Efficient Conversion of Inulin to ... - ACS Publications

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Efficient Conversion of Inulin to Inulooligosaccharides through Endoinulinase from Aspergillus niger Yanbing Xu,† Zhaojuan Zheng,‡ Qianqian Xu,‡ Qiang Yong,‡ and Jia Ouyang*,‡,§ †

College of Forestry and ‡College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, People’s Republic of China § Key Laboratory of Forest Genetics & Biotechnology of the Ministry of Education, Nanjing, People’s Republic of China S Supporting Information *

ABSTRACT: Inulooligosaccharides (IOS) represent an important class of oligosaccharides at industrial scale. An efficient conversion of inulin to IOS through endoinulinase from Aspergillus niger is presented. A 1482 bp codon optimized gene fragment encoding endoinulinase from A. niger DSM 2466 was cloned into pPIC9K vector and was transformed into Pichia pastoris KM71. Maximum activity of the recombinant endoinulinase, 858 U/mL, was obtained at 120 h of the high cell density fermentation process. The optimal conditions for inulin hydrolysis using the recombinant endoinulinase were investigated. IOS were harvested with a high concentration of 365.1 g/L and high yield up to 91.3%. IOS with different degrees of polymerization (DP, mainly DP 3−6) were distributed in the final reaction products. KEYWORDS: inulooligosaccharide, inulin, endoinulinase, Aspergillus niger, Pichia pastoris

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inhibition.21 Moreover, the inhibition of the byproducts, such as glucose, caused lower yields and additional separation that made this process less profitable.22 An alternative enzyme for FOS production is endoinulinase (EC 3.2.1.7). As the composition of native inulin is GFn molecules, accompanied by several Fm molecules, thereby, FOS can be synthesized by endoinulinase hydrolysis of the internal linkages of inulin. As a result, inulotriose (F3), inulotetraose (F4), and inulopentaose (F5) were released as the major products. Simultaneously, it supervened on GFn molecules.23 There have been some reports of IOS production employing various endoinulinases under different reaction conditions. For example, a dual endoinulinase system originating from Xanthomonas sp. and Pseudomonas sp. was used to produce IOS from pure inulin.24 The synthesis of the IOS in an aqueous−organic system using inulinase from Kluyveromyces marxianus and Aspergillus niger was studied as well.25 As the endoinulinase gas gained increasing industrial interest in recent years, its production has drawn extensive attention. Microorganisms are the best sources for commercial production of endoinulinases because of their easy cultivation and high yields. Up to now, many endoinulinases from various microorganisms, such as Arthrobacter sp.,26 Penicillium sp.,27 Pseudomonas sp.,28 Aspergillus ficuum,29 and A. niger30 have been isolated and characterized. Among them, A. niger was mostly chosen for production of recombinant endoinulinase in consideration of safety concerns and high activities. Although it has been found that the native strains can produce high yields of endoinulinase, it still could not meet the needs of industrial application.31 In addition, the isolation of endoinulinase from wild strains is complicated. Therefore, cloning and heterolo-

INTRODUCTION Inulin is a storage homopolysaccharide existing in the tubers of Jerusalem artichoke and roots of chicory, yacon. and dahlia. The content of inulin is >50% in their dried tubers and roots.1 Inulin consists of linear chains of β-2,1-linked D-fructose molecules terminated by a D-glucose residue through a sucrosetype linkage at the nonreducing end.2 Because inulin represents an inexpensive, renewable, abundant raw material and the hydrolytic product of inulin is a mix of fermentable sugars, inulin has received increasing attention for production of valueadded products, such as ultrahigh-fructose syrup,3 bioethanol,4 2,3-butanediol,5 single-cell protein,6 single-cell oil,7 highoptical-purity L-lactate,8 citric acid,9 and other chemicals.10 Instead of hydrolysis of inulin to monosaccharides, inulin can also be partially hydrolyzed to inulooligosaccharides (IOS),11 one kind of oligosaccharide. Oligosaccharides are relatively new functional food ingredients that have great potential to improve the quality of many foods as nondigestible prebiotics.12 Both the production and the applications of food-grade oligosaccharides are increasing rapidly, such as isomalto-oligosaccharides,13 cello-oligosaccharides,14 xylo-oligosaccharides,15 galacto-oligosaccharides,16 and fructooligosaccharides (FOS).17 Among them, FOS can be synthesized by transfructosylation from sucrose or by enzymatic hydrolysis of inulin under controlled conditions.18 Specifically, the FOS derived from inulin are referred to as IOS. The main enzyme for commercial FOS production is fructosyltransferase (EC 2.4.1.9). Fructosyltransferases act by breaking a sucrose molecule and then transferring the liberated fructose molecule to an acceptor molecule such as sucrose or another oligosaccharide to elongate the fructooligosaccharide.19 The FOS produced by fructosyltransferase were constituted of 1-kestose (GF2), nystose (GF3), and fructofuranosyl nystose (GF4).20 Current FOS production from sucrose using immobilized fructosyltransferases of fungi was disadvantageous due to a large loss of enzyme activity by end-product © 2016 American Chemical Society

Received: Revised: Accepted: Published: 2612

January 10, 2016 March 9, 2016 March 10, 2016 March 10, 2016 DOI: 10.1021/acs.jafc.5b05908 J. Agric. Food Chem. 2016, 64, 2612−2618

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Journal of Agricultural and Food Chemistry

presence of the optimized endoinulinase gene in the transformants was confirmed by PCR using yeast genomic DNA as template. For expression, the colonies were grown in 10 mL of YPD medium at 30 °C for 24 h, then inoculated into 100 mL of BMGY medium, and shaken (200 rpm) at 30 °C until OD600 nm between 2.0 and 6.0. The cells were collected by centrifugation at 1500−3000g for 5 min at room temperature. The supernatant was decanted and the cell pellets were resuspended in 25 mL of BMMY medium for further cultivation at 28 °C with constant shaking at 250 rpm. To maintain induction, 100% methanol was added to the culture to a final concentration of 1% (v/v) every 24 h throughout the induction phase. The recombinant P. pastoris strain with the highest endoinulinase yield in ab erlenmeyer flask was used to scale up fermentation. Batch fermentation was carried out in a 3 L fermenter (Bioflo 110, New Brunswick Scientific, USA) containing 1.5 L of BSM medium and 6.5 mL of PTM1 trace salts solution at 30 °C and pH 5.0. The pH was controlled by automatically adding ammonium hydroxide. After the level of dissolved oxygen increased, continuous glycerol feeding was carried out until the cellular yield of 180−220 g/L wet cells. When the dissolved oxygen increased again, a 100% methanol feeding (see the Supporting Information) containing 12 mL of PTM1 trace salts per liter of methanol was added to the medium for inducing protein expression. The induction temperature was maintained at 28 °C, and the level of dissolved oxygen concentration was maintained above 20% throughout the induction phase. The entire methanol induction phase lasted approximately 120 h until the highest level of endoinulinase activity. Determination of the Recombinant Endoinulinase Activity. The hydrolytic activity of endoinulinase was measured depending on the concentration of reducing sugars released from inulin.36 The reaction mixture containing 50 μL of diluted crude enzyme and 450 μL of 5% (w/v) inulin solution (dissolved in 0.1 M sodium acetate buffer, pH 4.6) was incubated at 60 °C for 10 min. All experiments were performed in three independent determinations. A separate blank of inactivated enzyme was set up for each sample to correct the nonenzymatic release of sugars. Then the produced reducing sugar was estimated by the 3,5-dinitrosalicylic acid method.37 Absorbance was read at 520 nm. A high absorbance indicated a high level of reducing sugar produced and consequently a high enzyme activity. Inulooligosaccharide Production from Inulin. Inulin from Jerusalem artichoke (Qinghai Weide Biotechnology Co. Ltd., Qinghai, China) was used as substrate. Inulin hydrolysis was carried out under various conditions, including temperature, pH, substrate concentration, and enzyme dosage, in stoppered 100 mL flasks incubated on a rotary shaker. At different intervals, 1 mL reaction mixtures were taken and incubated at 100 °C for 5 min to inactivate the enzyme. Each reaction uses three parallel samples to ensure the accuracy of the experiment. Samples were centrifuged at 10800g force for 5 min, and the IOS were quantitatively analyzed by high-performance anion exchange chromatography quantitatively coupled with pulsed ampere detection (HPAEC-PAD). HPAEC-PAD Analysis Method. The analysis method for IOS determination was developed on HPAEC-PAD. It was performed on a CarboPac PA200 column (250 mm × 3 mm, Dionex, Sunnyvale, CA, USA) with a gradient elution of 200 mM NaOH and 500 mM NaAc as the mobile phase: 0−5 min, 60% water and 40% 200 mM NaOH solution; 5−25 min, 48% water, 40% 200 mM NaOH, and 0−12% 500 mM NaAc solution; 25−30 min, 48% water, 40% 200 mM NaOH, and 12% 500 mM NaAc solution; 30−33 min, 20% water, 40% 200 mM NaOH, and 40% 500 mM NaAc solution; 33−35 min, 100% 200 mM NaOH solution; 35−50 min, 60% water and 40% 200 mM NaOH solution. The column was set at 30 °C, and the flow rate was 0.3 mL/ min. The column temperature was set at 30 °C, and the flow rate was 0.4 mL/min.38 The method showed that IOS had a good linear relationship within 0.1−10 mg/L concentration range. The yield of IOS was calculated as the ratio of total IOS (g) to the inulin (g).

gous expression of endoinulinase in recombinant strains, such as Escherichia coli,29 Saccharomyces cerevisiae,32 Yarrowia lipolytica,26 and Pichia pastoris,33 is a better choice. Furthermore, the methylotrophic yeast P. pastoris is the most widely used eukaryotic expression system for the production of a variety of heterologous proteins from eukaryotes.34,35 In this study, the gene encoding an endoinulinase from A. niger DSM 2466 was cloned and expressed in P. pastoris KM71. To achieve high-level expression of recombinant endoinulinase in P. pastoris, the codon of the gene was optimized while the signal peptide was removed. Thereafter, the high cell density and high expression level of recombinant enzyme were achieved in 3 L fermenter. The conditions for IOS production from inulin employing endoinulinases were optimized, including temperature, pH, substrate concentration, and enzyme dosage. In addition, the degree of polymerization (DP) of IOS was determined. Combing all of these results, an endoinulinase-based method for large-scale production of IOS was established.

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MATERIALS AND METHODS

Strains, Primers, and Plasmids. A. niger DSM 2466 was purchased from Deutsche Sammlung von Mikroorganismen and Zellkulturen (DSMZ, Braunschweig, Germany) and used for amplifying the endoinulinase gene with primers P1 (5′-GAATTCCAGTCTAATGATTACCGTCCTTCATACCA-3′) and P2 (5′GCGGCCGCTCATTCAAGTGAAACACTGCGCACGT-3′). The primers were designed according to the endoinulinase gene sequence of A. niger AF10 (AF369388). Two restriction sites, EcoRI and NotI, were added to the front and end terminals of P1 and P2, respectively. P. pastoris strain KM71 (Invitrogen) was used as a heterologous host for expression of the endoinulinase gene. The expression vector pPIC9K (Invitrogen) was used for protein expression in P. pastoris. All strains and plasmids were stored at −80 °C. Medium and Culture Conditions. A. niger DSM 2466 was cultured in potato−dextrose medium. All P. pastoris transformants were cultured in YPD broth containing 100 μg/mL Geneticin (Invitrogen) at 30 °C. The MD medium was used to screen the Mut phenotype. YPD medium containing each concentration of Geneticin were used to screen resistant colonies. BSM and PTM1 trace salts solution were used for the high-density cells fermentation of P. pastoris. All media were prepared according to the instructions for the cultivation of yeast from Pichia Expression Kit (Invitrogen, USA, catalog no. K1740-01; see the Supporting Information). In addition, the high-cell-density fermentation of the P. pastoris used in the work was according to Pichia fermentation process guidelines of Invitrogen. Codon Usage Optimization and Construction of the Expression Vectors. The gene encoding endoinulinase without the signal peptide (EnInu) was cloned from A. niger DSM 2466 and sequenced by Generay Biotech Co. (Shanghai, China). Afterward, EnInu was inserted into the pPIC9K expression vector between the EcoRI and NotI restriction sites, yielding the recombinant expression vector pPIC9K-EnInu. The codons were optimized on the basis of the codon preference table containing a preference for each codon in the P. pastoris genome (at Web site http://www.kazusa.or.jp/codon). Modifications were made throughout the sequence. The optimized endoinulinase gene (EnInuop) was synthesized by Generay Biotech Co. and inserted into EcoRI and NotI of pPIC9K, yielding the recombinant expression vector pPIC9K-EnInuop. Transformation and Expression of Endoinulinase in P. pastoris. The recombinant expression vectors pPIC9K-EnInu and pPIC9K-EnInuop were linearized by SacI and transformed into P. pastoris for insertion at 5′AOX1 (KM71, Muts) by electroporation using Gene Pulser Xcell (Bio-Rad, Hercules, CA, USA). The recombinant clones were grown on MD medium at 30 °C for 3 days and further screened on YPD medium containing increasing concentrations of Geneticin for 3−5 days. The recombinant P. pastoris with multiple copies of the endoinulinase gene was obtained. The 2613

DOI: 10.1021/acs.jafc.5b05908 J. Agric. Food Chem. 2016, 64, 2612−2618

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Figure 1. DNA sequence alignment comparison of the EcoRI and NotI fragments of the EnInu gene (top) and the EnInuop gene (bottom). Without changing the amino acid sequence, several codons of the EnInu sequence were exchanged (shaded) into the new EnInuop sequence with highly used codons in P. pastoris for heterologous protein expression emprovement.

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RESULTS AND DISCUSSION Design and Synthesis of the Optimized Endoinulinase Gene for Expression in P. pastoris. The nucleotide sequence analysis showed that the gene length was 1482 bp encoding a protein of 493 amino acids. The homology analysis indicated that it exhibited >95% identity to that of A. niger CBS 513.88 (Genbank accession no. DQ233221), A. niger 9891 (Genbank accession no. AF546870), and A. ficuum JNSP5-06 (Genbank accession no. FJ984582). Although P. pastoris has been developed as an excellent expression host, the discrepancy of codon usage bias and base composition variations between host and target gene might decrease the heterologous protein expression. In this study, the heterologous production and secretion of both the original and optimized endoinulinase were carried out in P. pastoris. For the optimized sequence, the rare codons were replaced by preferred ones, which appear more commonly in P. pastoris. As shown in Figure 1, a total of 359 nucleotides were changed without modifying the encoding amino acids, and the GC content was decreased from 54.3 to 44.2%. The optimized endoinulinase gene showed 75.78% identity with the wild-type endoinulinase. Expression and Production of Recombinant Endoinulinase in P. pastoris. For expression, the recombinants were screened in MD and YPD/G418 plates, and the inset was identified by PCR. The P. pastoris KM71 transformed with vector pPIC9K was used as control. No endoinulinase activity was detected in the strain with the original gene. The expression efficiency of the engineered P. pastoris harboring pPIC9K-EnInuop/KM71 was further explored in a 3 L fermenter. As shown in Figure 2, the enzyme activity rapidly increased before 96 h and then stabilized. With methanol feeding, the concentration of cells gradually increased. This means that methanol was utilized by the recombinant P. pastoris

Figure 2. High-density fermentation of recombinant P. pastoris KM71/pPIC9K-EnInuop in a 3 L fementor. Initiation of induction. The temperatures of growth phase and induction phase were controlled at 30 and 28 °C, respectively; pH was controlled at 5.0, and methanol solution was added to maintain the level of dissolved oxygen concentration.

cells. After methanol induction for 120 h, the endoinulinase activity in the culture supernatant was 858 U/mL and the protein concentration in the culture medium was 3024.3 μg/ mL. SDS-PAGE analysis showed that there was one major band of protein, approximately 66.2 kDa, secreted into the culture medium (Figure 3). Previously, several endoinulinase genes have been identified, cloned, and expressed in various hosts including bacteria and molds.39 The endoinulinase gene (EnIA) from Arthrobacter sp. S37 was ligated into the expression vector pINA1317 and overexpressed in Yarrowia lipolytica Po1h. It was found that the endoinulinase activity and specific endoinulinase activity produced by the transformant 1317-EnIA were 16.7 U/ mL and 93.4 U/mg, respectively.26 An endoinulinase gene from A. niger 9819 (CGMCC0991) was expressed in P. pastoris 2614


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of endoinulinase was 291 U/ml, 273 times higher than that from the original strain.40 In this study, the activity of recombinant endoinulinase reached 858 U/mL after codon optimization in high-cell-density fermentation, which was much higher than the expression systems mentioned above. It is worth mentioning that the activity of recombinant endoinulinase from A. niger CICIM F0620 was higher. However, the report did not set blank control of inactivated enzyme to correct the nonenzymatic release of sugars.41 Optimization of IOS Production from Inulin. To increase the efficiency of IOS production, the hydrolysis conditions, including temperature, pH, substrate concentration, enzyme dosage, and reaction time, were investigated. To study the effect of temperature on IOS production, enzyme reactions were conducted using the same substrate concentration (200 g/ L) and the same amount of enzyme (40 units/g inulin) at pH 6.0 for 8 h. As shown in Figure 4A, the optimum temperature for inulin hydrolysis by endoinulinase was 60 °C, with the maximum yield of 86.7% and the maximum concentration of 161.3 g/L, which was acceptable in commercial production. Figure 4B shows that the yield of IOS increased from 38.9 to

Figure 3. SDS-PAGE analysis of culture supernatant of P. pastoris KM71/pPIC9K-EnInuop in a 3 L fementor during the induction phase. Lanes: 1−6, culture supernatant of recombinant P. pastoris after methanol induction for 0, 24, 48, 72, 96, or 120 h, respectively; M, protein weight maker.

GS115 using pPIC9 vector. The recombinant endoinulinase was highly expressed, and the optimization of the expression in a 7 L of fermentor was investigated. In the fermented broth, the concentration of protein secreted was 2.15 mg/mL. The activity

Figure 4. Effects of temperature, pH, substrate concentration, and enzyme dosage on the production of IOS by the recombinant endoinulinase. (A) Effect of temperature on IOS production. The reactions were incubated for 8 h at different temperatures, under conditions of 200 g/L inulin as substrate, pH 6.0, and enzyme dosage of 40 units per gram of inulin. (B) Effect of pH on IOS production. The reactions were incubated for 8 h at different pH values, using 200 g/L inulin as substrate, 60 °C, and enzyme dosage of 40 units per gram inulin. (C) Effect of substrate concentration on IOS production. The reactions were incubated for 8 h at different substrate concentration, under conditions of pH 6.0, 60 °C, and enzyme dosage of 40 units per gram inulin. (D) Effect of enzyme dosage on IOS production. The reactions were incubated for 8 h at different enzyme dosages, under conditions of 400 g/L inulin as substrate, pH 6.0, and 60 °C. Error bars correspond to the standard deviation of three independent determinations. 2615


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Figure 5. Chromatographic follow-up (using quantitative HPAEC and histogram representation) of inulin hydrolysis with EnInuop.

Figure 6. HPAEC of inulin hydrolysis with EnInuop.

Distribution of Reaction Products. To study the change of product composition during the hydrolysis, enzyme reactions were carried out under the optimal conditions obtained above: temperature of 60 °C, pH 6.0, substrate concentration of 400 g/L, and enzyme dosage of 40 U/g substrate. During the course of inulin hydrolysis by endoinulinase, aliquots of the reaction mixture were withdrawn and analyzed for the hydrolysis products by HPAEC. The distribution of products were harvested at different times (Figure 5). IOS can be observed at 0 h because the incubation at 100 °C before detection caused a part of the inulin to degrade. When the hydrolysis time was 4 h, IOS with higher DP, namely, GF4, F4, and GF5, were gradually hydrolyzed after reaching their maxima due to the presence of endoinulinase. At the same

87.4% when the reaction pH was increased from 4.0 to 6.0, whereas the yield was obviously decreased at higher pH. The effect of the substrate concentration was also investigated to determine the optimal range. The hydrolysis of inulin was carried out using the same amount of enzyme (40 units/g inulin) under six different substrate concentrations at 60 °C and pH 6.0 for 8 h. As observed in Figure 4C, the yields of IOS were all >80% within the inulin concentrations of 100− 400 g/L and then decreased. However, the concentration of IOS was continuously increased within the scope of this investigation. In previous studies, the low yields may lead to waste of substrate. Therefore, when endoinulinase was used for IOS production, 400 g/L inulin was optimal. To determine the optimal enzyme dosage, enzyme reactions were carried out with several enzyme concentrations using 400 g/L inulin at 60 °C and pH 6.0 for 8 h. As shown in Figure 4D, the maximum concentration and yield of IOS was found at 40 U/g substrate. The time course of inulin hydrolysis by endoinulinase and reaction products needed to be investigated. 2616


Journal of Agricultural and Food Chemistry time, the production of GF2, GF3, and F3 was gradually increased. When the reaction time was >8 h, the production of GF2, GF3, and F3 was no longer increased. This means that the endoinulinase does not affect GF2, GF3, and F3. In addition, the distribution of hydrolysis products at 4 h is shown in Figure 6. Under the optimal reaction conditions, the DP of IOS in reaction mixtures ranged from DP 3 to DP 6, where the major IOS were 8.84% GF2, 18.05% GF3, 30.18% F3, 15.67% GF4, 10.58% F4, and 7.97% GF5 oligomer, respectively. It was reported that purified endoinulinase originating from A. ficuum could hydrolyze up to 70% of inulin in 72 h.42 The hydrolysates by Pseudomonas sp. consisted of IOS ranging from DP 2 to DP 7, mainly DP 2 and DP 3, and the maximum yield of 75.6% in total IOS was achieved.28 The endoinulinase from Pseudomonas mucidolens expressed on the cell surface of S. cerevisiae cell could reach 71.2% yield after reaction for 30 h, and the major product was F4.43 By comparison, the EnInuop from A. niger DSM 2466 in this study can effectively hydrolyze inulin with a higher yield of 91.3%. The DP of IOS was evenly distributed in the final reaction products. Because of the high product concentration and short reaction time described above, the recombinant endoinulinase produced by P. pastoris was better for IOS production. In summary, an endoinulinase gene from A. niger DSM 2466 was cloned. Systematic codon usage optimization of the endoinulinase gene makes high-level expression of this gene in P. pastoris possible. SDS-PAGE proved the successful expression of endoinulinase after codon optimization in P. pastoris KM71. After high-cell-density fermentation of recombinant P. pastoris, the activity of endoinulinase reached 858 U/mL and the protein concentration in the culture media was 3024.3 μg/mL. Furthermore, the recombinant endoinulinase was used for enzymatic hydrolysis of inulin to produce IOS, with the maximum yield of IOS 91.3% and the concentration of IOS 365.1 g/L. The IOS production process developed in this study is inexpensive, efficient, and simple to scale up to industrial production.

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ABBREVIATIONS USED

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REFERENCES

IOS, inulooligosaccharides; FOS, fructooligosaccharides; DP, degree of polymerization; GFn, inulin molecule with a terminal glucose moiety; Fm, inulin molecule with fructose molecules only; GF2, 1-kestose; GF3, nystose; GF4, fructofuranosyl nystose; F3, Inulotriose; F4, inulotetraose; F5, inulopentaose; YPD, yeast peptone dextrose medium; MD, minimal dextrose medium; BSA, basal salts media; BMGY, buffered glycerol complex medium; BMMY, buffered methanol complex medium

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b05908. Medium formulas and methanol feeding strategy (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(J.O.) Mail: College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, People’s Republic of China. Phone: 86-025-85427129. Fax: 86-025-85427587. Email: [email protected]. Funding

This study was supported by the NSFC-NRCT project (51561145015), the National Natural Science Foundation of China (31300487), the Natural Science Foundation of Jiangsu Province of China (BK20130970), the National Hi-Tech Research and Development Program of China (2012AA022301), the Key Research and Development Program of Jiangsu Province of China (BF2015007), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Notes

The authors declare no competing financial interest. 2617


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