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Electronic Journal of Biotechnology

versão On-line ISSN 0717-3458

Electron. J. Biotechnol. vol.19 no.6 Valparaíso nov. 2016

http://dx.doi.org/10.1016/j.ejbt.2016.10.004 

RESEARCH ARTICLE

Over-expression of Mycobacterium neoaurum 3-ketosteroid-∆1-dehydrogenase in Corynebacterium crenatum for efficient bioconversion of 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione

 

Xian Zhanga, Dan Wua,Taowei Yanga, Meijuan Xua, Zhiming Raoa,b,*

a The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, PR China 
b State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi,Jiangsu 214122, PR China


ABSTRACT

Background: 3-Ketosteroid-∆1-dehydrogenase (KSDD), a flavoprotein enzyme, catalyzes the bioconversion of 4-androstene-3,17-dione (AD) to androst-1,4-diene-3,17-dione (ADD). To date, there has been no report about characterization of KSDD from Mycobacterium neoaurum strains, which were usually employed to produce AD or ADD by fermentation.

Results: In this work, Corynebacterium crenatum was chosen asa new host for heterologous expression of KSDD from M. neoaurum JC-12 after codon optimization of the KSDD gene. SDS-PAGE and western blotting results indicated that the recombinant C. crenatum harboring the optimized ksdd (ksddn) gene showed significantly improved ability to express KSDD. The expression level of KSDD was about 1.6-fold increased C. crenatum after codon optimization. After purification of the protein, we first characterized KSDD from M. neoaurum JC-12, and the results showed that the optimum temperature and pH for KSDD activity were 30°C and pH 7.0, respectively. The Km and Vmax values of purified KSDD were 8.91 µM and 6.43 mM/min. In this work, C. crenatum as a novel whole-cell catalyst was also employed and validated for bioconversion of AD to ADD. The highest transformation rate of AD to ADD by recombinant C. crenatum was about 83.87% after 10 h reaction time, which was more efficient than M. neoaurum JC-12 (only 3.56% at 10 h).

Conclusions: In this work, basing on the codon optimization, overexpression, purification and characterization of KSDD, we constructed a novel system, the recombinant C. crenatum SYPA 5-5 expressing KSDD, to accumulate ADDfromADefficiently. This work provided new insights into strengthening sterol catabolism by overexpressing the key enzyme KSDD, for efficient ADD production.

Keywords: Androst-1,4-diene-3,17-dione, Bioconversion, Codon optimization, Flavoprotein enzyme, Heterologous expression, Mycobacterium neoaurum,  Overexpression, Recombinant Corynebacterium, Sterol catabolism, Whole-cell catalyst.


 

1. Introduction

Steroid drug intermediates are widely used for the commercial production of pharmaceutical steroid drugs. Compared with the chemical synthesis process, bioprocess of transforming sterols to steroid drug intermediates has its obvious advantage and has been widely used as a common and economical alternative method in the pharmaceutical industry. The microbial transformation of steroids has long prevailed in the pharmaceutical industry since the 1950s. Degradation of steroids can yield much valuable steroidal derivatives, such as 4-androstene-3,17-dione (AD), androst-1,4-diene-3,17-dione (ADD), 9α-OH-AD, and 9α-OH-ADD. ADD has been acknowledged to be a worthwhile precursor in the synthesis of steroid pharmaceuticals such as oestrogens, contraceptive agents and progestogens. Chemical synthesis has been the main method of ADD production in the pharmaceutical industry for a long time. Nevertheless, the substantial consumption of organic chemicals and the production of chemical waste, make it an environmentally unfriendly approach. As an alternative and modest synthesis method, biocatalytic production of ADD has become a good alternative, mainly because it provides a superb combination of cost-effectiveness, sustainability and scalability.

3-Ketosteroid-∆1-dehydrogenase (KSDD) [EC 1.3.99.4] catalyzes the insertion of a double bond between the C1 and C2 atoms of the chemically stable 3-ketosteroid A-ring (Fig. 1).Several steroid-degrading bacteria with KSDD activity have been reported, including Mycobacterium, Rhodococcus, Comamonas, and Arthrobacter. The constructive N-terminal flavin adenine dinucleotide (FAD)-binding site was coincided with the sequence G-S-G-(A/G)-(A/ G)-(A/G)-X17-E. According to the crystal structure of the KSDD from Rhodococcus erythropolis, the enzyme does not have any trans-membrane helices, and the protein behaves as a soluble protein.

Fig. 1. Bioconversion of AD to ADD by S-ketosteroid-∆1 dehydrogenase.

Much work has been done on strain amelioration, enhancing or limiting KSDD expression by molecular or mutagenesis methods. In our previous research, we isolated and identified Mycobacterium neoaurum JC-12 with the capacity of converting phytosterol to the product ADD in our laboratory. The ksdd gene was amplified and expressed in Bacillus subtilis 168 using plasmid pMA5. Regrettably, the expression level of KSDD was very low for purification and no high yields of ADD were obtained. Nevertheless KSDD is a key enzyme for producing and accumulating ADD during steroid degradation. So far, there was no detail report that the KSDD from M neoaurum has been successfully characterized. This lack of knowledge limits, both, the knowledge on the properties of KSDD and the optimum use of this enzyme for converting AD to ADD.

In this work, Corynebacterium crenatum was chosen as a new host for heterologous expression of KSDD. As the native gene ksdd showed a poor codon usage bias for C. crenatum, we suspected that it should be one of the limiting factors leading to the inefficient expression. In order to improve the expression of KSDD, we designed and constructed a full-length synthetic gene by optimizing the codon usage without changing its amino acid sequence. We also investigated the purification and characterization of the KSDD from M. neoaurum JC-12. Under optimal conditions, the efficient transformation of AD to ADD by the whole-cell of the recombinant C. crenatum SYPA 5-5 was achieved.

2. Materials and methods

2.1. Bacterial strains, plasmids and culture conditions

C. crenatum strain SYPA 5-5 was screened and stored in our laboratory. It is an aerobic, Gram-positive, non-sporulating coryneform bacterium and a high-yield strain of amino acids in the industry.

M. neoaurum JC-12 and the vector pXMJ19 were preserved in our laboratory (Table 1). The strains C. crenatum SYPA 5-5/pXMJ19, M. neoaurum JC-12 and the recombinant C. crenatum SYPA 5-5 were cultivated at 30°C and 160 rpm in Luria-Bertani broth containing 0.5% glucose(LBG). Chloroamphenicol (50 mg/mL) was added to the growth medium if necessary. 4-Androstene-S,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD) were supplied by the Sigma-Aldrich Chemical Co. Inc. (USA). Hydroxypropyl-β-cyclodextrin (HP-β-CD) was purchased from Zhiyuan Bio-Technology Co., Ltd. (Binzhou, China). All other chemicals with the analytical grade could be purchased.

2.2. General cloning techniques

T4 DNA ligase, Mini Plasmid Rapid Isolation Kit and Mini DNA Rapid Purification kit were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Restriction enzymes were bought from TaKaRa Co. (Dalian, China).

2.3. Codon optimization of ksdd gene

According to the sequence of ksdd gene from M. neoaurum JC-12, the optimization of its codon was subjected to the Codon Adaptation Tool (http://www.jcat.de/) on the basis of codon preference of Corynebacterium glutamicum ATCC1S0S2. The high GC content which did not favor for gene expressing was reduced. The modified gene was synthesized by Sangon Biotech Co., Ltd., (Shanghai, China).

2.4. Construction of recombinant plasmid pXMJ19-ksdd and transformation of C. crenatum SYPA 5-5

The modified gene ksddII and native ksdd were amplified with the primers ksddI R/ksddII Fand ksddI R/ksddI F (Table 1). Both the modified and native genes were cloned into the plasmid pXMJ19 by designed primers with Hind III and BamH I restriction sites (underline). Then the recombinant plasmids were transformed into C. crenatum SYPA 5-5 to obtain engineered C. crenatum strains by the electroporation methods described by Tauch et al. Chloramphenicol was chosen as the selectable marker to screen the recombinant C. crenatum,and then verified by DNA sequencing.

Table 1
Bacterial strains, plasmids, and primers.

2.5. Expression of ksddII and ksddI in C. crenatum SYPA 5-5 with pXMJ19 and preparation ofcell extracts

The recombinant plasmids pXMJ19-ksddII and pXMJ19-ksddI were introduced into C. crenatum SYPA 5-5. Transformants were obtained after growing 72 h on selective LBG agar plate added with chloroamphenicol. The recombinant cells were grown in 50 mL LBG medium for 14 h with 50 uL IPTG (200 mg/mL) to induce the enzyme expression. Cell pellets (8000 g; 10 min; 4°C) were washed with 10 mL 50 mM Tris-HCl buffer (pH 7.0). The supernatant of culture was used for KSDD enzyme activity assay. Pellets were suspended in 5 mL Tris-HCl buffer and sonicated for 15 min. 0.1 mM dithiothreitol (DTT) was added to protect the enzyme. In order to remove the cell fragments, cell extracts were centrifuged for 30 min at 10,000 x g in an SIGMA SK-15 centrifuge. The supernatant of cell extraction was used for KSDD enzyme activity assay, SDS-PAGE (12% acrylamide) analysis or storage at -20°C with 10% glycerol.

2.6. SDS-PAGE analysis and determination of protein concentration

The samples used for SDS-PAGE were mixed with 2x SDS loading buffer (100 mM Tris-HCl, pH 6.8, 200 mM DTT, 0.4 g/L SDS, 0.02 g/L bromophenol blue, and 20% (v/v) glycerol) with ratio 1:1 (v/v). The mixture was then boiled in water for 10 min, and centrifuged for 30 min at 10,000 x g. The samples were run on a SDS-PAGE as described by Laemmli. Expression of KSDD was also detected by western blotting with a mouse monoclonal anti-His6 antibody. The samples were treated as described by Bao et al., and the bands were detected by chemiluminescence with luminol and peroxide with the help of a Bio-Rad Chemidoc XRS. Bradford method was employed to determine the protein concentration by using BSA as standard protein.

2.7. KSDD enzyme activity assay

Enzyme activity of KSDD was determined spectrophotometrically at 30°C using 2,6-dichlorophenolindophenol (DCPIP) and phenazine methosulphate (PMS). The reaction mixture (1 mL) consisted of 50 mM Tris-HCl buffer (pH 7.0), 1.5 mM PMS, 40 µM DCPIP, appropriate concentration of the supernatant or cell extract, and 500 uM AD in methanol (2%). Cofactor (FAD) was added when necessary. Activity was expressed as units per milligram of protein; 1 U is defined as the reduction of 1 µmol/min DCPIP (s600 nm = 18.7 x 103 cm-1 M-1). No activity was found in reaction mixtures without 4-androstene-3,17-dione (AD).

2.8. Purification and characterization of the recombinant KSDD

The recombinant KSDD with histidine-tag was expressed in C. crenatum and purified by affinity chromatography on a Ni-NTA sepharose prepacked column His Trap HP (GE Healthcare Life Sciences, USA). Purification was performed according to the instructions of His Trap TM HP column. The purified enzymes were subsequently assayed by SDS-PAGE. Bradford method was employed to determine the protein concentration. The eluted fractions containing the target protein were collected and assayed for KSDD activity. The purified enzyme can be stored at -20°C about half of month with 10% glycerol, 0.01 mM FAD and 0.1 mM DTT to maintain its stability.

To determine the optimal temperature for KSDD activity, the purified KSDD was assayed at pH 7.0 for 15 min at different temperatures (0°C-60°C). The thermal stability was assayed by incubating the purified enzyme at temperatures from 0°C to 60°C for 2 h. The residual enzyme activities were measured under standard assay conditions. The pH optimum of KSDD was examined at S0°C for 15 min at pH range from 3.0 to 10.0 (pH 3.0-pH 6.0, 0.05 M citrate-sodium citrate buffer; pH 6.0-pH 9.0, 0.05 M Tris-HCl buffer; pH 9.0-pH 10.0, 0.05 M borax-sodium hydroxide buffer). The pH stability was determined by incubating the enzyme in different buffers at 0°C for 2 h and the residual activity was measured at pH 7.0 and 30°C.

Fig. 2. SDS-PAGE analysis of cell-free extract and purified KSDD. (a) Western blot result of KSDD expressed in C. crenatum.Lane 1: C. crenatum SYPA 5-5/pXMJ19; lane 2: C. crenatum SYPA 5-5/pXMJ19-ksdd11; lane S: C crenatum SYPA 5-5/pXMJ19-ksdd1. (b) SDS-PAGE analysis of KSDD expressed in C. crenatum. Lane 1: cell-free extract of control C. crenatum SYPA 5-5/pXMJ19; lane 2: cell-free extract of C. crenatum SYPA 5-5/ pXMJ19-ksdd"; lane S: cell-free extract of C. crenatum SYPA 5-5/pXMJ19-ksddI; lane M: protein marker (Takara Biotechnology Co., Ltd., Dalian, China). (c) SDS-PAGE analysis of purified KSDD. Lane 1,2: 10 µL of purified mature His6-KSDD treated; lane M: protein marker (Takara Biotechnology Co., Ltd., Dalian, China).

The influence of selected metal ions (K+,Na+,Ag+,Ca2+,Mg2+, Mn2+, Cu2+, Fe3+) and ethylenediaminetetraacetic acid (EDTA) at 1mM final concentration on the activity of the purified KSDD was investigated. Most chemicals are chloric compounds except AgNO3-. Thus, we used NaNO3 as a control experiment to investigate whether NOS- affect the results. Relative activity was assayed as a percentage of the activity without agents. Under different concentration of AD, Kinetic parameters were investigated with PMS as electron acceptor at a fixed concentration of 1.5 mM. The Km and Vmax values were determined from Lineweaver-Burk plots.

2.9. Bioconversion of AD by recombinant strains C. crenatum/pXMJI9-ksdd

The bioconversion of AD was carried out in 250 Ml shake flasks with the recombinants C. crenatum and M. neoaurum JC-12. The cultural conditions of C. crenatum recombinants and M. neoaurum JC-12 were described previously. Cells were collected by an SIGMA SK-15 centrifuge at late exponential phase (OD600 4- 6). Cell pellets were washed twice with 100 mL 50 mM Tris-HCl buffer (pH 7.0). After the pellets were resuspended in 50 mL Tris-HCl buffer (added with 1 mM K+,Na+ and Ca2+), AD (1% (w/v)) and

Table 2
The enzyme activities of KSDD from M. neoaurum JC-12 and recombinant C. crenatum.

Table 3
Purification of recombinant KSDD from C. crenatum SYPA 5-5/pXMJ19-ksddII

hydroxypropyl-β-cyclodextrin (HP-β-CD, S% (w/v)) were added into the biotransformation system, which was carried out for 24 h. The extraction of steroids from the medium (1 mL) by ethyl acetate was analyzed by high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC). Steroids were analyzed by HPLC (column: reversed phase Diamonsil C18, UV254 nm detection, liquid phase: methanol: water (7:3), column temperature: 30°C, flowrate: 1mL min-1) and TLC (F254 10 x 10 cm in petroleum ether/ethyl acetate (6:4), staining fluid: 20% sulfuric acid).

3. Results

3.1. Construction of the recombinant C. crenatum SYPA 5-5

The native ksddI gene sequence was codon optimized for efficient translation. The synthesized gene ksddII and native gene ksddI were cloned onto an expression vector to generate two recombinant plasmids pXMJ19-ksddII and pXMJ19-ksddksddI. They were verified by digestion with Hind III and BamH I into two DNA fragments. In order to generate the recombinant strain C. crenatum SYPA 5-5, the two recombinant plasmids were subsequently transformed into C. crenatum SYPA 5-5. The recombinant strains were selected with chloramphenicol and verified by DNA sequencing.

3.2. Overexpression and purification of KSDD

The possible expression of KSDD in C. crenatum SYPA 5-5 was investigated as follows. Crude cell extract was assayed by SDS-PAGE and western blotting; the molecular weight of the expressed protein was about 60 kDa (Fig. 2a, b). The intracellular and extracellular KSDD activity from C. crenatum SYPA 5-5 was assayed (Table 2). In the recombinant strains C. crenatum SYPA 5-5/pXMJ19-ksddII,the intracellular KSDD had much higher specificactivitythan M. neoaurum JC-12 and C. crenatum SYPA 5-5/pXMJ19-ksddI.From this we concluded that, compared with the wild type strain, the KSDD expression level of the mutant strain had been increased by about 1.6 fold (Table 2). It was also observed that there was no secretion of KSDD in M. neoaurum JC-12, C. crenatum SYPA 5-5/pXMJ19 and the recombinant strains. In conclusion, a high activity of KSDD has achieved in C. crenatum SYPA 5-5. The expression of KSDD has been significantly improved by codon optimization. Purification of the recombinant KSDD was carried out by using the Ni-NTA affinity chromatography. The result of SDS-PAGE analysis of the purified enzyme is given (Fig. 2c). The recombinant enzyme showed one band consistent with a molecular mass of about 60 kDa. The purification resulted in a yield of 7.S7% and a purification of 5.02-fold (Table 3 ).

3.3. Characterization of KSDD

The purified KSDD was assayed at pH 7.0 for 15 min at different temperatures (0-60°C) to determine the optimal temperature for its reaction. It revealed that the optimal temperature of KSDD was S0°C (Fig. 3a). However, thermal stability profiles of the purified KSDD revealed that it was unstable at temperatures exceeding S0°C. The enzyme lost more than half of its activity after incubation at S0°C for 2h (Fig. 3b), which corroborated previous report (KSDD from Nocardia corallina). The purified enzyme was also assayed at S0°C for 15 min at different pH (ranging from pH S.0 to pH 10.0) to determine its pH optimum. The result showed that the optimal pH of KSDD was 7.0 (Fig. 4a). Furthermore, the profile of pH stability revealed that KSDD was fairly stable within a broad pH range for it retained more than 75% of its activity in pH ranging from 4.0-10.0 (Fig. 4b).

The effect ofselected metal ions and EDTA on KSDD activity was also discussed in this study (Fig. 5). The results showed that KSDD activity was strongly stimulated by 1 mM K+,Na+ and Ca2+, which had been revealed about other ∆1-dehydrogenation reaction. However, in the presence of 1 mM Ag+, KSDD activity decreased to 28.05% compared to the control experiment. While subjected to the preferred reaction conditions, using AD as substrate, the purified KSDD exhibited typical Michaelis-Menten kinetics. The Km and Vmax values were 8.91 µM and 6.4S mM/min, respectively.

3.4. Efficient production of ADD by the recombinant C. crenatum

When using the whole-cell of the recombinant C. crenatum pXMJ19-ksddI and C. crenatum pXMJ19-ksddII as biocatalysts, the transformation from AD to ADD was assayed by HPLC (Fig. 51a) and TLC (Fig 51b) The results showed that the recombinant C. crenatum strains, which could over-express KSDD, were surely identified to have the capability to produce ADD when using AD as substrate. As a control, there was no ADD accumulated by the strain C. crenatum SYPA 5-5 harboring pXMJ19. By using recombinant C. crenatum pXMJ19-ksddII as biocatalyst, the production of ADD was improved to 8.S9 g/L (Fig. 6). The overexpressed KSDD stimulated a shortened reaction duration about 1S-fold, from 1S2 h to 10 h. As shown in Table 4, the recombinant strains C. crenatum pXMJ19-ksddII and C. crenatum pXMJ19-ksddI showed the maximum conversion rates from AD to ADD about 83.87% and 58.72% at 10 h. However, M. neoaurum JC-12 showed the conversion rate from AD to ADD only 3.56% at 10 h, and the maximum conversion rate of 23% at 132 h (Table 4 and Fig. 6). The results proved that the recombinant C. crenatum pXMJ19-ksddII cells could efficiently catalyze the transformation from AD to ADD.

Fig. 3. Effect oftemperature on KSDD activity (a) and stability (b).

Fig. 4. Effect of pH on KSDD activity (a) and stability (b).

4. Discussion

It has been reported that genes encoding KSDD1, KSDD2 and KSDDS were found in the genome of M. neoaurum. However, KSDDS performed the main function in steroid pathway and showed specific activities toward to the substrate AD. Studies have tried to clone and heterologous express of KSDD from M. neoaurum in R. erythropolis, Escherichia coli and Streptomyces lividans. Unfortunately, the low expression level of recombinant KSDD made it difficult using biocatalyst to transform AD to ADD. On the other hand, missing of the characterization of KSDD restricted the use of this enzyme, although it had been purified. In this work, we successfully cloned and overexpressed KSDD from M. neoaurum in C. crenatum after codon adaption and optimization. The results of SDS-PAGE and western blotting indicated that a high expression level of recombinant KSDD ( C. crenatum pXMJ19-ksddIIwas achieved. The expression level of KSDD was improved approximately 1.65-fold in C. crenatum after codon optimization. After purification of KSDD, this work first characterized this enzyme and made the recombinant C. crenatum pXMJ19-ksddII as a biocatalyst for transforming AD to ADD. After characterization of KSDD, there was a huge loss of activity, with only 7.S7% activity remaining for a 5-fold purification, because this enzyme was not very stable. It has been reported that this enzyme was not sensitive to carbonyl reagents and little sensitive to metal chelating agents. Till now, no useful protectant has been reported. As reported that ions such as K+,Na+,Ca2+,Fe2+ and Mg2+ could stimulate the A1-dehydrogenation, we investigated the effect of mental ions on KSDD activity. The results showed that KSDD activity was strongly stimulated by 1 mM K+,Na+ and Ca2+. The redox-active ions such as Ag+, Mn2+, Cu2+ and Fe2+ might interfere with KSDD activity assay, and that was why they appeared inhibition effect on KSDD catalyzed reaction. On account of KSDD was very unstable above 30°C, in the following whole-cell biocatalysis, the strains were cultivated at 30°C and pH 7.0, which is favorable to maintain the integrity of cell and KSDD activity. Because of the low solubility of AD, HP-β-CD was added as an accessory solvent to increase the concentration of substrate. In order to further increase the conversion rate of AD to ADD, the stimulating metal ions for KSDD activity were also added.

Fig. 5. Effects of metal ions and EDTA (1 mM) on KSDD activity. CK represents control experiment: no metal ion or EDTA added.

Fig.6.Time course of ADD accumulation from AD by C. crenatum SYPA5-5/pXMJ19-ksddII, C. crenatum SYPA 5-5/pXMJ19-ksddI, C. crenatum SYPA 5-5/pXMJ19 and M. neoaurum JC-12. (Allassays were performed by three independent biological experiments, and the standard deviations of the biological replicateswere represented byerror bars).

Table 4
The whole-cell transformation from AD to ADD.

In this work, C. crenatum, which has been widely employed as a safe microorganism in the industry, was employed as a novel whole-cell catalyst for bioconversion of AD to ADD. E. coli and B. subtilis have been employed for heterologous expression of KSDD. Compared to E. coli and B. subtilis expression systems, C. crenatum produced more soluble protein after code optimization of ksdd gene, and this feature made it easy to purify and study the characterization of KSDD. The recombinant C. crenatum pXMJ19-ksddII showed a good performance of bioconversion from AD to ADD, and might be a promising strain in steroid industry. In the further work, site-specific mutagenesis of ksdd gene will be taken into consideration to improve the thermostability and the specific activity of recombinant KSDD. The protectants for maintaining KSDD activity will be selected and their mixture will be optimized on the ration. On the other hand, the optimization of the transformation system, including two-phase system, aqueous two-phase system and cloud point system will be applied to further improve ADD production.

Financial support

This work was supported by the High-tech Research and Development Programs of China (2011AA02A211, 2015AA021004), the National Natural Science Foundation of China (S1570085, S1500065), Jiangsu Province Science Fund for Distinguished Young Scholars (BK20150002), the China Postdoctoral Science Foundation Funded Project (2015M570407, 2016T90421), Natural Science Foundation of Jiangsu Province (BK20150142), the Program of the Key Laboratory of Industrial Biotechnology, Ministry of Education, China (KLIB-KF201406), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, the 111 Project (No. 111-2-06), and the Jiangsu province "Collaborative Innovation Center for Advanced Industrial Fermentation" industry development program.

Conflict of interest

The authors declare that they have no competing interests.

 

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Article history: Received 21 July 2016 Accepted 11 October 2016 Available online 26 October 2016

* Corresponding author. E-mail addresses: raozhm@jiangnan.edu.cnzxshengwu@126.com (Z. Rao).

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