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Gene cloning, heterologous expression and characterization of a Coprinopsis cinerea endo-β-1,3(4)-glucanase
Accepted Manuscript Gene cloning, heterologous expression and characterization of a Coprinopsis cinerea endo-β-1,3(4)-glucanase Jun Wang, Liqin Kang, Zhonghua Liu, Sheng Yuan PII:
S1878-6146(16)30143-X
DOI:
10.1016/j.funbio.2016.09.003
Reference:
FUNBIO 770
To appear in:
Fungal Biology
Received Date: 25 January 2016 Revised Date:
5 September 2016
Accepted Date: 6 September 2016
Please cite this article as: Wang, J., Kang, L., Liu, Z., Yuan, S., Gene cloning, heterologous expression and characterization of a Coprinopsis cinerea endo-β-1,3(4)-glucanase, Fungal Biology (2016), doi: 10.1016/j.funbio.2016.09.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Gene cloning, heterologous expression and characterization of a Coprinopsis cinerea endo-β-1,3(4)-glucanase
Jun Wang, Liqin Kang, Zhonghua Liu*, Sheng Yuan* Jiangsu Key Laboratory for Microbes and Microbial Functional Genomics, Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources,
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College of Life Science, Nanjing Normal University, Nanjing 210023, PR China
* Co-corresponding author:
Cell Pnone: 86-135-8409-3709; E-mail: [email protected].
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*(Liu, Z.,)
[email protected]. College of Life Science Nanjing Normal University
Xianlin University Park
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PR China
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Nanjing, 210023
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1 Wenyuan Rd
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*(Yuan, S.,) Phone: 86-25-8589-1067; Fax: 86-25-85891067; E-mail:
Running Head: endo-β-1,3(4)-glucanase for nutrition
Highlights:
ENG16A highly expresses during the mycelium stage Adjacent β-1,4- bonds favors ENG16A hydrolysis of β-1,3-glycosidic bonds Adjacent β-1,6- bonds hinders ENG16A hydrolysis of β-1,3-glycosidic bonds An endo-β-1,3(4)-glucanase corresponds to nutrient degradation.
ACCEPTED MANUSCRIPT Abstract A gene coding endo-β-1,3(4)-glucanase (ENG16A) was cloned from Coprinopsis cinerea and heterologously expressed in Pichia pastoris. ENG16A only acts on substrates containing β-1, 3 glycosidic bonds but not on substrates containing only β-1,4- or β-1,6- glycosidic bonds.
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Interestingly, compared to the activity of this enzyme towards carboxymethyl (CM)-pachyman containing only β-1,3-glycosidic bonds, its activity towards barley β-glucan containing both β-1,3-glycosidic and β-1,4-glycosidic bonds was increased by 64.72 %,, its activity towards laminarin containing both β-1,3-glycosidic and β-1,6-glycosidic bonds was
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decreased by 50.83%. In addition, ENG16A has a higher Km value and Vmax for barley β-glucan than laminarin, which may be related to the fact that barley β-glucan contains
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mainly β-1,4-glycosidic bonds mixed with a few β-1,3-glycosidic bonds, whereas laminarin mainly contains β-1,3-glycosidic bonds with a few β-1,6-branched glucose residues. The adjacent β-1,4-glycosidic bond promotes ENG16A to hydrolyze β-1,3-glycosidic bonds, leading to an increased Vmax; the nearby β-1,6-glycosidic bonds inhibited its hydrolysis of β-1,3-glycosidic bonds, resulting in a decreased Vmax. This property is suggested to be
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related to the mechanism that C. cinerea uses to degrade and utilize hemicellulose in straw medium and to protect its cell wall during the mycelium growth stage.
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autolysis
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Key words: Endo-β-1,3(4)-glucanase, barley β-glucan, laminarin, nutrition degradation, wall
ACCEPTED MANUSCRIPT Introduction C. cinerea is an ideal model organism with which to study the growth and development of basidiomycete fruiting bodies (Kües 2000). Similar to all other agarics, C. cinerea exhibits obvious fruiting body autolysis (Fukuda et al. 2008; Kües 2000; Kawakami et al. 2004;
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Sakamoto et al. 2012; Tao et al. 2013). The autolysis of the fruiting body pileus of C. cinerea are mainly resulted from action of various hydrolases which were synthesized and secreted to degrade the cell walls for the release of basidiospores during maturation of fruiting body (Hammad et al. 1993; Iten 1969; Kües 2000; Miyake et al. 1980; Moore 2003). We recently
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reported that a group of glucanases with different action mode were isolated from the pileus of mature fruiting body of C. cinerea including an endo-β-1,3-glucanase, an β-glucosidase (Zhou et al.
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exo-β-1,3-glucanase, a β-1,3-glucosidase, as well as a possible
2015). These glucanases degrade the β-1,3/1,6-glucans in the cell walls of the pileus in a synergistic manner. Furthermore, through mutagenesis, we obtained one mutant of C. cinerea in which the pileus does not undergo autolysis (Liu et al. 2015). A comparative study of the wild type and non-autolysis mutant of C. cinerea revealed that during the processes of pileus
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opening and hydrolysis, the glucanase activity in the extract of the wild type C. cinerea pileus was significantly higher than in the extract of the mutant C. cinerea pileus, suggesting that glucanase plays a major role in the autolysis of the C. cinerea pileus. When the expression
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levels of 43 glucanases, which mainly act on β-1,3-glycosidic bonds, were examined in the C. cinerea genome, we found that the expression levels of the four glucanases mentioned above
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that we previously purified and an additional glucanase were significantly higher than the expression levels of other 38 glucanases in the wild type C. cinerea in pileus during its opening process and that their expression levels were significantly suppressed in the mutant C. cinerea pileus, which does not undergo autolysis, during its opening process. This result suggests that these five glucanase genes may play key roles in the pileus autolysis process of C. cinerea (Liu et al. 2015). Apart from the five glucanase genes that may be directly associated with pileus autolysis, there are another 38 functionally similar glucanase genes of the 43 glucanase genes of C. cinerea that mainly act on β-1,3-glycosidic bonds. Their expression levels are not significantly different between wild type and mutant C. cinerea, and most of them belong to
ACCEPTED MANUSCRIPT glycoside hydrolase families 2 and 16 (Liu et al. 2015). Generally, it is thought that β-glucanase not only plays a role in degrading the cell wall (Fontaine et al. 1997; Liu et al. 2015; Mouyna et al. 2002; Zhou et al. 2015) and remodeling cell wall components and structures (Cole and Hung 2001; Delgado et al. 2003; Hung et al. 2001; Mouyna et al. 2000)
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during the growth, development and morphogenesis of fungi, but also involve in degrading the nutritional components in the culture medium to maintain the energy and nutrition needed for cell growth (Fukuda et al. 2008; Larriba et al. 1993; Nombela et al. 1988). The synthesis of some specific β-glucanases was reported to depend on the induction of some specific
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polysaccharides in the medium (Jayus et al., 2002). To determine wheather those rest of the 38 glucanases play a roles in the degradation of culture medium or the other physiological
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process during the growth and development of C. cinerea, we selected a glucanase from the 38-glucanases, an endo-β-1,3(4)-glucanase (ENG16A) (GenBank accession: XP_001828985), which belongs to glycoside hydrolase family 16, as the study subject. After cloning, the selected gene was heterologously expressed in yeast Pichia pastoris, and the properties and
Materials and methods
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possible physiological functions of this enzyme were studied.
Construction of the expression strain
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The mycelia and fruiting bodies of C. cinerea were cultured in a straw medium containing 88% dry rice straw, 5% wheat bran, 3% corn flour, 2% fertilizer, 1% lime powder, 1%
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sucrose and water quantum satis, as described by Zhou et al. (2015). The total RNA was extracted from the fruiting body of C. cinerea using the Spin Column Fungal Total RNA Purification Kit (Bio Basic Inc., Amherst, NY, USA) and the synthesis of the cDNA was carried out using the HiScript II Q RT SuperMix for qPCR kit (+gDNA wiper) (Vazyme, Nanjing, China). The upstream primer eng16A-F and downstream primer eng16A-R were used to amplify the cDNA of the eng16A gene from the the total cDNA by PCR, in which the first 72 bp nucleotide sequence encoding a signal peptide of 24 amino acids was removed. The PCR product was purified using the Spin Column Gel Extraction Kit (Takara, Beijing, China) and identified by DNA sequencing.
ACCEPTED MANUSCRIPT The eng16A cDNA fragment and the plasmid pPICZαA (ThermoFisher Scientific, Grand Island, USA) double digested respectively with EcoRI and NotI were ligated to form the plasmid pPICZαA-eng16A following the instruction manual of the ClonExpress® Entry One Step Cloning Kit (Vazyme, Nanjing, China). The plasmid pPICZαA-eng16A was transformed into Escherichia coli DH5α cells for amplification. After producing an expanded culture of E. coli DH5α, the pPICZαA-eng16A plasmid was isolated from E. coli cells and singly digested
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by SacI to linearize it. The linearized plasmid was transformed into P. pastoris GS115 cells by electroporation. The transformants were allowed to grow on YPDS + Zeocin™ (ThermoFisher Scientific, USA) plates for three days, and single colonies were picked to confirm that the eng16A gene was successfully integrated into the genome of P. pastoris
(ThermoFisher Scientific, Grand Island,USA). Expression of the recombinant enzyme
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GS115 at the AOX1 site according to the instructions of the Pichia Expression Kit
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The P. pastoris GS115 cells with the integrated eng16A gene were inoculated to a 100 mL flask containing 15 mL of BMGY medium and cultured for 18-20 h at 30˚C with shaking at 220 rpm. When the OD600 reached 2-6, the yeast cells were harvested by centrifugation at 1000×g for 10 min. The yeast cells were resuspended and transferred to a 500 mL flask containing 100 mL BMMY medium for induction cultivation at 30˚C with shaking at 220 rpm for three days; the medium was supplemented with methanol at 0.5% of the total volume
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every 24 h to induce expression.
Purification of the recombinant enzyme
The culture medium was centrifuged at 12000×g at 4˚C to remove yeast cells. The supernatant was dialyzed at 4˚C overnight against equilibrium buffer (300 mM NaCl, 50 mM
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NaH2PO4, 10 mM imidazole, 10 mM Tris base, pH 8.0). The Ni-affinity chromatography method was then used to purify the recombinant enzyme from the culture supernatant by the manual of the ProteinIso® Ni-NTA Resin (Transgen, Beijing, China). All of the purification
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steps were carried out at 4˚C.
Protein analysis of the recombinant enzyme The purity and molecular weight of the purified recombinant enzyme were determined by SDS-PAGE (Laemmli 1970). The protein concentration of the recombinant enzyme was determined using the Bradford method with Coomassie Brilliant Blue (Bradford 1976), and bovine serum albumin was used as the standard. The amino acid sequencing of the purified protein was determined by MALDI-TOF/TOF Ms (Zhou et al., 2015). Determination of the activity of the recombinant enzyme The hydrolysis activity of the recombinant enzyme was determined by measuring the amount of reducing sugars released after it hydrolyzed the substrate. The amount of reducing sugars was determined by the 3,5-dinitrosalicylic acid method (DNS) (Miller 1959). Briefly, 200 µL
ACCEPTED MANUSCRIPT of reaction solution containing an appropriate amount of enzyme, 1% barley β-glucan (Megazyme, Ireland), carboxymethyl (CM)-pachyman (Megazyme, Ireland), laminarin (TCI, Japan), periodate-oxidized laminarin (performed as described Goldstein et al. (1965) ), pustulan (Megazyme, Ireland), oat spelt xylan (Sigma, USA), avicel (Megazyme, Ireland), sodium carboxyl methyl cellulose (CMC-Na) (Sigma, USA), xyloglucan (TCI, Japan), wheat arabinoxylan (Sigma, USA) or p-nitrophenyl-β-D-glucopyranoside (pNPG) (Sigma, USA)
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and 50 mM acetate buffer (pH 6.0) was incubated at 50˚C with shaking at 800 rpm for 20 min. Then, 200 µL of DNS solution was added, and the mixture was heated at 100˚C for 10 min to stop the reaction. The solution was cooled in an ice bath for 2 min, and the supernatant was used directly or after a brief spin to determine its absorbance at 520 nm. One enzyme activity
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unit was defined as the amount of enzyme required to release 1.0 µmol of reducing sugars (glucose was used as a standard) per minute under the reaction conditions. All assays were repeated three times.
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The effect of metal ions and chemical reagents on the activity of the recombinant enzyme
To determine whether various metal ions and chemical reagents could influence the activity of the recombinant enzyme, NaCl, KCl, CaCl2, MgSO4, MnSO4, FeSO4, FeCl3, CuSO4, AlCl3, BaCl2, NiSO4, CoCl2, β-mercaptoethanol, EDTA and SDS were added to final concentrations of 1 or 2 mM to the 200 µL reaction solutions described above. The reaction solution without
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any added metal ions or chemical reagents was used as a control.
Determination of the optimum temperature and pH of the recombinant enzyme To determine the effect of pH on the activity of the recombinant enzyme, 200 µL of reaction solutions containing an appropriate amount of enzyme, 1% barley β-glucan and 50 mM
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buffers of various pHs (NaAc-Ac buffer pH 3.0-6.0, Tris-HCl buffer pH 7.0-9.0) was incubated and measured as described above. When determining the optimum pH for stability of the recombinant enzyme, the enzyme solution at various pHs was first incubated at 37˚C
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for 30 min, and then determined for their residual activity under above conditions. To determine the effect of reaction temperature on the activity of the recombinant enzyme, 200 µL reaction solutions were incubated at temperatures between 20 and 80˚C (at 10˚C intervals) for 20 min, and the DNS method was then used to determine the amount of reducing sugars. When determining the temperature stability of the recombinant enzyme, the enzyme solution was first incubated at various temperatures (20-80˚C at intervals of 10˚C) for 30 min, and the enzyme activity was then determined under above conditions.
Enzyme kinetics
ACCEPTED MANUSCRIPT For determination of enzyme kinetics, 200 µL the reaction solutions contained different concentrations of barley β-glucan (1-10 mg ml-1) or laminarin (1-50 mg ml-1) were incubated and measured as described above. Lineweaver-Burk plots were generated from which the Michaelis constant (Km) and maximum reaction rate (Vmax) of the recombinant enzyme were calculated. Analysis of hydrolysis products
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To analyze the hydrolysis products, 10 µL reaction solution containing 1% of laminarioligosaccharides (Megazyme, Ireland) and 0.8 µg recombinant enzyme, 50 mM NaAc-Ac (pH 6.0) was incubated at 37˚C with shaking at 800 rpm for 20 min, or 200µL reaction solution containing 1% of laminarin or barley β-glucan and 1.6 µg recombinant enzyme, 50 mM NaAc-Ac (pH 6.0) was incubated at 50˚C with shaking at 800 rpm for 20
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min. In the end, the samples were heated at 100˚C for 10 min. The reaction mixtures were subjected to thin-layer chromatography (TLC) analysis using TLC Silica gel 60 plates (Merck Millipore, Hong Kong, China). The composition of the eluent for the TLC analysis was
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n-butanol: formic acid: water = 2: 3: 1 (v: v: v). After the separation process was complete, the plate was dried and sprayed with developing solution (concentrated sulfuric acid: ethanol = 2: 8, v: v), and was then heated at 110˚C for 10 min to develop the color (Kumagai et al. 2014). Standard sugars of laminarioligosaccharides (glucose, laminaribiose, laminaritriose, aminaritetraose
laminaripentaose
and
laminarihexaose)
and
standard
sugars
of
celloligosaccharides (glucose, cellobiose, cellotriose, cellotetrose, cellopentose and
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cellohexaose) were used as standard markers on the TLC. Analysis of the transcription levels of the eng16A
The total RNA was extracted respectively from fresh mycelium or fruiting bodies (30 mm) grown on the straw medium using a Spin Column Fungal Total RNA Purification Kit
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(SK8659, BBI). The total RNA was reverse-transcripted into cDNA and used as a template for Quantitative PCR with the SYBR Premix ExTaqII (Tli RNase H Plus) mix (Takara, Beijing, China). The β-tubulin was used as the reference for mRNA level. The reactions
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were run on the StepOnePlus Real-Time PCR System (Applied Biosystems, Beijing, China). Relative mRNA levels were expressed as the 2-∆CT calculated by the comparative CT value method:
CT = (CTtarget-CTβ-tubulin) (Casarini et al. 2006; Ponchel et al. 2003).
Results Expression and purification of the recombinant enzyme The sequence that encodes the signal peptide (MYSLQALPVYLLLVVAQGITFAEA) was removed from the eng16A gene so its extracellular secretion was mediated by the N-terminal α-factor signal peptide of the vector. The recombinant mature ENG16A formed a fusion
ACCEPTED MANUSCRIPT protein with the vector’s C-terminal 6X histidines. After three days of induction cultivation, the hydrolysis activity of the culture medium of the recombinant expression strain with the eng16A reached to 9.27 U ml-1 using the barley β-glucan substrate. The recombinant enzyme was purified from the culture medium by Ni-affinity chromatography, and an imidazole concentration gradient was used to elute the protein (Fig 1(a)). The fraction with the highest barley β-glucan hydrolysis activity appeared in the eluate
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when the imidazole concentration was at 100 mM. The yield of the recombinant enzyme was 0.048 mg ml-1 culture medium, and the specific activity of the enzyme was 118.8 U mg-1 protein.
The SDS-PAGE analysis showed that there was a protein band at approximately 60 kDa
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in the culture medium of the recombinant expression strain with the eng16A compared to the control strain with empty vector. The purified enzyme from the culture medium by Ni-affinity chromatography also appeared at this position as a single band on SDS-PAGE (Fig. 1(b)).
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When this protein band was analyzed by MALDI TOF/TOF Ms, the amino acid sequence of the trypsinized protein fragment was confirmed to be consistent with the amino acid sequence of the putative endo-β-1,3(4)-glucanase (GenBank accession: XP_001828985), suggesting that the ENG16A had been successfully expressed in P. pastoris GS115. Substrate specificity
As shown in Table 1, the recombinant ENG16A could only act on substrates containing β-1, 3
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glycosidic bonds but could not act on substrates containing only β-1,4-bonds (CM-cellulose) or β-1,6- glycosidic bonds (Pustulan). Interestingly, the activity of this enzyme towards CM-pachyman, which only contains β-1,3-glycosidic bonds, was 72.09 U mg-1, however, its activity towards barley β-glucan, which contains both β-1,3-glycosidic and β-1,4-glycosidic
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bonds (more β-1,4-glycosidic bonds), was significantly higher, reaching 118.75 U mg-1 (a 64.72 % increase over its activity towards CM-pachyman). In contrast, its activity towards the substrate laminarin, which contains both β-1,3-glycosidic and β-1,6-glycosidic bonds (more
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β-1,3-glycosidic bonds), was relatively low at only 35.45 U mg-1 (a 50.83% decrease compared with its activity towards CM-pachyman). Furthermore, this enzyme activity towards the specific endo-glucanase substrates periodate-oxidized laminarin (Goni et al., 2011) was similar to that towards laminarin, and had no activity towards the exoglucanase substrate pNPG. This results indicated the Eng16A is an endo-beta-1,3-glucanase. The hydrolysis products of the recombinant enzyme TLC analysis of the hydrolysis products from the reactions of the recombinant ENG16A with the substrates laminarioligosaccharides, laminarin and barley β-glucan showed that ENG16A had almost no hydrolytic effect on laminaribiose, but it could hydrolyze laminaritriose, laminaritetraose, laminaripentaose, and laminarihexaose into oligosaccharides with even lower degrees of oligomerization (the smallest hydrolysis product was a disaccharide, but there was no
ACCEPTED MANUSCRIPT glucose production). ENG16A hydrolyzed laminarin and barley β-glucan to produce a series of corresponding oligosaccharides; the oligosaccharides with the lowest degrees of oligomerization were trisaccharides (Fig. 2).
Effects of metal ions and chemical reagents on the recombinant enzyme The effects of various metal ions and chemical reagents on the activity of ENG16A are shown in Table 2. Na+, K+, Fe2+ and Ni2+ did not have significant impacts on the activity of ENG16A.
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At a concentration of 1 mM, Ca2+ and Mg2 + did not have significant effects on enzyme activity; however, when their concentration reached 2 mM, the activity of ENG16A increased to 250%. Even at a concentration of 1 mM, Mn2+ increased ENG16A’s activity to 200%; at a concentration of 2 mM, it increased the activity to 240%. At concentrations of 1 mM, Fe3+,
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Al3+ and Co2+ did not significantly increase the activity of ENG16A; however, when their concentrations reached 2 mM, all of them were able to increase the activity to 180%. The presence of Cu2+ significantly inhibited the activity of ENG16A; at a concentration of 1 mM,
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it caused a 95% reduction in the activity of ENG16A, and at 2 mM, the enzyme activity was reduced by 98%. Ba2+, β-mercaptoethanol, EDTA and SDS inhibited the activity of ENG16A to varying degrees. These results suggest that ENG16A has a high sensitivity to these metal ions and chemical reagents. However, the response of the activity of ENG16A to different metal ions is different from reported other microbial β-1,3(4)-glucanase (Chen et al., 2012; Hua et al., 2011; Meng et al., 2016).
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The optimum temperature and pH of the recombinant enzyme
When the reaction temperature was between 20 and 60˚C, the activity of ENG16A increased as the temperature increased; however, when the reaction temperature reached 70˚C, the activity of ENG16A dropped rapidly to 20% of its maximum activity. The temperature
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stability test results showed that ENG16A could maintain its stability up to 40˚C; when the temperature was at 50˚C or above, the activity of ENG16A decreased dramatically (Fig. 3a). The optimum pH for ENG16A was found to be pH 6.0. In a reaction system in which the pH
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was between 5.0 and 9.0, the activity of ENG16A could reach over 70% of its maximum activity; however, only in the pH range 5.0-6.0 did the enzyme maintain better stability (above 70% of its maximum activity) (Fig. 3b). Enzyme kinetics
The initial reaction velocity of ENG16A was determined at 50˚C and pH 6.0. Using the initial velocity of the reactions containing ENG16A and various concentrations of substrate, a Michaelis-Menten hyperbolic curve and the corresponding Lineweaver-Burk plots were generated (Fig. 4). From the Lineweaver-Burk plots, when barley β-glucan was used as the substrate, ENG16A exhibited a Km of 20.84 mg ml-1 and a Vmax of 384.62 µmol mg-1 min-1; when laminarin was used as the substrate, ENG16A exhibited a Km of 6.49 mg ml-1 and a Vmax of 57.47 µmol mg-1 min-1.
ACCEPTED MANUSCRIPT Expression of the eng16A during Coprinopsis cinerea development By comparing differences in eng16A gene transcription of C. cinerea during its mycelium and fruiting body development stages in the straw medium, we found that during the mycelium stage, the mRNA level of eng16A was relatively high, but after entering the fruiting body growth and development stage, the mRNA level of eng16A decreased to one third of its transcript level in the mycelium stage (Fig. 5).
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Discussion
Initially, E. coli Rosetta was used as the host strain to heterologously express ENG16A, but was unsuccessful due to its lacking of appropriate posttranslational modifications of this recombinant protein (data not shown) (Morton and Potter 2000). Finally we succeeded
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expressed ENG16A in yeast P. pastoris, which futheer proved that P. pastoris is an good host for the heterologous expression of recombinant proteins with the advantages of being easy to culture, capable of posttranslationally modifying proteins and forming di-sulfide bonds, and
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folding proteins into their correct conformation (Shumiao et al. 2010).
The present study found that when a substrate contained β-1,4-glycosidic bonds in addition to β-1,3-glycosidic bonds, the activity of ENG16A was higher than when the substrate
only
contained
β-1,3-glycosidic
bonds.
When
the
substrate
contained
β-1,6-glycosidic bonds in addition to β-1,3-glycosidic bonds, the activity of ENG16A was lower than when the substrate only contained β-1,3-glycosidic bonds. Furthermore, if the
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substrate only contained β-1,4-glycosidic bonds or β-1,6-glycosidic bonds, ENG16A did not exhibit any hydrolysis activity. This result indicates that ENG16A can only hydrolyze substrates containing β-1,3-glycosidic bonds, but adjacent β-1,4-glycosidic bonds may providing a more conducive structure for enzyme access, whereas nearby β-1,6-glycosidic
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bond branch may provides a space barrier for enzyme access to hydrolyze β-1,3-glycosidic bonds. These findings are significantly different than our previous findings on the hydrolytic
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characteristics of the highly expressed endo-β-1,3-glucanase ENG extracted from the mature fruiting body pileus of C. cinerea. For endo-β-1,3-glucanase ENG, the presence of either β-1,4-glycosidic or β-1,6-glycosidic bonds in the substrate resulted in higher hydrolytic activity than when the substrate only contained β-1,3-glycosidic bonds (Zhou et al. 2015). In addition, the Km value of ENG16A with barley β-glucan was larger than with laminarin, suggesting that its affinity for barley β-glucan is lower than its affinity for laminarin. This observation may be related to the fact that barley β-glucan contains mainly β-1,4-glycosidic bonds mixed with β-1,3-glycosidic bonds (Fry et al. 2008; Shibuya and Iwasaki 1985), whereas the main chain of laminarin contains β-1,3-glycosidic bonds with β-1,6-branched glucose residues (Read et al. 1996); therefore, as a β-1,3(4)-glucanase, ENG16A has a stronger affinity for laminarin and a weaker affinity for barley β-glucan. However, although
ACCEPTED MANUSCRIPT its affinity for barley β-glucan is lower, the adjacent β-1,4-glycosidic bond is favor for ENG16A to hydrolyze β-1,3-glycosidic bonds, leading to an increased Vmax; similarly, the nearby β-1,6-glycosidic bond hinders its hydrolysis of β-1,3-glycosidic bonds, resulting in a decreased Vmax. It has been reported that a β-1,3(4)-glucanase from basidiomycete Phanerochaete chrysosporium more effectively hydrolyzed laminarin than a β-1,3/1,4-glucan, lichenan (Kawai et al. 2006) and the activity of a β-1,3(4)-glucanase from fungal
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Trichoderma sp. towards barley mixed-linkage β-1,3/1,4-glucan was only 9-41% of activity towards laminarin (Kuge et al. 2015), in contrast, the activity for laminarin of the β-1,3(4)-glucanase from fungal Penicillium pinophilum (Chen et al. 2012) or Paecilomyces sp. (Hua et al. 2011) was only 12.6 %, 22.3 % of the activity for barley mixed-linkage
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1,3/1,4-β-glucan, respectively. However, so far the physiological significance of the different effects of the types of glycosides adjacent to the β-1,3-glycoside in β-1,3-glucans on the hydrolytic activity of β-1,3-glucanases has not been explored. We hypothesize that the
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differences of substrate specificity between different β-1,3(4)-glucanase are favour for them to performance different physiological functions.
In this study, we used straw medium containing 88 % dry rice straw, 5 % wheat bran, 3 % corn flour to cultivate C. cinerea, all of these medium components contain mixed-linkage β-1,3/1,4-glucans (Buckeridge et al. 1999; Li et al. 2006; Shibuya and Iwasaki 1985). Based on the observation that ENG16A is highly expressed during the mycelium stage and its
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hydrolysis is favour for β-1,3/1,4-glucans, we speculate that during the early growth stage of C. cinerea, the mycelium must degrade the cellulose, hemicellulose and lignin in the culture medium to provide the materials and energy for self-growth, and this process may be completed by the concerted action of ENG16A and a series of related enzymes. We
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previously reported that the endo-β-1,3-glucanase ENG, which is highly expressed when the pileus is undergoing autolysis, has a higher hydrolytic activity towards β-1,6-glycosidic bond
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branch-containing β-1,3-glucans to adapt to its need to degrade the cell wall during the pileus autolysis process (Zhou et al. 2015). However, the major function of ENG16A reported here is to correspond to degradation of the mixed-linkage β-1,3/1,4-glucan in the culture medium. It is known that the core structure of the cell walls of most fungi, including C. cinerea, is a complex in which a β-1,6-glycosidic bond branch-containing β-1,3-glucan is connected to chitin (Fleet 1991; Fontaine et al. 2000; Kollár et al. 1995; Pérez and Ribas 2004). We hypothesize that, to avoid damaging the cell wall components of C. cinerea via the secretion of ENG16A outside the cells, ENG16A has evolved to have lower hydrolytic activity towards β-1,6-glycosidic bond branch-containing β-1,3/1,6-glucans; therefore, even when its expression level is higher in the mycelium stage, it is still difficult for ENG16A to cause damage to the cell walls of C. cinerea itself.
ACCEPTED MANUSCRIPT Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 31570046), the Priority Academic Development Program of Jiangsu Higher Education Institutions and the Scientific Innovation, the program of Natural Science Research of Jiangsu
Province of China (Project BK20140918).
References
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Higher Education Institutions of China (Grant No. 14KJB180013), and the NSF of Jiangsu
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Bradford MM, 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry 72: 248-254. Buckeridge MS, Vergara CE, Carpita NC, 1999. BIOCHEMISTRY AND MACROMOLECULAR STRUCTURE-The Mechanism of Synthesis of a Mixed-Linkage (1--> 3),(1-> 4) bD-Glucan in Maize. Evidence for Multiple Sites of Glucosyl Transfer in the Synthase Complex. Plant Physiology 120: 1105-1116.
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Casarini A, Pinto E, Jallad R, Giorgi R, Giannella-Neto D, Bronstein M, 2006. Dissociation between tumor shrinkage and hormonal response during somatostatin analog treatment in an acromegalic patient: preferential expression of somatostatin receptor subtype 3. Journal of endocrinological investigation 29: 826-830. Chen X, Meng K, Shi P, Bai Y, Luo H, Huang H, Yuan T, Yang P, Yao B, 2012. High-level expression of a novel Penicillium endo-1,3(4)-β-D-glucanase with high specific activity in
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Pichia pastoris. Journal of industrial microbiology and biotechnology 39: 869-876.
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Cole G, Hung C-Y, 2001. The parasitic cell wall of Coccidioides immitis. Medical mycology 39: 31-40. Delgado N, Xue J, Yu J-J, Hung C-Y, Cole GT, 2003. A recombinant β-1, 3-glucanosyltransferase homolog of Coccidioides posadasii protects mice against coccidioidomycosis. Infection and immunity 71: 3010-3019. Fleet G, 1991. Cell walls. The yeasts 4: 199-277. Fontaine T, Hartland RP, Diaquin M, Simenel C, Latgé JP, 1997. Differential patterns of activity displayed by two exo-beta-1, 3-glucanases associated with the Aspergillus fumigatus cell wall. Journal of bacteriology 179: 3154-3163.
ACCEPTED MANUSCRIPT Fontaine T, Simenel C, Dubreucq G, Adam O, Delepierre M, Lemoine J, Vorgias CE, Diaquin M, Latgé J-P, 2000. Molecular organization of the alkali-insoluble fraction of Aspergillus fumigatus cell wall. Journal of Biological Chemistry 275: 27594-27607.
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Fry SC, Nesselrode BH, Miller JG, Mewburn BR, 2008. Mixed-linkage (1→ 3, 1→ 4) -β-d-glucan is a major hemicellulose of Equisetum (horsetail) cell walls. New Phytologist 179: 104-115. Fukuda K, Hiraga M, Asakuma S, Arai I, Sekikawa M, Urashima T, 2008. Purification and characterization of a novel exo-β-1, 3-1, 6-glucanase from the fruiting body of the edible mushroom Enoki (Flammulina velutipes). Bioscience, biotechnology, and biochemistry 72: 3107-3113.
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Goldstein IS, Hay GW, Lewis BA, Smith F, 1965. Controlled degradation of polysaccharides by periodate oxidation reduction and hydrolysis. In: Whistler, R.L. (Ed.), Methods in Carbohydrate Chemistry, vol. 5. Academic Press, New York, pp. 361–370. Goni O, Sanchez-Ballesta MT, Merodio C, Escribano MI, 2011. A cryoprotective and cold-adapted 1,3-β-endoglucanase from cherimoya (Annona cherimola) fruit. Phytochemistry 72: 844-854.
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Hammad F, Ji J, Watling R, Moore D, 1993. Cell population dynamics in Coprinus cinereus: co-ordination of cell inflation throughout the maturing basidiome. Mycological Research 97: 269-274.
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Hua C, Yi H, Jiao L, 2011. Cloning and expression of the endo-1,3 (4)-β-glucanase gene from Paecilomyces sp. FLH30 and characterization of the recombinant enzyme. Bioscience, biotechnology, and biochemistry 75: 1807-1812.
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Hung C-Y, Yu J-J, Lehmann PF, Cole GT, 2001. Cloning and expression of the gene which encodes a tube precipitin antigen and wall-associated β-glucosidase of Coccidioides immitis. Infection and immunity 69: 2211-2222. Iten W, 1969. Zur Funktion hydrolytischer Enzyme bei der Autolyse von Coprinus. Diss. Naturwiss. ETH Zürich, Nr. 4247, 0000. Ref.: Matile, P.; Korref.: Frey-Wyssling, A. Iten W, Matile P, 1970. Role of chitinase and other lysosomal enzymes of Coprinus lagopus in the autolysis of fruiting bodies. Journal of General Microbiology 61: 301-309. Jayus, McDougall BM, Seviour RJ (2002) Factors affecting the synthesis of (1→3) and (1→6)- β-glucanases by the fungus Acremonium sp. IMI 383068 grown in batch culture. Enzyme and Microbial Technology 31: 289–299
ACCEPTED MANUSCRIPT Kawai R, Igarashi K, Yoshida M, Kitaoka M, Samejima M, 2006. Hydrolysis of β-1, 3/1, 6-glucan by glycoside hydrolase family 16 endo-1, 3 (4)-β-glucanase from the basidiomycete Phanerochaete chrysosporium. Applied microbiology and biotechnology 71: 898-906.
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Kawakami S, Nomura K, Tsuchida H, Mizuno M, 2004. An exo b-1, 3-glucanase synthesized de novo degrades lentinan during storage of Lentinule edodes and diminishes immunomodulating activity of the mushroom. CARBOHYDRATE POLYMERS 56: 279-286. Kollár R, Petráková E, Ashwell G, Robbins PW, Cabib E, 1995. Architecture of the yeast cell wall the linkage between chitin and β (13)-glucan. Journal of Biological Chemistry 270: 1170-1178.
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Kües U, 2000. Life history and developmental processes in the basidiomycete Coprinus cinereus. Microbiology and molecular biology reviews 64: 316-353.
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Kuge T, Nagoya H, Tryfona T, Kurokawa T, Yoshimi Y, Dohmae N, Tsubaki K, Dupree P, Tsumuraya Y, Kotake T, 2015. Action of an endo-β-1, 3 (4)-glucanase on cellobiosyl unit structure in barley β-1, 3: 1, 4-glucan. Bioscience, biotechnology, and biochemistry 79: 1810-1817.
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Kumagai Y, Satoh T, Inoue A, Ojima T, 2014. A laminaribiose-hydrolyzing enzyme, AkLab, from the common sea hare Aplysia kurodai and its transglycosylation activity. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 167: 1-7. Laemmli U, 1970. Most commonly used discontinuous buffer system for SDS electrophoresis. Nature 227: 680-685.
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Li W, Cui SW, Kakuda Y, 2006. Extraction, fractionation, structural and physical characterization of wheat β-D-glucans. CARBOHYDRATE POLYMERS 63: 408-416. Liu Z, Niu X, Wang J, Zhang W, Yang M, Liu C, Xiong Y, Zhao Y, Pei S, Qin Q, 2015. Comparative Study of Nonautolytic Mutant and Wild-Type Strains of Coprinopsis cinerea Supports an Important Role of Glucanases in Fruiting Body Autolysis. Journal of agricultural and food chemistry 63: 9609-9614. Meng D, Wang B, Ma X, Ji S, Lu M, Li F, 2016. Characterization of a thermostable endo-1,3(4)-β-glucanase from Caldicellulosiruptor sp. strain F32 and its application for yeast lysis. Applied microbiology and biotechnology. 100: 4923-4934. Miller GL, 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical chemistry 31: 426-428.
ACCEPTED MANUSCRIPT Miyake H, Takemaru T, Ishikawa T, 1980. Sequential production of enzymes and basidiospore formation in fruiting bodies of Coprinus macrorhizus. Archives of Microbiology 126: 201-205. Moore D, 2003. Fungal morphogenesis. Cambridge University Press.
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Morton CL, Potter PM, 2000. Comparison of Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Spodoptera frugiperda, and COS7 cells for recombinant gene expression. Molecular biotechnology 16: 193-202.
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Mouyna I, Fontaine T, Vai M, Monod M, Fonzi WA, Diaquin M, Popolo L, Hartland RP, Latgé J-P, 2000. Glycosylphosphatidylinositol-anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall. Journal of Biological Chemistry 275: 14882-14889.
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Mouyna I, Sarfati J, Recco P, Fontaine T, Henrissat B, Latgé J-P, 2002. Molecular characterization of a cell wall-associated β (1–3) endoglucanase of Aspergillus fumigatus. Medical mycology 40: 455-464. Nombela C, Molina M, Cenamor R, Sanchez M, 1988. Yeast beta-glucanases: a complex system of secreted enzymes. Microbiological sciences 5: 328-332.
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Pérez P, Ribas JC, 2004. Cell wall analysis. Methods 33: 245-251.
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Ponchel F, Toomes C, Bransfield K, Leong FT, Douglas SH, Field SL, Bell SM, Combaret V, Puisieux A, Mighell AJ, 2003. Real-time PCR based on SYBR-Green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions. BMC biotechnology 3: 18.
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Read SM, Currie G, Bacic A, 1996. Analysis of the structural heterogeneity of laminarin by electrospray-ionisation-mass spectrometry. Carbohydrate research 281: 187-201. Sakamoto Y, Nakade K, Konno N, Sato T, 2012. Senescence of the Lentinula edodes fruiting body after harvesting. INTECH Open Access Publisher. Shibuya N, Iwasaki T, 1985. Structural features of rice bran hemicellulose. Phytochemistry 24: 285-289. Shumiao Z, Huang J, Zhang C, Deng L, Hu N, Liang Y, 2010. High-level expression of an Aspergillus niger endo-beta-1, 4-glucanase in Pichia pastoris through gene codon optimization and synthesis. Journal of microbiology and biotechnology 20: 467-473. Tao Y, Xie B, Yang Z, Chen Z, Chen B, Deng Y, Jiang Y, van Peer AF, 2013. Identification and expression analysis of a new glycoside hydrolase family 55 exo-β-1,
ACCEPTED MANUSCRIPT 3-glucanase-encoding gene in Volvariella volvacea suggests a role in fruiting body development. Gene 527: 154-160.
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Zhou Y, Zhang W, Liu Z, Wang J, Yuan S, 2015. Purification, characterization and synergism in autolysis of a group of 1, 3-β-glucan hydrolases from the pilei of Coprinopsis cinerea fruiting bodies. Microbiology 161: 1978-1989.
Fig. 1 Purification (a) and SDS-PAGE analysis (b) of the recombinant ENG16A. Lanes: M, standard protein molecular weight markers; 1, the culture medium of control stain; 2, the
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culture medium of recombinant expression strain; 3, the purified recombinant ENG16A Fig. 2 TLC analysis products of ENG16A action on Laminarioligosaccharides (1-5), Laminarin (6) and Barley β-glucan (7). ST(L), standard sugars of Laminarioligosaccharides; G1, glucose; L2-L6, Laminaribiose to Laminarihexaose; ST(C), standard sugars of
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celloligosaccharides; G1, glucose; C2-C6, cellobiose to cellohexaose. Fig. 3 The optimal temperature and pH of purified recombinant ENG16A. a, Temperature
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effect and Thermostability of ENG16A activity towards barley β-glucan. b, pH effect and pH stability of ENG16A activity towards barley β-glucan. Fig. 4 a, Effect of barley β-glucan concentration on the activity of ENG16A. b, Effect of Laminarin concentration on the activity of ENG16A. Fig. 5 Relative mRNA level of eng16A in mycelium and fruiting body.
ACCEPTED MANUSCRIPT Table 1 Substrate specificity of ENG16A Substrate
Major linkage type
Substrate specificity (U mg-1) a
β-1,3; β-1,4
118.75±2.66
CM-pachyman
β-1,3
72.09±3.37
Laminarin
β-1,3; β-1,6
35.45±2.23
Periodate-oxidized laminarin
β-1,3; β-1,6
39.80±1.84
Pustulan
β-1,6
0
Oat spelt xylan
β-1,4
0
Avicel
β-1,4
CMC-Na
β-1,4
Xyloglucan
β-1,4
Wheat arabinoxylan
β-1,4
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0
0
0
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Values represent the means ± SD (n=3)
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a
0
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pNPG
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Barley β-glucan
ACCEPTED MANUSCRIPT Table 2 Effect of metal ions and chemical reagents on the hydrolytic activity of ENG16A Relative activity(%)a
Chemicals
2 mM
None
100
100
Na+
102.36±1.27
104.64±0.45
K+
103.28±2.33
Ca2+
129.91±3.32
Mg2+
113.34±0.87
Mn2+
214.95±3.67
Fe2+
103.73±1.53
Fe3+
116.27±0.86
Al3+ Ba2+
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EDTA SDS
253.60±4.11
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238.73±2.67
112.43±0.77 180.02±0.89 1.87±0.22
109.69±1.78
198.36±3.67
95.32±2.69
59.23±3.89
98.24±1.65
93.27±2.44
143.04±1.37
184.82±2.53
93.23±0.75
77.49±1.44
84.29±2.07
50.44±1.68
72.23±1.43
25.95±2.11
Values represent the means ± SD (n=3) relative to the untreated control samples
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a
252.97±2.73
5.31±0.47
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Ni2+
β-Mercaptoethanol
109.04±1.78
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Cu2+
Co2+
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1 mM
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S1878-6146(16)30143-X
DOI:
10.1016/j.funbio.2016.09.003
Reference:
FUNBIO 770
To appear in:
Fungal Biology
Received Date: 25 January 2016 Revised Date:
5 September 2016
Accepted Date: 6 September 2016
Please cite this article as: Wang, J., Kang, L., Liu, Z., Yuan, S., Gene cloning, heterologous expression and characterization of a Coprinopsis cinerea endo-β-1,3(4)-glucanase, Fungal Biology (2016), doi: 10.1016/j.funbio.2016.09.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Gene cloning, heterologous expression and characterization of a Coprinopsis cinerea endo-β-1,3(4)-glucanase
Jun Wang, Liqin Kang, Zhonghua Liu*, Sheng Yuan* Jiangsu Key Laboratory for Microbes and Microbial Functional Genomics, Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources,
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College of Life Science, Nanjing Normal University, Nanjing 210023, PR China
* Co-corresponding author:
Cell Pnone: 86-135-8409-3709; E-mail: [email protected].
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*(Liu, Z.,)
[email protected]. College of Life Science Nanjing Normal University
Xianlin University Park
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PR China
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Nanjing, 210023
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1 Wenyuan Rd
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*(Yuan, S.,) Phone: 86-25-8589-1067; Fax: 86-25-85891067; E-mail:
Running Head: endo-β-1,3(4)-glucanase for nutrition
Highlights:
ENG16A highly expresses during the mycelium stage Adjacent β-1,4- bonds favors ENG16A hydrolysis of β-1,3-glycosidic bonds Adjacent β-1,6- bonds hinders ENG16A hydrolysis of β-1,3-glycosidic bonds An endo-β-1,3(4)-glucanase corresponds to nutrient degradation.
ACCEPTED MANUSCRIPT Abstract A gene coding endo-β-1,3(4)-glucanase (ENG16A) was cloned from Coprinopsis cinerea and heterologously expressed in Pichia pastoris. ENG16A only acts on substrates containing β-1, 3 glycosidic bonds but not on substrates containing only β-1,4- or β-1,6- glycosidic bonds.
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Interestingly, compared to the activity of this enzyme towards carboxymethyl (CM)-pachyman containing only β-1,3-glycosidic bonds, its activity towards barley β-glucan containing both β-1,3-glycosidic and β-1,4-glycosidic bonds was increased by 64.72 %,, its activity towards laminarin containing both β-1,3-glycosidic and β-1,6-glycosidic bonds was
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decreased by 50.83%. In addition, ENG16A has a higher Km value and Vmax for barley β-glucan than laminarin, which may be related to the fact that barley β-glucan contains
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mainly β-1,4-glycosidic bonds mixed with a few β-1,3-glycosidic bonds, whereas laminarin mainly contains β-1,3-glycosidic bonds with a few β-1,6-branched glucose residues. The adjacent β-1,4-glycosidic bond promotes ENG16A to hydrolyze β-1,3-glycosidic bonds, leading to an increased Vmax; the nearby β-1,6-glycosidic bonds inhibited its hydrolysis of β-1,3-glycosidic bonds, resulting in a decreased Vmax. This property is suggested to be
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related to the mechanism that C. cinerea uses to degrade and utilize hemicellulose in straw medium and to protect its cell wall during the mycelium growth stage.
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autolysis
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Key words: Endo-β-1,3(4)-glucanase, barley β-glucan, laminarin, nutrition degradation, wall
ACCEPTED MANUSCRIPT Introduction C. cinerea is an ideal model organism with which to study the growth and development of basidiomycete fruiting bodies (Kües 2000). Similar to all other agarics, C. cinerea exhibits obvious fruiting body autolysis (Fukuda et al. 2008; Kües 2000; Kawakami et al. 2004;
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Sakamoto et al. 2012; Tao et al. 2013). The autolysis of the fruiting body pileus of C. cinerea are mainly resulted from action of various hydrolases which were synthesized and secreted to degrade the cell walls for the release of basidiospores during maturation of fruiting body (Hammad et al. 1993; Iten 1969; Kües 2000; Miyake et al. 1980; Moore 2003). We recently
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reported that a group of glucanases with different action mode were isolated from the pileus of mature fruiting body of C. cinerea including an endo-β-1,3-glucanase, an β-glucosidase (Zhou et al.
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exo-β-1,3-glucanase, a β-1,3-glucosidase, as well as a possible
2015). These glucanases degrade the β-1,3/1,6-glucans in the cell walls of the pileus in a synergistic manner. Furthermore, through mutagenesis, we obtained one mutant of C. cinerea in which the pileus does not undergo autolysis (Liu et al. 2015). A comparative study of the wild type and non-autolysis mutant of C. cinerea revealed that during the processes of pileus
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opening and hydrolysis, the glucanase activity in the extract of the wild type C. cinerea pileus was significantly higher than in the extract of the mutant C. cinerea pileus, suggesting that glucanase plays a major role in the autolysis of the C. cinerea pileus. When the expression
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levels of 43 glucanases, which mainly act on β-1,3-glycosidic bonds, were examined in the C. cinerea genome, we found that the expression levels of the four glucanases mentioned above
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that we previously purified and an additional glucanase were significantly higher than the expression levels of other 38 glucanases in the wild type C. cinerea in pileus during its opening process and that their expression levels were significantly suppressed in the mutant C. cinerea pileus, which does not undergo autolysis, during its opening process. This result suggests that these five glucanase genes may play key roles in the pileus autolysis process of C. cinerea (Liu et al. 2015). Apart from the five glucanase genes that may be directly associated with pileus autolysis, there are another 38 functionally similar glucanase genes of the 43 glucanase genes of C. cinerea that mainly act on β-1,3-glycosidic bonds. Their expression levels are not significantly different between wild type and mutant C. cinerea, and most of them belong to
ACCEPTED MANUSCRIPT glycoside hydrolase families 2 and 16 (Liu et al. 2015). Generally, it is thought that β-glucanase not only plays a role in degrading the cell wall (Fontaine et al. 1997; Liu et al. 2015; Mouyna et al. 2002; Zhou et al. 2015) and remodeling cell wall components and structures (Cole and Hung 2001; Delgado et al. 2003; Hung et al. 2001; Mouyna et al. 2000)
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during the growth, development and morphogenesis of fungi, but also involve in degrading the nutritional components in the culture medium to maintain the energy and nutrition needed for cell growth (Fukuda et al. 2008; Larriba et al. 1993; Nombela et al. 1988). The synthesis of some specific β-glucanases was reported to depend on the induction of some specific
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polysaccharides in the medium (Jayus et al., 2002). To determine wheather those rest of the 38 glucanases play a roles in the degradation of culture medium or the other physiological
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process during the growth and development of C. cinerea, we selected a glucanase from the 38-glucanases, an endo-β-1,3(4)-glucanase (ENG16A) (GenBank accession: XP_001828985), which belongs to glycoside hydrolase family 16, as the study subject. After cloning, the selected gene was heterologously expressed in yeast Pichia pastoris, and the properties and
Materials and methods
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possible physiological functions of this enzyme were studied.
Construction of the expression strain
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The mycelia and fruiting bodies of C. cinerea were cultured in a straw medium containing 88% dry rice straw, 5% wheat bran, 3% corn flour, 2% fertilizer, 1% lime powder, 1%
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sucrose and water quantum satis, as described by Zhou et al. (2015). The total RNA was extracted from the fruiting body of C. cinerea using the Spin Column Fungal Total RNA Purification Kit (Bio Basic Inc., Amherst, NY, USA) and the synthesis of the cDNA was carried out using the HiScript II Q RT SuperMix for qPCR kit (+gDNA wiper) (Vazyme, Nanjing, China). The upstream primer eng16A-F and downstream primer eng16A-R were used to amplify the cDNA of the eng16A gene from the the total cDNA by PCR, in which the first 72 bp nucleotide sequence encoding a signal peptide of 24 amino acids was removed. The PCR product was purified using the Spin Column Gel Extraction Kit (Takara, Beijing, China) and identified by DNA sequencing.
ACCEPTED MANUSCRIPT The eng16A cDNA fragment and the plasmid pPICZαA (ThermoFisher Scientific, Grand Island, USA) double digested respectively with EcoRI and NotI were ligated to form the plasmid pPICZαA-eng16A following the instruction manual of the ClonExpress® Entry One Step Cloning Kit (Vazyme, Nanjing, China). The plasmid pPICZαA-eng16A was transformed into Escherichia coli DH5α cells for amplification. After producing an expanded culture of E. coli DH5α, the pPICZαA-eng16A plasmid was isolated from E. coli cells and singly digested
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by SacI to linearize it. The linearized plasmid was transformed into P. pastoris GS115 cells by electroporation. The transformants were allowed to grow on YPDS + Zeocin™ (ThermoFisher Scientific, USA) plates for three days, and single colonies were picked to confirm that the eng16A gene was successfully integrated into the genome of P. pastoris
(ThermoFisher Scientific, Grand Island,USA). Expression of the recombinant enzyme
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GS115 at the AOX1 site according to the instructions of the Pichia Expression Kit
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The P. pastoris GS115 cells with the integrated eng16A gene were inoculated to a 100 mL flask containing 15 mL of BMGY medium and cultured for 18-20 h at 30˚C with shaking at 220 rpm. When the OD600 reached 2-6, the yeast cells were harvested by centrifugation at 1000×g for 10 min. The yeast cells were resuspended and transferred to a 500 mL flask containing 100 mL BMMY medium for induction cultivation at 30˚C with shaking at 220 rpm for three days; the medium was supplemented with methanol at 0.5% of the total volume
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every 24 h to induce expression.
Purification of the recombinant enzyme
The culture medium was centrifuged at 12000×g at 4˚C to remove yeast cells. The supernatant was dialyzed at 4˚C overnight against equilibrium buffer (300 mM NaCl, 50 mM
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NaH2PO4, 10 mM imidazole, 10 mM Tris base, pH 8.0). The Ni-affinity chromatography method was then used to purify the recombinant enzyme from the culture supernatant by the manual of the ProteinIso® Ni-NTA Resin (Transgen, Beijing, China). All of the purification
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steps were carried out at 4˚C.
Protein analysis of the recombinant enzyme The purity and molecular weight of the purified recombinant enzyme were determined by SDS-PAGE (Laemmli 1970). The protein concentration of the recombinant enzyme was determined using the Bradford method with Coomassie Brilliant Blue (Bradford 1976), and bovine serum albumin was used as the standard. The amino acid sequencing of the purified protein was determined by MALDI-TOF/TOF Ms (Zhou et al., 2015). Determination of the activity of the recombinant enzyme The hydrolysis activity of the recombinant enzyme was determined by measuring the amount of reducing sugars released after it hydrolyzed the substrate. The amount of reducing sugars was determined by the 3,5-dinitrosalicylic acid method (DNS) (Miller 1959). Briefly, 200 µL
ACCEPTED MANUSCRIPT of reaction solution containing an appropriate amount of enzyme, 1% barley β-glucan (Megazyme, Ireland), carboxymethyl (CM)-pachyman (Megazyme, Ireland), laminarin (TCI, Japan), periodate-oxidized laminarin (performed as described Goldstein et al. (1965) ), pustulan (Megazyme, Ireland), oat spelt xylan (Sigma, USA), avicel (Megazyme, Ireland), sodium carboxyl methyl cellulose (CMC-Na) (Sigma, USA), xyloglucan (TCI, Japan), wheat arabinoxylan (Sigma, USA) or p-nitrophenyl-β-D-glucopyranoside (pNPG) (Sigma, USA)
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and 50 mM acetate buffer (pH 6.0) was incubated at 50˚C with shaking at 800 rpm for 20 min. Then, 200 µL of DNS solution was added, and the mixture was heated at 100˚C for 10 min to stop the reaction. The solution was cooled in an ice bath for 2 min, and the supernatant was used directly or after a brief spin to determine its absorbance at 520 nm. One enzyme activity
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unit was defined as the amount of enzyme required to release 1.0 µmol of reducing sugars (glucose was used as a standard) per minute under the reaction conditions. All assays were repeated three times.
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The effect of metal ions and chemical reagents on the activity of the recombinant enzyme
To determine whether various metal ions and chemical reagents could influence the activity of the recombinant enzyme, NaCl, KCl, CaCl2, MgSO4, MnSO4, FeSO4, FeCl3, CuSO4, AlCl3, BaCl2, NiSO4, CoCl2, β-mercaptoethanol, EDTA and SDS were added to final concentrations of 1 or 2 mM to the 200 µL reaction solutions described above. The reaction solution without
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any added metal ions or chemical reagents was used as a control.
Determination of the optimum temperature and pH of the recombinant enzyme To determine the effect of pH on the activity of the recombinant enzyme, 200 µL of reaction solutions containing an appropriate amount of enzyme, 1% barley β-glucan and 50 mM
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buffers of various pHs (NaAc-Ac buffer pH 3.0-6.0, Tris-HCl buffer pH 7.0-9.0) was incubated and measured as described above. When determining the optimum pH for stability of the recombinant enzyme, the enzyme solution at various pHs was first incubated at 37˚C
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for 30 min, and then determined for their residual activity under above conditions. To determine the effect of reaction temperature on the activity of the recombinant enzyme, 200 µL reaction solutions were incubated at temperatures between 20 and 80˚C (at 10˚C intervals) for 20 min, and the DNS method was then used to determine the amount of reducing sugars. When determining the temperature stability of the recombinant enzyme, the enzyme solution was first incubated at various temperatures (20-80˚C at intervals of 10˚C) for 30 min, and the enzyme activity was then determined under above conditions.
Enzyme kinetics
ACCEPTED MANUSCRIPT For determination of enzyme kinetics, 200 µL the reaction solutions contained different concentrations of barley β-glucan (1-10 mg ml-1) or laminarin (1-50 mg ml-1) were incubated and measured as described above. Lineweaver-Burk plots were generated from which the Michaelis constant (Km) and maximum reaction rate (Vmax) of the recombinant enzyme were calculated. Analysis of hydrolysis products
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To analyze the hydrolysis products, 10 µL reaction solution containing 1% of laminarioligosaccharides (Megazyme, Ireland) and 0.8 µg recombinant enzyme, 50 mM NaAc-Ac (pH 6.0) was incubated at 37˚C with shaking at 800 rpm for 20 min, or 200µL reaction solution containing 1% of laminarin or barley β-glucan and 1.6 µg recombinant enzyme, 50 mM NaAc-Ac (pH 6.0) was incubated at 50˚C with shaking at 800 rpm for 20
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min. In the end, the samples were heated at 100˚C for 10 min. The reaction mixtures were subjected to thin-layer chromatography (TLC) analysis using TLC Silica gel 60 plates (Merck Millipore, Hong Kong, China). The composition of the eluent for the TLC analysis was
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n-butanol: formic acid: water = 2: 3: 1 (v: v: v). After the separation process was complete, the plate was dried and sprayed with developing solution (concentrated sulfuric acid: ethanol = 2: 8, v: v), and was then heated at 110˚C for 10 min to develop the color (Kumagai et al. 2014). Standard sugars of laminarioligosaccharides (glucose, laminaribiose, laminaritriose, aminaritetraose
laminaripentaose
and
laminarihexaose)
and
standard
sugars
of
celloligosaccharides (glucose, cellobiose, cellotriose, cellotetrose, cellopentose and
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cellohexaose) were used as standard markers on the TLC. Analysis of the transcription levels of the eng16A
The total RNA was extracted respectively from fresh mycelium or fruiting bodies (30 mm) grown on the straw medium using a Spin Column Fungal Total RNA Purification Kit
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(SK8659, BBI). The total RNA was reverse-transcripted into cDNA and used as a template for Quantitative PCR with the SYBR Premix ExTaqII (Tli RNase H Plus) mix (Takara, Beijing, China). The β-tubulin was used as the reference for mRNA level. The reactions
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were run on the StepOnePlus Real-Time PCR System (Applied Biosystems, Beijing, China). Relative mRNA levels were expressed as the 2-∆CT calculated by the comparative CT value method:
CT = (CTtarget-CTβ-tubulin) (Casarini et al. 2006; Ponchel et al. 2003).
Results Expression and purification of the recombinant enzyme The sequence that encodes the signal peptide (MYSLQALPVYLLLVVAQGITFAEA) was removed from the eng16A gene so its extracellular secretion was mediated by the N-terminal α-factor signal peptide of the vector. The recombinant mature ENG16A formed a fusion
ACCEPTED MANUSCRIPT protein with the vector’s C-terminal 6X histidines. After three days of induction cultivation, the hydrolysis activity of the culture medium of the recombinant expression strain with the eng16A reached to 9.27 U ml-1 using the barley β-glucan substrate. The recombinant enzyme was purified from the culture medium by Ni-affinity chromatography, and an imidazole concentration gradient was used to elute the protein (Fig 1(a)). The fraction with the highest barley β-glucan hydrolysis activity appeared in the eluate
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when the imidazole concentration was at 100 mM. The yield of the recombinant enzyme was 0.048 mg ml-1 culture medium, and the specific activity of the enzyme was 118.8 U mg-1 protein.
The SDS-PAGE analysis showed that there was a protein band at approximately 60 kDa
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in the culture medium of the recombinant expression strain with the eng16A compared to the control strain with empty vector. The purified enzyme from the culture medium by Ni-affinity chromatography also appeared at this position as a single band on SDS-PAGE (Fig. 1(b)).
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When this protein band was analyzed by MALDI TOF/TOF Ms, the amino acid sequence of the trypsinized protein fragment was confirmed to be consistent with the amino acid sequence of the putative endo-β-1,3(4)-glucanase (GenBank accession: XP_001828985), suggesting that the ENG16A had been successfully expressed in P. pastoris GS115. Substrate specificity
As shown in Table 1, the recombinant ENG16A could only act on substrates containing β-1, 3
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glycosidic bonds but could not act on substrates containing only β-1,4-bonds (CM-cellulose) or β-1,6- glycosidic bonds (Pustulan). Interestingly, the activity of this enzyme towards CM-pachyman, which only contains β-1,3-glycosidic bonds, was 72.09 U mg-1, however, its activity towards barley β-glucan, which contains both β-1,3-glycosidic and β-1,4-glycosidic
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bonds (more β-1,4-glycosidic bonds), was significantly higher, reaching 118.75 U mg-1 (a 64.72 % increase over its activity towards CM-pachyman). In contrast, its activity towards the substrate laminarin, which contains both β-1,3-glycosidic and β-1,6-glycosidic bonds (more
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β-1,3-glycosidic bonds), was relatively low at only 35.45 U mg-1 (a 50.83% decrease compared with its activity towards CM-pachyman). Furthermore, this enzyme activity towards the specific endo-glucanase substrates periodate-oxidized laminarin (Goni et al., 2011) was similar to that towards laminarin, and had no activity towards the exoglucanase substrate pNPG. This results indicated the Eng16A is an endo-beta-1,3-glucanase. The hydrolysis products of the recombinant enzyme TLC analysis of the hydrolysis products from the reactions of the recombinant ENG16A with the substrates laminarioligosaccharides, laminarin and barley β-glucan showed that ENG16A had almost no hydrolytic effect on laminaribiose, but it could hydrolyze laminaritriose, laminaritetraose, laminaripentaose, and laminarihexaose into oligosaccharides with even lower degrees of oligomerization (the smallest hydrolysis product was a disaccharide, but there was no
ACCEPTED MANUSCRIPT glucose production). ENG16A hydrolyzed laminarin and barley β-glucan to produce a series of corresponding oligosaccharides; the oligosaccharides with the lowest degrees of oligomerization were trisaccharides (Fig. 2).
Effects of metal ions and chemical reagents on the recombinant enzyme The effects of various metal ions and chemical reagents on the activity of ENG16A are shown in Table 2. Na+, K+, Fe2+ and Ni2+ did not have significant impacts on the activity of ENG16A.
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At a concentration of 1 mM, Ca2+ and Mg2 + did not have significant effects on enzyme activity; however, when their concentration reached 2 mM, the activity of ENG16A increased to 250%. Even at a concentration of 1 mM, Mn2+ increased ENG16A’s activity to 200%; at a concentration of 2 mM, it increased the activity to 240%. At concentrations of 1 mM, Fe3+,
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Al3+ and Co2+ did not significantly increase the activity of ENG16A; however, when their concentrations reached 2 mM, all of them were able to increase the activity to 180%. The presence of Cu2+ significantly inhibited the activity of ENG16A; at a concentration of 1 mM,
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it caused a 95% reduction in the activity of ENG16A, and at 2 mM, the enzyme activity was reduced by 98%. Ba2+, β-mercaptoethanol, EDTA and SDS inhibited the activity of ENG16A to varying degrees. These results suggest that ENG16A has a high sensitivity to these metal ions and chemical reagents. However, the response of the activity of ENG16A to different metal ions is different from reported other microbial β-1,3(4)-glucanase (Chen et al., 2012; Hua et al., 2011; Meng et al., 2016).
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The optimum temperature and pH of the recombinant enzyme
When the reaction temperature was between 20 and 60˚C, the activity of ENG16A increased as the temperature increased; however, when the reaction temperature reached 70˚C, the activity of ENG16A dropped rapidly to 20% of its maximum activity. The temperature
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stability test results showed that ENG16A could maintain its stability up to 40˚C; when the temperature was at 50˚C or above, the activity of ENG16A decreased dramatically (Fig. 3a). The optimum pH for ENG16A was found to be pH 6.0. In a reaction system in which the pH
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was between 5.0 and 9.0, the activity of ENG16A could reach over 70% of its maximum activity; however, only in the pH range 5.0-6.0 did the enzyme maintain better stability (above 70% of its maximum activity) (Fig. 3b). Enzyme kinetics
The initial reaction velocity of ENG16A was determined at 50˚C and pH 6.0. Using the initial velocity of the reactions containing ENG16A and various concentrations of substrate, a Michaelis-Menten hyperbolic curve and the corresponding Lineweaver-Burk plots were generated (Fig. 4). From the Lineweaver-Burk plots, when barley β-glucan was used as the substrate, ENG16A exhibited a Km of 20.84 mg ml-1 and a Vmax of 384.62 µmol mg-1 min-1; when laminarin was used as the substrate, ENG16A exhibited a Km of 6.49 mg ml-1 and a Vmax of 57.47 µmol mg-1 min-1.
ACCEPTED MANUSCRIPT Expression of the eng16A during Coprinopsis cinerea development By comparing differences in eng16A gene transcription of C. cinerea during its mycelium and fruiting body development stages in the straw medium, we found that during the mycelium stage, the mRNA level of eng16A was relatively high, but after entering the fruiting body growth and development stage, the mRNA level of eng16A decreased to one third of its transcript level in the mycelium stage (Fig. 5).
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Discussion
Initially, E. coli Rosetta was used as the host strain to heterologously express ENG16A, but was unsuccessful due to its lacking of appropriate posttranslational modifications of this recombinant protein (data not shown) (Morton and Potter 2000). Finally we succeeded
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expressed ENG16A in yeast P. pastoris, which futheer proved that P. pastoris is an good host for the heterologous expression of recombinant proteins with the advantages of being easy to culture, capable of posttranslationally modifying proteins and forming di-sulfide bonds, and
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folding proteins into their correct conformation (Shumiao et al. 2010).
The present study found that when a substrate contained β-1,4-glycosidic bonds in addition to β-1,3-glycosidic bonds, the activity of ENG16A was higher than when the substrate
only
contained
β-1,3-glycosidic
bonds.
When
the
substrate
contained
β-1,6-glycosidic bonds in addition to β-1,3-glycosidic bonds, the activity of ENG16A was lower than when the substrate only contained β-1,3-glycosidic bonds. Furthermore, if the
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substrate only contained β-1,4-glycosidic bonds or β-1,6-glycosidic bonds, ENG16A did not exhibit any hydrolysis activity. This result indicates that ENG16A can only hydrolyze substrates containing β-1,3-glycosidic bonds, but adjacent β-1,4-glycosidic bonds may providing a more conducive structure for enzyme access, whereas nearby β-1,6-glycosidic
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bond branch may provides a space barrier for enzyme access to hydrolyze β-1,3-glycosidic bonds. These findings are significantly different than our previous findings on the hydrolytic
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characteristics of the highly expressed endo-β-1,3-glucanase ENG extracted from the mature fruiting body pileus of C. cinerea. For endo-β-1,3-glucanase ENG, the presence of either β-1,4-glycosidic or β-1,6-glycosidic bonds in the substrate resulted in higher hydrolytic activity than when the substrate only contained β-1,3-glycosidic bonds (Zhou et al. 2015). In addition, the Km value of ENG16A with barley β-glucan was larger than with laminarin, suggesting that its affinity for barley β-glucan is lower than its affinity for laminarin. This observation may be related to the fact that barley β-glucan contains mainly β-1,4-glycosidic bonds mixed with β-1,3-glycosidic bonds (Fry et al. 2008; Shibuya and Iwasaki 1985), whereas the main chain of laminarin contains β-1,3-glycosidic bonds with β-1,6-branched glucose residues (Read et al. 1996); therefore, as a β-1,3(4)-glucanase, ENG16A has a stronger affinity for laminarin and a weaker affinity for barley β-glucan. However, although
ACCEPTED MANUSCRIPT its affinity for barley β-glucan is lower, the adjacent β-1,4-glycosidic bond is favor for ENG16A to hydrolyze β-1,3-glycosidic bonds, leading to an increased Vmax; similarly, the nearby β-1,6-glycosidic bond hinders its hydrolysis of β-1,3-glycosidic bonds, resulting in a decreased Vmax. It has been reported that a β-1,3(4)-glucanase from basidiomycete Phanerochaete chrysosporium more effectively hydrolyzed laminarin than a β-1,3/1,4-glucan, lichenan (Kawai et al. 2006) and the activity of a β-1,3(4)-glucanase from fungal
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Trichoderma sp. towards barley mixed-linkage β-1,3/1,4-glucan was only 9-41% of activity towards laminarin (Kuge et al. 2015), in contrast, the activity for laminarin of the β-1,3(4)-glucanase from fungal Penicillium pinophilum (Chen et al. 2012) or Paecilomyces sp. (Hua et al. 2011) was only 12.6 %, 22.3 % of the activity for barley mixed-linkage
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1,3/1,4-β-glucan, respectively. However, so far the physiological significance of the different effects of the types of glycosides adjacent to the β-1,3-glycoside in β-1,3-glucans on the hydrolytic activity of β-1,3-glucanases has not been explored. We hypothesize that the
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differences of substrate specificity between different β-1,3(4)-glucanase are favour for them to performance different physiological functions.
In this study, we used straw medium containing 88 % dry rice straw, 5 % wheat bran, 3 % corn flour to cultivate C. cinerea, all of these medium components contain mixed-linkage β-1,3/1,4-glucans (Buckeridge et al. 1999; Li et al. 2006; Shibuya and Iwasaki 1985). Based on the observation that ENG16A is highly expressed during the mycelium stage and its
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hydrolysis is favour for β-1,3/1,4-glucans, we speculate that during the early growth stage of C. cinerea, the mycelium must degrade the cellulose, hemicellulose and lignin in the culture medium to provide the materials and energy for self-growth, and this process may be completed by the concerted action of ENG16A and a series of related enzymes. We
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previously reported that the endo-β-1,3-glucanase ENG, which is highly expressed when the pileus is undergoing autolysis, has a higher hydrolytic activity towards β-1,6-glycosidic bond
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branch-containing β-1,3-glucans to adapt to its need to degrade the cell wall during the pileus autolysis process (Zhou et al. 2015). However, the major function of ENG16A reported here is to correspond to degradation of the mixed-linkage β-1,3/1,4-glucan in the culture medium. It is known that the core structure of the cell walls of most fungi, including C. cinerea, is a complex in which a β-1,6-glycosidic bond branch-containing β-1,3-glucan is connected to chitin (Fleet 1991; Fontaine et al. 2000; Kollár et al. 1995; Pérez and Ribas 2004). We hypothesize that, to avoid damaging the cell wall components of C. cinerea via the secretion of ENG16A outside the cells, ENG16A has evolved to have lower hydrolytic activity towards β-1,6-glycosidic bond branch-containing β-1,3/1,6-glucans; therefore, even when its expression level is higher in the mycelium stage, it is still difficult for ENG16A to cause damage to the cell walls of C. cinerea itself.
ACCEPTED MANUSCRIPT Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 31570046), the Priority Academic Development Program of Jiangsu Higher Education Institutions and the Scientific Innovation, the program of Natural Science Research of Jiangsu
Province of China (Project BK20140918).
References
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Higher Education Institutions of China (Grant No. 14KJB180013), and the NSF of Jiangsu
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Hung C-Y, Yu J-J, Lehmann PF, Cole GT, 2001. Cloning and expression of the gene which encodes a tube precipitin antigen and wall-associated β-glucosidase of Coccidioides immitis. Infection and immunity 69: 2211-2222. Iten W, 1969. Zur Funktion hydrolytischer Enzyme bei der Autolyse von Coprinus. Diss. Naturwiss. ETH Zürich, Nr. 4247, 0000. Ref.: Matile, P.; Korref.: Frey-Wyssling, A. Iten W, Matile P, 1970. Role of chitinase and other lysosomal enzymes of Coprinus lagopus in the autolysis of fruiting bodies. Journal of General Microbiology 61: 301-309. Jayus, McDougall BM, Seviour RJ (2002) Factors affecting the synthesis of (1→3) and (1→6)- β-glucanases by the fungus Acremonium sp. IMI 383068 grown in batch culture. Enzyme and Microbial Technology 31: 289–299
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ACCEPTED MANUSCRIPT 3-glucanase-encoding gene in Volvariella volvacea suggests a role in fruiting body development. Gene 527: 154-160.
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Zhou Y, Zhang W, Liu Z, Wang J, Yuan S, 2015. Purification, characterization and synergism in autolysis of a group of 1, 3-β-glucan hydrolases from the pilei of Coprinopsis cinerea fruiting bodies. Microbiology 161: 1978-1989.
Fig. 1 Purification (a) and SDS-PAGE analysis (b) of the recombinant ENG16A. Lanes: M, standard protein molecular weight markers; 1, the culture medium of control stain; 2, the
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culture medium of recombinant expression strain; 3, the purified recombinant ENG16A Fig. 2 TLC analysis products of ENG16A action on Laminarioligosaccharides (1-5), Laminarin (6) and Barley β-glucan (7). ST(L), standard sugars of Laminarioligosaccharides; G1, glucose; L2-L6, Laminaribiose to Laminarihexaose; ST(C), standard sugars of
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celloligosaccharides; G1, glucose; C2-C6, cellobiose to cellohexaose. Fig. 3 The optimal temperature and pH of purified recombinant ENG16A. a, Temperature
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effect and Thermostability of ENG16A activity towards barley β-glucan. b, pH effect and pH stability of ENG16A activity towards barley β-glucan. Fig. 4 a, Effect of barley β-glucan concentration on the activity of ENG16A. b, Effect of Laminarin concentration on the activity of ENG16A. Fig. 5 Relative mRNA level of eng16A in mycelium and fruiting body.
ACCEPTED MANUSCRIPT Table 1 Substrate specificity of ENG16A Substrate
Major linkage type
Substrate specificity (U mg-1) a
β-1,3; β-1,4
118.75±2.66
CM-pachyman
β-1,3
72.09±3.37
Laminarin
β-1,3; β-1,6
35.45±2.23
Periodate-oxidized laminarin
β-1,3; β-1,6
39.80±1.84
Pustulan
β-1,6
0
Oat spelt xylan
β-1,4
0
Avicel
β-1,4
CMC-Na
β-1,4
Xyloglucan
β-1,4
Wheat arabinoxylan
β-1,4
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0
0
0
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Values represent the means ± SD (n=3)
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a
0
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pNPG
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Barley β-glucan
ACCEPTED MANUSCRIPT Table 2 Effect of metal ions and chemical reagents on the hydrolytic activity of ENG16A Relative activity(%)a
Chemicals
2 mM
None
100
100
Na+
102.36±1.27
104.64±0.45
K+
103.28±2.33
Ca2+
129.91±3.32
Mg2+
113.34±0.87
Mn2+
214.95±3.67
Fe2+
103.73±1.53
Fe3+
116.27±0.86
Al3+ Ba2+
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253.60±4.11
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238.73±2.67
112.43±0.77 180.02±0.89 1.87±0.22
109.69±1.78
198.36±3.67
95.32±2.69
59.23±3.89
98.24±1.65
93.27±2.44
143.04±1.37
184.82±2.53
93.23±0.75
77.49±1.44
84.29±2.07
50.44±1.68
72.23±1.43
25.95±2.11
Values represent the means ± SD (n=3) relative to the untreated control samples
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a
252.97±2.73
5.31±0.47
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Ni2+
β-Mercaptoethanol
109.04±1.78
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Cu2+
Co2+
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