 85% deacetylated chitosan, glycol chitosan,

104

laminarin, sodium carboxyl methyl cellulose (CMC-Na), and p-nitrophenyl (pNP) derivatives of

105

chitin oligosaccharides ((GlcNAc)1−3-pNP) were purchased from Sigma-Aldrich Co. LLC (USA).

106

N-acetylglucosamine, chitinbiose, chitintriose, chitintetraose, chitinpentaose, and chitinhexaose

107

were purchased from Elicityl Oligotech (France).

108

Colloidal chitin was prepared from chitin powder from shrimp shells using the Sandhya et al.

109

method.18 Highly acetylated, high molecular-weight, and water-soluble chitosan (FA = 0.65) was

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prepared from chitin powder using the Sannan et al. method.19 Glycol chitin was prepared from

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glycol chitosan using the Lee et al. method.20

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Strains, Plasmid, and Culture Conditions. C. cinerea (5026 + 5172) ATCC 56838 was

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purchased from American Type Culture Collection (USA). Fruiting bodies were cultivated

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according to Zhou et al. method.14 Pichia pastoris GS115 and the expression vector pPICZαA

115

used for expression of the recombinant chitinase ChiE1 were purchased from Invitrogen (USA).

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Sequence and Structure Analysis of ChiE1. The sequence of chitinase ChiE1 was obtained

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from the C. cinerea okayama 7 #130 genome in GenBank (accession no. EAU80760) at the

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National Center for Biotechnology Information (NCBI, USA). The conserved domains of ChiE1

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were analyzed through NCBI's conserved domain database.21 The signal peptide was determined

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using the SignalP 4.1 server (http://www.cbs.dtu.dk/services/SignalP) and TargetP 1.1 server

121

(http://www.cbs.dtu.dk/services/TargetP/). Amino acid sequence alignment of ChiE1 and ChiB1

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were performed using M-COFFEE (http://www.tcoffee.org). Sequence identity was analyzed

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using the DNAMAN software (version 7.212, Lynnon Corp., Quebec, Canada). The

124

three-dimensional model structure of ChiE1 was predicted using I-TASSER. The structure model

125

with the highest C-score among the five predicted models was used for further accuracy analysis 5



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and identified by TM-align (http://zhang.bioinformatics.ku.edu/I-TASSER/),22 and visualized by

127

Chimera.23

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Cloning, Expression, and Purification of ChiE1. The extraction of total RNA from the apical

129

stipes of C. cinerea fruiting bodies, cDNA synthesis from DNA-free RNA and PCR amplification

130

of

131

(AGAGAGGCTGAAGCTGAATTCCGTGTGCCCACTGAACCGTCTCC) and reverse primer

132

(TGTTCTAGAAAGCTGGCGGCCGCGGCATCGGGCATCCCCTGC)

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according to Niu et al.17 The PCR fragment of chiE1 and the plasmid pPICZαA digested with

134

EcoRI and NotI were ligated to generate the plasmid pPICZαAChiE1 using the ClonExpressTM

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II/One Step Cloning Kit (Vazyme, China). Transformation of pPICZαAChiE1, selection and

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cultivation of transformants with pPICZαAChiE1, and expression and purification of recombinant

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ChiE1 were conducted according to Niu et al.17 To determine the the chitinase activity of the

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culture medium, 200 μL of a reaction mixture containing 50 μL of supernatant culture medium of

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the recombinant chiE1 expression strain and 0.5% colloidal chitin in 50 mM NaAc-HAc (pH 5.0)

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was incubated at 37 °C and 800 rpm for 4 h, released reducing sugars from colloidal chitin were

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measured with DNS method as described in following hydrolysis activity assay. One unit of

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chitinase activity was defined as the volume of the culture medium containing enzyme that

143

liberates the reducing sugar corresponding to 1 μmol of N-acetylglucosamine per min.

chiE1

cDNA

with

the

forward

were

primer

conducted

144

Protein Analysis. Purified recombinant ChiE1 was analyzed using SDS-PAGE.24 The protein

145

concentration was determined by the Bradford method.25 The amino acid sequences of partial

146

peptides of the purified recombinant ChiE1 were analyzed by MALDI-TOF/TOF MS.14

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Hydrolysis Activity Assays. The hydrolysis activity of the recombinant chitinase ChiE1

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towards chitin or related polysaccharides was determined as described by Niu et al.,16 and the

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amount of N-acetylglucosamine or reducing sugars released from the substrates by chitinases was

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measured by the 3,5-dinitrosalicylic acid (DNS) method.26 Briefly, after 200 μL of a reaction

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mixture containing 100 μg mL-1 ChiE1 and 0.5% chitin or related polysaccharides in 50 mM

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NaAc-HAc (pH 5.0) was incubated at 37 °C and 800 rpm for 4 h, the reaction mixture was mixed

153

with 200 μL of DNS reagent for determination of reducing sugars. The determination of the effect

154

of temperature on the hydrolysis activity of ChiE1 toward colloidal chitin was conducted as above 6


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procedures, except that the reaction mixtures were incubated at 20-90 °C. The determination of the

156

effect of the pH on the hydrolysis activity of ChiE1 toward colloidal chitin was conducted as

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above procedures, except that the reaction mixtures with pH in the range of 4-9 (using 50 mM

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NaAc-HAc buffer (pH 4.0-6.0), 50 mM Na2HPO4-NaH2PO4 buffer (pH 6.0-8.0), and 50 mM

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Tris-HCl buffer (pH 8.0-9.0)) were used. For determination of the stability of ChiE1, the reaction

160

mixtures without the substrate were first incubated at 20-90 °C or at pH 4.0-9.0 for 1 h and then

161

combined with colloidal chitin to react as described above procediures. For determination of the

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effect of metal ions or the metal ion-chelator EDTA on the hydrolysis activity of ChiE1 toward

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colloidal chitin, ChiE1 was first incubated in 50 mM NaAc-HAc (pH 5.0) containing 1 mM of the

164

indicated metal ion salt or 1 mM or 2 mM EDTA at 37 °C for 1 h and then combined with the

165

substrate of colloidal chitin to react, as described above procedures. For the determination of the

166

effect of the substrate concentration and reaction kinetics, glycol chitin concentration was varied

167

from 4 to 10 mg mL−1. The reaction rate V was plotted against glycol chitin concentration and Km

168

and Vmax were determined uising OriginPro 8 SR0 (OriginLab Corporation, Northampton, MA)

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to fit a hyperbola to the data.27 For determination of the synergistic action of ChiE1 and previously

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reported purified recombinant ChiIII from C. cinerea,16 200-μL aliquots of reaction mixtures that

171

contained 0.5% chitinous substrates and 100 μg mL-1 of ChiE1, or 100 μg mL-1 of ChiIII, or 100

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μg mL-1 of ChiE1 plus 100 μg mL-1 of ChiIII (for chitin powder, glycol chitin, and 85%

173

deacetylated chitosan), or 10 μg mL-1 of ChiE1, or 100 μg mL-1 of ChiIII, or 10 μg mL-1 of ChiE1

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plus 100 μg mL-1 of ChiIII (for colloidal chitin) in 50 mM NaAc-HAc (pH 5.0), with other

175

parameters as listed above, were used. One unit of chitinase activity was defined as the amount of

176

enzyme that liberates the reducing sugar corresponding to 1 μmol of N-acetylglucosamine per min.

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The hydrolysis activity toward chitin oligosaccharides by ChiE1 was determined by

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HPAEC-PAD analysis.16 Briefly, after 20 μL of reaction mixtures containing 5 mM chitin

179

oligosaccharide and 50 μg mL-1 ChiE1 in 50 mM NaAc-HAc (pH 5.0) were incubated at 37 °C for

180

30 min, the amount of chitin oligosaccharides released from substrates were quantified by

181

measuring peak areas on a CarboPac PA-1 column of the HPAEC-PAD analysis, comparing to the

182

peak areas of known concentrations of the standard chitin oligosaccharides. One unit of chitinase

183

activity was defined as the amount of enzyme that liberated 1 μmol of chitin oligosaccharides per 7



184

min.

185

The hydrolysis activity toward pNP derivatives of chitin oligosaccharides by ChiE1 was

186

determined according to the manufacturer’s protocol in the chitinase assay kit (Sigma CS0980,

187

USA). Briefly, after 100 μL reaction mixtures containing 1 mg mL-1 (GlcNAc)1-3-pNP and 10 μg

188

mL-1 ChiE1 in assay buffer were incubated at 37 °C for 30 min, the mixtures absorbance was

189

measured at 405 nm. One unit of chitinase activity was defined as the amount of enzyme that

190

released 1.0 μmol of p-nitrophenyl from (GlcNAc)1-3-pNP per min.

191

Chromatographic Analysis of Hydrolysis Products. For the high performance anion

192

exchange chromatography with pulsed amperometric detection (HPAEC-PAD) analysis of

193

ChiE1-digested products of chitin substrates, 200 μL of a reaction mixture containing 0.5% chitin

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powder or colloidal chitin and 100 μg mL-1 (for chitin powder) or 10 μg mL-1 (for colloidal chitin)

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ChiE1 in 50 mM NaAc-HAc (pH 5.0) were incubated at 37 °C and 800 rpm in the Thermomixer

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comfort of Eppendorf for the indicated times, then boiled water at 100 °C was added up to a final

197

volume of 1 mL and heated at 100 °C for 10 min. For HPAEC-PAD analysis of the

198

ChiE1-digested products of chitin oligosaccharides, 20 μL of a reaction mixture containing the

199

appropriate amount of chitin oligosaccharide (see text for details) and 5 μg mL-1 ChiE1 in 50 mM

200

NaAc-HAc (pH 5.0) were incubated at 37 °C for 30 min. They were then combined with boiled

201

water at 100 °C to a final volume of 1 mL and heated at 100 °C for 10 min. The above reaction

202

mixtures were centrifuged, and the supernatants were filtered through a 0.22-μm filter and loaded

203

and eluted with ultrapure water (18.2 MΩ cm−1) on a CarboPac PA-1 column (4 × 250 mm,

204

Dionex) with a PA-1 guard column preconditioned at 25 °C based on a five-step method,28 which

205

was equipped with a 940 Professional IC Vario system with an IC Amperometric detector

206

(Metrohm). Standard GlcNAc)n (n = 1-6) were used to calibrate the retention time.

207

For size-exclusion chromatography analysis of chitooligosaccharides released from chitosan

208

(FA = 0.65) by ChiE1,6 10 mg of the chitosan was dissolved in 1 mL H2O, mixed with 1 mL

209

buffer (80 mM NaAc-HAc, 0.2 M NaCl, pH 5.0) and 0.2 mg BSA, and then pre-incubated at

210

37 °C. After adding 10 μg ChiE1, the reactions were carried out for 5 min to 1 week at 37 °C and

211

800 rpm in the Thermomixer comfort of Eppendorf. At the indicated time, the reactions were

212

stopped by lowering the pH to 2.5 through addition of 1.0 M HCl, and then immersing the samples 8


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213

in boiling water for 10 min. After centrifugation and filtration, the reaction solutions were loaded

214

and eluted at 0.80 mL min-1 with 0.15 M ammonium acetate (pH 4.5) on three XK26 columns in

215

series together, packed with SuperdexTM 30 (GE Healthcare) with an overall dimension of 2.60 ×

216

180 cm. The relative amounts of oligomers were detected with an online refractive index detector

217

(Agilent 1100 Series G1362A RID) and the data were logged with an Agilent HPLC 1100

218

ChemStore. The parallel reaction solutions were lyophilized, dissolved in D2O, and the pD was

219

adjusted to 4 with DCl. The average mole fraction of N-acetylated units (FA) and the average

220

degree of polymerization (DPn, the degree of scission α = 1/DPn) of ChiE1-degraded chitosans

221

were determined by 1H-NMR spectroscopy (Bruker AscendTM 400 NMR spectrometer) as

222

described by Varum et al.29 and Sørbotten et al.7

223

Characterization of Oligosaccharides. For characterization of transglycosylation products

224

released from chitin oligosaccharides by ChiE1, 1 μL of the above reaction mixture was combined

225

with 2 μL of 30% acetonitrile containing 15 mg mL-1 2,5-dihydroxybenzoic acid and then spotted

226

onto a target plate and dried. For characterization of oligomers in the products of hydrolysis of

227

chitosan (FA = 0.65) by ChiE1, the above fractions of oligomers eluted from the XK26 columns

228

packed with SuperdexTM 30 were respectively collected, lyophilized, and dissolved in 1 mL of

229

ultrapure water; 1 μL of each oligomer fraction was mixed with 2 μL of 30% acetonitrile

230

containing 15 mg mL1 -cyano-4-hydroxycinnamic acid and then spotted onto a target plate and

231

dried.

232

Above samples on the target plate were subjected to analysis by an UltrafleXtreme

233

MALDI-TOF/TOF mass spectrometer (Bruker) with gridless ion optics under control of

234

Flexcontrol 4.1.17.

235

Insoluble Chitin Binding Assays. Insoluble chitin binding assay was performed as described

236

by Niu et al.17 and Neeraja et al.30 Briefly, 40-200 μg mL-1 of ChiE1 were incubated with 1 mg

237

mL-1 chitin power or colloidal chitin in 1 mL of 50 mM sodium acetate buffer (pH 5.0) at 4 °C and

238

450 rpm in the Thermomixer comfort of Eppendorf for 1 h. After incubation, the unbound protein

239

in the supernatant of the reaction solution obtained by centrifugation was analyzed by detection of

240

the absorbance at 280 nm in spectrophotometer. The reaction solutions without incubation were

241

taken as controls. The bound protein was calculated as the initial total protein minus the unbound 9



242

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protein detected.

243 244

RESULTS AND DISCUSSION

245

Cloning, Heterologous Expression and Purification of ChiE1. The putative chitinase ChiE1

246

(EAU80760.2) from C. cinerea okayama 7 #130 consists of 497 amino acids with a calculated

247

molecular

248

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

249

(http://www.cbs.dtu.dk/services/TargetP/)

250

(MLKAVTWSSTSTAPLGFLLTAIIFFKAGQYSA) at the N-terminal is a signal peptide (Fig 1A).

251

The analysis of conserved domains in NCBI database shows that ChiE1 has a catalytic domain

252

without carbohydrate-binding module (CBM). Fig 3C of sequence alignment shows that ChiE1

253

displays 29.5% identity to previously reported single catalytic domain chitinse ChiB1 belonging to

254

exo-acting class V chitinases from C. cinerea.15 However, it only has 13.3% sequence identity to

255

reported C. cinerea ChiIII, which contains two CBMs, ChtBD3 and ChiC-BD, belonging to

256

endo-acting class III chitinases.16 This is consistent with the phylogenetic analysis of eight

257

putative chitinases from C. cinerea, which shows that ChiE1 and exochitinase ChiB1 are in the

258

same subgroup.16 The protein structure of ChiE1 predicted by I-TASSER (Iterative Threading

259

ASSEmbly Refinement, http://zhanglab.ccmb.med.umich.edu/I-TASSER/) shows that similar to

260

reported exochitinases ChiA and ChiB from S. marcescens,8 ChiE1 has an α + β fold insertion

261

domain between strand 7 and helix 7 of the TIM-barrel fold, and a deep substrate-binding cleft in

262

which some aromatic amino acids are exposed on its surface (Fig. 1B).

mass

of

55384

Da.

Analysis

using

and show

the

Signal

TargetP that

the

4.1

1.1 32

amino

server server acids

263

For construction of the expression plasmid, the nucleotide sequence coding the signal peptide

264

was removed from chiE1 so that extracellular secretion of the recombinant mature ChiE1 was

265

mediated by the N-terminal α-factor signal peptide of the plasmid pPICZαAChiE1. Six histidines

266

were fused to the C-terminal of the recombinant ChiE1 by a linker from the plasmid

267

pPICZαAChiE1 with a calculated molecular mass of 55290 Da. After six days of induction

268

cultivation, the hydrolysis activity of the culture medium of the recombinant chiE1 expression

269

strain towards colloidal chitin reached 30.3 mU mL-1.

270

The recombinant ChiE1 was purified by Ni-affinity chromatography from the culture medium 10


Page 11 of 32


271

(Fig 1D). The yield of recombinant ChiE1 was 0.05 mg mL-1 culture medium and the specific

272

activity of unpurified and purified recombinant ChiE1 toward colloidal chitin was 1.44 mU mg-1

273

protein and 373 mU mg-1 protein, respectively.

274

SDS-PAGE analysis showed a extra broad protein band at approximately 66 kDa in the culture

275

medium of the recombinant chiE1 expression strain compared to that for the control strain with an

276

empty plasmid. The purified recombinant ChiE1 from the culture medium still exhibited this

277

broad band on the polyacrylamide gel, however, a narrow dark protein band at approximately 66

278

kDa in the both of control and recombinant strain culture mediums disappeared in the purified

279

recombinant ChiE1on the polyacrylamide gel (Fig. 1E). The broad protein band was confirmed to

280

be the putative chitinase ChiE1 by MALDI TOF/TOF Ms analysis of its trypsin-digested

281

fragments (Fig 1F). The higher apparent molecular weight and the broad protein band on the

282

polyacrylamide gel contributed to glycosylation of the recombinant protein in Pichia pastoris.31

283

Enzymatic Features. ChiE1 hydrolyzed insoluble chitin powder, colloidal chitin, glycol chitin,

284

and 85% deacetylated chitosan, but not glycol chitosan, CMC-Na, or laminarin (Table 1). Of note,

285

previously reported ChiB115 and ChiEn117 from C. cinerea did not hydrolyze chitin powder, and

286

only ChiIII from C. cinerea showed hydrolysis activity toward chitin powder.16 Furthermore, the

287

specific hydrolysis activities of ChiE1 toward chitin powder, colloidal chitin, and 85%

288

deacetylated chitosan are apparently higher than that of endochitinase ChiIII.16 Interestingly, the

289

specific hydrolysis activity of ChiE1 toward colloidal chitin is more than 10 times higher than that

290

toward chitin powder, whereas we previously reported that the specific hydrolytic activity of

291

ChiIII toward colloidal chitin was only 82.9% higher than that toward chitin powder.16 Suzuki et

292

al.32 reported that chitinases ChiA, ChiB, and ChiC1 from S. marcescens show higher hydrolytic

293

activity toward colloidal chitin than that toward chitin powder. Colloidal chitin is more accessible

294

to the chitinases because it has undergone acidic hydrolysis pretreatment, which reduces its

295

crystallinity and results in a much larger exposed surface area per unit weight.1 This results in

296

more rapid breakdown of colloidal chitin by chitinases. Apparently, the sites on the chitin attacked

297

by ChiE1 are different from that attacked by ChiIII.

298

ChiE1 could degrade chitintriose or longer chitin oligosaccharides, but not degrade chitinbiose

299

(Table 1). The hydrolytic activity of ChiE1 toward (GlcNAc)4-6 was more than three times its 11



300

hydrolytic activity toward (GlcNAc)3. ChiE1 hydrolyzed (GlcNAc)3-pNP and (GlcNAc)2-pNP to

301

release the detected color pNP, but could not hydrolyze GlcNAc-pNP (Table 1). The activity of

302

ChiE1 towards (GlcNAc)2-pNP was 3.2 times the activity toward (GlcNAc)3-pNP. The hydrolysis

303

pattern of ChiE1 on (GlcNAc)1-3-pNP is different from C. cinerea ChiB1 which only hydrolyzes

304

(GlcNAc)2-pNP15 whereas similar to a chitinase Chit42 from Trichoderma harzianum homologous

305

to bacterial exochitinases.33 It is suggested that glucosaminidase, chitobiosidase (exochitinase) and

306

endochitinase activities are determined through measuring the pNP released from GlcNAc-pNP,

307

(GlcNAc)2-pNP and (Glc-NAc)3-pNP, respectively,34,

308

very strict for characterization of the mode of action of chitinases.34 Some exochitinases were

309

reported to degrade both (GlcNAc)2-pNP and (Glc-NAc)3-pNP, but their hydrolytic activity

310

toward (GlcNAc)2-pNP is usually higher than that toward (Glc-NAc)3-pNP.36 Therefore, above

311

results suggest that ChiE1 is an exochitinase.

35

however, the chromogenic assay is not

312

The optimal pH for the hydrolytic activity of ChiE1 toward colloidal chitin was 5.0. The pH

313

stability test showed that ChiE1 activity almost did not change after the pre-incubation of ChiE1

314

over a broad pH range of 4.0-9.0 for 1h (Fig. 2A). The optimal temperature for the hydrolytic

315

activity of ChiE1 toward colloidal chitin was 40 °C. The temperature stability test showed that the

316

preincubation of ChiE1 at 20-40 °C for 1 h essentially did not affect ChiE1 activity while

317

preincubation over 40 °C resulted in the loss of its activity (Fig. 2B). The optimal reaction

318

conditions for ChiE1 are similar to those for ChiB1,15 ChiIII,16 and ChiEn117 in the same

319

chitinolytic system of C. cinerea. The effect of glycol chitin concentration on the hydrolysis

320

activity of ChiE1 is shown in Fig. 2C, with a Km of 8.90 mg mL-1, a kcat of 0.06 s-1, and a Vmax of

321

0.08 mol min-1 mg protein-1.

322

The detected metal ions did not enhance the hydrolysis activity of ChiE1 toward colloidal chitin

323

and 1 mM or 2 mM of the metal ion chelator EDTA almost had no effect on the hydrolysis activity

324

of ChiE1 toward colloidal chitin (Table S1). Therefore, metal ions are not essential for ChiE1

325

activity. This is similar to that for C. cinerea ChiB115 but different from that for C. cinerea

326

ChiIII16 and ChiEn1.17

327

Hydrolysis Products. The time course for enzyme hydrolysis of chitin showed that ChiE1

328

degraded insoluble chitin powder to produce (GlcNAc)2 as a dominant product and GlcNAc as a 12


Page 12 of 32

Page 13 of 32


329

minor product at the initial 0.5 h; at 1 h, (GlcNAc)3 as a minor product started to be observed in

330

the reaction solution; at 24 h, the minor (GlcNAc)3 completely disappeared and only the dominant

331

(GlcNAc)2 and a small amount of GlcNAc were observed (Fig. 3A). In contrast, ChiE1 degraded

332

colloidal chitin to produce (GlcNAc)2 as a dominant product and (GlcNAc)3 as a minor product at

333

the initial 0.5 h; at 1 h, a minor amount of GlcNAc appeared in the reaction solution; at 24 h, the

334

dominant (GlcNAc)2, as well as the small amount of both (GlcNAc)3 and GlcNAc were present in

335

the reaction solution (Fig. 3B). The hydrolysis activity of ChiE1 toward insoluble chitin powder

336

was only 4% of the hydrolysis activity toward (GlcNAc)3, whereas the hydrolysis activity of

337

ChiE1 toward colloidal chitin was 41.4% of the hydrolysis activity toward (GlcNAc)3 (see Table

338

1). Therefore, the minor intermediate (GlcNAc)3 released from insoluble chitin powder by ChiE1

339

was prone to be immediately degraded into (GlcNAc)2 and GlcNAc.

340

ChiE1 did not degrade (GlcNAc)2, while it degraded (GlcNAc)3 to (GlcNAc)2 and GlcNAc.

341

When (GlcNAc)4-6 were used as the substrates ChiE1 degraded (GlcNAc)4-6 to yield (GlcNAc)2

342

and (GlcNAc)n-2, as well as minor longer chitin oligosaccharides (GlcNAc)n+2 and (GlcNAc)n+2+2

343

(Fig, 4A). The lack of a high dimer peak for (GlcNAc)5 and (GlcNAc)6 compared to (GlcNAc)3

344

and (GlcNAc)4 suggests that the (GlcNAc)2 released from substrate (GlcNAc)5 or (GlcNAc)6 is

345

transferred to the other same substrate or its product to yield longer chitin oligosaccharides.

346

Because of the lack of commercial chitin oligosaccharide standards larger than DP6, the presence

347

of long-chain transglycosylation products, (GlcNAc)7-10, was further confirmed by MALDI-TOF

348

MS analysis (Fig 4B), supporting that ChiE1 has a transglycosylation activity. Notably, only a

349

minor amount of (GlcNAc)3 were observed in the ChiE1-hydrolysis products of (GlcNAc)4 and

350

(GlcNAc)6 which is one of distinctive characteristics of the exochitinase hydrolysis patterns of

351

chitin oligosaccharides. 15, 32, 37

352

The size distributions of chitooligosaccharides produced at various stages of degradation of a

353

highly acetylated, high molecular weight, and water-soluble chitosan (FA = 0.65) by ChiE1 were

354

investigated for elucidation of the mode of action and the processivity of ChiE1.6, 7 As shown in

355

Fig 4C, the slow disappearance of the void volume peak of the substrate and larger products and

356

the early appearance of only shorter oligomer products indicate that ChiE1 degraded initially the

357

chitosan chains from their ends.6, 7 The initial products of the chitosan (FA = 0.65) reacted with 13



358

ChiE1 consisted almost exclusively of even numbers of sugar units (see α ≤ 0.15), whereas the

359

final products contained a continuum of odd- and even-numbered oligomers (see α = 0.36) with

360

the AA dimer as a dominant product, and the void peak disappeared completely only after 7 days

361

of reaction. These results indicate that ChiE1 acts in an exo-fashion and that it has a processive

362

mode of action, similar to exochitinase ChiA and ChiB with CBM from S. marcescens.6, 7 This is

363

the first study to report the single catalytic domain of chitinase with a processive mode of action.

364

Capacity of ChiE1 to Bind to and Synergistically Act on Chitin. Although ChiE1 has no

365

CBM, it binds to the chitin substrates. As shown in Table 2, after incubation of varying

366

concentration of ChiE1 with 1 mg mL-1 chitin power or colloidal chitin at 4 °C for 1 h, at a low

367

protein concentration (40 μg mL-1) 33.3% and 66.7% of ChiE1 proteins were bound to chitin

368

powder and colloidal chitin, respectively; at a high protein concentration (200 μg mL-1), 17.5%

369

and 55.6% of ChiE1 proteins were bound to chitin powder and colloidal chitin, respectively,

370

exhibiting a higher affinity to colloidal chitin than chitin powder. The carbohydrate-binding

371

module in chitinases is suggested to play a role in the capacity to bind to substrates and the

372

processive mode of action of chitinases. However, chitinases lacking the carbohydrate-binding

373

module often bind to substrates via the aromatic residues exposed on the surface of

374

substrate-binding cleft.38-41 The predicted protein structure of ChiE1 shows that more aromatic

375

amino acids are exposed on the surface of the substrate-binding cleft (Fig 1B), similar to the single

376

catalytic domain SpChiD from S. proteamaculans, which could bind to insoluble chitin powder

377

and colloidal chitin.40 Notably, at the same concentration of substrates and enzyme, approximately

378

70% SpChiD was bound to chitin powder and colloidal chitin, respectively, after incubation for 1

379

h at 4°C, and the specific activity of SpChiD toward colloidal chitin was only a slightly higher

380

than that toward chitin powder. Apparently, the more-than 10 times higher specific hydrolytic

381

activity of ChiE1 toward colloidal chitin than toward chitin powder is due to a higher affinity of

382

ChiE1 to colloidal chitin than to chitin powder. The higher affinity of ChiE1 to the substrate could

383

obtain more chance to attack the sites of the substrate for hydrolysis of chitin.

384

It has been reported that microbial chitinases could synergistically act on the degradation of

385

insoluble chitin substrates which is due to their different and complementary catalytic features,

386

endo-mode of action vs exo-mode of action, processivity vs non-processivity, and initially 14


Page 14 of 32

Page 15 of 32


387

cleaving at the reducing end vs nonreducing end of chitin chains.8,17,32 To examine whether ChiE1

388

acts synergistically with the previously characterized endochitinase ChiIII from C. cinerea on

389

chitinous substrates, the amount of reducing sugar released from chitinous polysaccharides by a

390

combination of ChiE1 and ChiIII, or the same concentration of ChiE1 or ChiIII alone after 4 h

391

incubation at 37 °C were determined. As shown in Table 3, a clear synergistic effect was observed.

392

The amount of reducing sugars released from insoluble chitin powder or colloidal chitin by a

393

combination of ChiE1 and ChiIII increased by 120% and 49.7%, respectively, compared to the

394

sum of the amount of reducing sugars released by the same concentration of ChiE1 or ChiIII alone.

395

However, the amount of reducing sugars released from soluble glycol chitin and 85% deacetylated

396

chitosan by the combination of ChiE1 and ChiIII decreased by 13.0% and 13.1%, respectively,

397

compared to the sum of the amount of reducing sugars released by the same concentration of

398

ChiE1 or ChiIII alone. The decreased hydrolytic activity of the combination of ChiEn1 and ChiIII

399

toward soluble glycol chitin and 85% deacetylated chitosan compared to the summary hydrolytic

400

activity of ChiIII or ChiEn1 alone may be due to their competitively binding to completely soluble

401

and fully accessible chitinous substrates.

402

The synergistic actions of different chitinases such as endo- and exo-chitinases on the

403

degradation of insoluble chitin substrates have been reported. Brurberg et al.34 reported that when

404

colloidal chitin was treated with combined ChiA and ChiB both of which are exochitinases but

405

degrade the chitin polymer from different ends,8 from bacterium S. marcescens, the hydrolytic

406

activity was increased by 54.8% compared to the sum of these two enzymes alone. Suzuki et al.32

407

reported that when chitin powder was treated with a combination of these two exochitinases ChiA

408

and ChiB, or a combination of exochitinases ChiA and endochitinase ChiC1, or a combination of

409

these three chitinases, ChiA，ChiB, and ChiC1 from S. marcescens, the hydrolysis activities were

410

increased by 80%, 45%, 100%, respectively, compared to the sum of each chitinase alone;

411

whereas, the combination of exochitinase ChiB and endochitinase ChiC1 did not show any

412

synergistic effect. Orikoshi et al.42 reported that when chitin powder was treated with the

413

combination of four chitinases, ChiA, ChiB, ChiC, and ChiD from the marine bacterium

414

Alteromonas sp. strain O-7, the hydrolytic activity was approximately increased by 1.0-fold

415

compared to the sum of all the individual chitinase activities. We previously reported that when 15



416

chitin powder was treated with the combination of so-called endochitinase ChiEn1 and

417

endochitinase ChiIII from C. cinerea, the hydrolytic activity was increased by 105% compared to

418

the sum of ChiEn1 and ChiIII individual activity.17 This study shows that the hydrolysis activity of

419

the combination of exochitinase ChiE1 and endochitinase ChiIII from C. cinerea toward chitin

420

powder is increased by approximately 120% compared to the sum of ChiE1 and ChiIII alone. The

421

synergistic action of exochitinase ChiE1 and endochitinase ChiIII from C. cinerea on degradation

422

of chitin powder is apparently higher than previously reported synergism of chitinases from

423

bacteria and fungi. The chitinase conversion of chitin to chitin oligosaccharides typically requires

424

endo-acting non-processive chitinases and exo-acting processive chitinases that act in a synergistic

425

manner.43 Therefore, the recombinant chitinase ChiE1 expressed in P. pastoris may be used as a

426

synergistic chitinase for a reconstituted chitinolytic system to efficiently hydrolyze the crystalline

427

crab/shrimp shell chitin to produce the chitin oligosaccharides for potential agricultural, food/feed,

428

biomedical, and environmental applications.44-46

429 430

Supporting Information

431

Effects of Metal Ions and the Ion Chelator EDTA on C. cinerea Chitinase ChiE1 Activity toward

432

Colloidal Chitin

433 434

Acknowledgments

435

This study was supported by the National Natural Science Foundation of China (No. 31570046),

436

the Priority Academic Development Program of Jiangsu Higher Education Institutions, and the

437

Scientific Innovation.

438 439

Notes

440

The authors declare no competing financial interest.

441 442

Reference

443

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444

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plant/pathogen interactions. Curr. Genet. 2016, 62, 243-254.

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single chitooligosaccharides. Carbohyd. Polym. 2016, 139, 178-190.

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chitin-binding domain in Bacillus chitinases improves the substrate binding affinity and

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565

22


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Table 1. Chitinase ChiE1 Activity toward Various Substrates Substrate

Specific activitya

Polysaccharides

mU mg-1 proteinb

chitin powder (Sigma C7170)

36.2 ± 0.61

colloidal chitin (made in the laboratory by Sandhya et al.)

373 ± 2.51

glycol chitin (made in the laboratory by Li et al.)

27.3 ± 0.14

85% deacetylated chitosan (Sigma C3646)

79.1 ± 0.81

CMC-Na (Sigma C5013)

0 ± 0.35

glycol chitosan (Sigma G7753)

0 ± 0.68

Laminarin (Sigma 9634)

0 ± 1.00

Oligosaccharides

U mg-1 proteinc

chitinbiose (OligoTechGLU432)

0

chitintriose (OligoTech GLU433)

0.90 ± 0.03

chitintetraose (OligoTech GLU434)

3.30 ± 0.04

chitinpentose (OligoTech GLU435)

3.52 ± 0.03

chitinhexaose (OligoTech GLU436)

4.24 ± 0.05

chitohexaose(ZB-10010)

0

(GlcNAc)1−3-pNP

U mg-1 proteind

4-nitrophenylN-acetyl-β-D-glucosaminide (GlcNAc-pNP) (Sigma, N9376) 4-nitrophenylN,N′-diacetyl-β-D-chitobioside

0

6.04 ± 0.02

((GlcNAc)2-pNP) (Sigma, N6133) 4-nitrophenyl-β-D-N,N′,N″-triacetylchitotriose

1.89 ± 0.01

((GlcNAc)3-pNP) (Sigma, N8638) 568

a Data

569

b

570

sugar corresponding to 1 μmol of N-acetylglucosamine per min.

571

c

572

chitin oligosaccharides per min.

573

d

574

p-nitrophenol from (GlcNAc)1-3-pNP per min.

are means±SD of three replicates.

One unit of chitinase activity was defined as the amount of enzyme that liberates the reducing One unit of chitinase activity was defined as the amount of enzyme that liberated 1 μmol of One unit of chitinase activity was defined as the amount of enzyme that released 1 μmol of

575

23



576

Table 2. Insoluble Chitin-Binding Capacity of ChiE1

577

Total proteina

Protein bound to chitin (% total protein)b

578

(μg mL-1)

Chitin powderc

Colloidal chitinc

579

40

33.3 ± 14.4*

66.7 ± 14.4*

580

80

33.3 ± 8.2*

55.6 ± 11.1*

120

33.3 ± 4.1**

54.8 ± 4.1**

160

29.6 ± 3.2***

55.6 ± 0.0***

200

17.5 ± 2.7***

55.6 ± 5.5***

581 582 583 584

a Total

585

b The

586

reaction solution after 1 h of incubation with chitin substrates. Data are means±SD of three

587

replicates.

588

c

589

colloidal chitin at P≤0.05*, P≤0.01**, and P≤0.001***, respectively.

protein represents the protein amount of ChiE1 added in the binding reaction solution.

bound protein was calculated as the total protein minus the unbound protein measured in the

T-test indicated significant differences in binding capacities of ChiE1 of chitin powder and

590 591 592 593

24


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Page 25 of 32

594


Table 3. Synergistic Action of Chitinase ChiE1 and ChiIII on Chitinous Polysaccharides Chitinase in

Reducing sugar released from chitinous polysaccharides (μmol mL-1)a 85% Deacetylated

reaction

Chitin powder

Colloidal chitin

Glycol chitin

ChiE1

0.682 ± 0.008

0.724 ± 0.008

0.643 ± 0.004

0.938 ± 0.006

ChiIII

0.690 ± 0.010

0.654 ± 0.005

0.662 ± 0.004

0.739 ± 0.010

ChiE1+ChiIII

3.016 ± 0.019

2.063 ± 0.027

1.136 ± 0.013

1.458 ± 0.058

Synergism (%)b

120%***

49.7%***

-13.0%*

-13.1%**

chitosan

595

a Data

596

b

597

indicated chitinous polysaccharide by combination of ChiE1 and ChiIII compared to the sum of

598

reducing sugars released from the same indicated chitinous polysaccharide by the same

599

concentration of ChiE1 or ChiIII alone. T-test showed that these synergisms were significant at

600

P≤0.05*, P≤0.01**, and P≤0.001***, respectively.

are means±SD of three replicates.

Synergism represents increased or decreased amount (%) of reducing sugars released from the

25



602

Legend

603

Fig. 1 A. The entire amino acid sequence that is predicted from the gene sequence of a putative

604

ChiE1 annotated in the C. cinerea genome (locus/protein identifier accession EAU80760 in

605

GenBank). The underlined N-terminal amino acid sequence is a signal peptide; and the shadowed

606

amino acid sequences exhibit the sequences of the two partial peptide fragments from the

607

recombinant ChiE1 with trypsin determined using MALDI-TOF/TOF MS analysis (F). B. The

608

protein structure of ChiE1 was predicted by the I-TASSER based on the 33 - 497 amino acids of

609

ChiE1 without signal peptide. The crystal structure of Aspergillus fumigatus chitinase B1 (2A3E)

610

was used as the template for model building. B1, ChiE1 shows a TIM (α/β)8 barrel fold consisting

611

of eight α-helices (blue) and eight parallel β-strands (red), and an α + β insertion domain (yellow).

612

B2, The predicted substrate binding cleft of ChiE1 shows the D173XD175XE177 catalytic motif

613

(red) and the aromatic amino acids, W50, W379, W251, W137, W314, Y53, Y139, and Y178

614

(blue) exposed on its surface. C. Amino acid sequence alignment of ChiE1 and ChiB1 from C.

615

cinerea. D. Ni-affinity chromatography of the recombinant ChiE1 in the culture medium. Solid

616

line, A280; dotted line, chitinase activity toward colloidal chitin. E. SDS-PAGE analysis of the

617

recombinant chitinase ChiE1. Lanes: M, standard protein molecular weight markers; 1, 20 μL of

618

culture medium of control stain; 2, 20 μL of culture medium of recombinant expression strain; 3, 5

619

μg purified recombinant ChiE1. F. The spectra of MALDI TOF/TOF MS of the trypsinized

620

peptide fragment 1 (F1) and peptide fragment 2 (F2) from the recombinant ChiE1 in A. F1, the

621

sequence with Mascot ion score of 192 and m/z of 3115.4258; F2, the sequence with Mascot ion

622

score of 163 and m/z of 2348.0822.

623

26


Page 26 of 32

Page 27 of 32


624

Fig. 2. A, The pH effect (solid line) and pH stability (dotted line) of ChiE1 activity towards

625

colloidal chitin. B, The temperature effect (solid line) and temperature stability (dotted line) of

626

ChiE1 activity towards colloidal chitin. C, The effect of glycol chitin concentration on ChiE1

627

activity. The reaction rate v was plotted directly against glycol chitin concentration. Data are

628

expressed as means ± SD from three experiments.

629 630

Fig. 3 HPAEC-PAD analysis of the hydrolysis products from A, 0.5% chitin powder or B, 0.5%

631

colloidal chitin incubated with ChiE1 (100 μg mL-1 for chitin power and 10 μg mL-1 for colloidal

632

chitin) for 0.5 to 24 h. Chitin oligosaccharides (GlcNAc)1-6 were used as standards.

633 634

Fig. 4 A, HPAEC-PAD analysis of hydrolysis products of 5 mM chitin oligosaccharides

635

(GlcNAc)2,4-6 or 1 mM (GlcNAc)3 reacted with 5 μg mL-1 ChiE1 for 30 min. B, MALDI-TOF MS

636

spectra of the reaction mixture of (GlcNAc)4 (B1), (GlcNAc)5 (B2), and (GlcNAc)6 (B3)

637

incubated with ChiE1 for 30 min as described in Fig. 4A. C, the size-exclusion chromatography of

638

chitooligosaccharides produced during the degradation of 0.5% chitosan (FA = 0.65) by 5 μg mL-1

639

ChiE1 for 5 min (α = 0.07), 60 min (α = 0.15), 120 min (α = 0.20), 180 min (α = 0.23), 7 d (α =

640

0.36) on XK26 columns packed with SuperdexTM 30. The α-values denote the degree of scission

641

(α = 1/DPn). The peaks are marked by the degree of polymerization (DP) or by the sequence (for

642

the one known compound) of the oligomers they contain. AA and DA are (GlcNAc)2 and

643

GlcNGlcNAc, respectively. Undegraded chitosan and chitosan fragments with a DP > 40 elutes in

644

the void volume of the column.

645 27



646

Figure 1.

647 648

28


Page 28 of 32

Page 29 of 32

649


Figure 2.

650 651

29



652

Figure 3.

653 654

30


Page 30 of 32

Page 31 of 32

655


Figure 4.

656 657

31



658

TOC graphic

659

32


Page 32 of 32

A ChiE1 from Coprinopsis cinerea is characterized as a processive

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