Bioproduction and characterization of extracellular melanin-like pigment from industrially polluted metagenomic library equipped Escherichia coli

Science of the Total Environment 635 (2018) 323–332

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Bioproduction and characterization of extracellular melanin-like pigment from industrially polluted metagenomic library equipped Escherichia coli Shivani Amin a, Rajesh P. Rastogi a,b,⁎, Ravi R. Sonani a, Arabinda Ray c, Rakesh Sharma d, Datta Madamwar a,⁎ a

Post-Graduate Department of Biosciences, UGC-Centre of Advanced Study, Satellite Campus, Vadtal Road, Sardar Patel University, Bakrol, 388315, Anand, Gujarat, India Ministry of Environment, Forests & Climate Change, Indira Paryavaran Bhawan, Jor Bagh Road, New Delhi 110 003, India c Advanced Organic Chemistry Department, P. D. Patel Institute of Applied Sciences, CHARUSAT, Changa 388421, Gujarat, India d CSIR-Institute of Genomics and Integrated Biology (IGIB), Sukhdev Vihar, Mathura Road, New Delhi 110 020, India b

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Metagenomic library from the industrially stressed environment was constructed. • Melanin-like pigment producing clone was sub-cloned, sequenced, over expressed. • Pigment was purified, characterized for its physic-chemical, structural properties. • Homology-based structure and function prediction of melanin producing enzyme HPPD.

a r t i c l e

i n f o

Article history: Received 23 November 2017 Received in revised form 6 April 2018 Accepted 7 April 2018 Available online 24 April 2018 Editor: F Frederic Coulon Keywords: Over expression Fosmid vector Amlakhadi canal E. coli EPI300™-T1R pET-28a(+) vector 4-Hydroxyphenylpyruvate-dioxygenase

a b s t r a c t To explore the potential genes from the industrially polluted Amlakhadi canal, located in Ankleshwar, Gujarat, India, its community genome was extracted and cloned into E. coli EPI300™-T1R using a fosmid vector (pCC2 FOS™) generating a library of 3,92,000 clones with average size of 40 kb of DNA-insert. From this library, the clone DM1 producing brown colored melanin-like pigment was isolated and characterized. For over expression of the pigment, further sub-cloning of the clone DM1 was done. Sub-clone containing 10 kb of the insert was sequenced for gene identification. The amino acids sequence of a protein 4-Hydroxyphenylpyruvate dioxygenase (HPPD), which is know to be involved in melanin biosynthesis was obtained from the gene sequence. The sequence-homology based 3D structure model of HPPD was constructed and analyzed. The physico-chemical nature of pigment was further analysed using 1H and 13C NMR, LC-MS, FTIR and UV–visible spectroscopy. The pigment was readily soluble in DMSO with an absorption maximum around 290 nm. Based on the genetic and chemical characterization, the compound was confirmed as melanin-like pigment. The present results indicate that the metagenomic library from industrially polluted environment generated a microbial tool for the production of melanin-like pigment. © 2018 Elsevier B.V. All rights reserved.

⁎ Corresponding authors. E-mail addresses: [email protected] (R.P. Rastogi), [email protected] (D. Madamwar).

https://doi.org/10.1016/j.scitotenv.2018.04.107 0048-9697/© 2018 Elsevier B.V. All rights reserved.

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1. Introduction

2. Materials and methods

Microbes are very flexible and thus, their population heterogeneity is being shaped by environmental factors they reside in (Daniel, 2005; Freedman and Zak, 2015). The genomic pools of microbial populations residing at industrially polluted environments are armed with several stress response genes (Devpura et al., 2017). Therefore, genome mining may offer the selection of new genes having the unique ability to mitigate the stress conditions. Construction and screening of a metagenomic clone library is being widely used to identify the potential genes encoding novel stress response or bioremediation enzymes or secondary bioactive compounds (Martín et al., 2006; Desai et al., 2010; Bouhajja et al., 2017). The Amlakhadi canal near Ankleshwar, Gujarat, in India, is an aquatic highly polluted body with the waste of nearby chemical and textile industries. Several efforts have been made to understand the composition, dynamics, and functions of microbial communities inhabiting such a polluted environment. For instance, Vaidya et al. (2017), Patel et al. (2014) and Pandit et al. (2013) have isolated and characterized the hydrocarbon degrading, chromium resistant and other heavy metal resistant organisms (bacteria/fungi), respectively, from the same site. However, the coverage of these studies is limited either to cultivable microbes or to the prediction of functional genes of uncultivable microbes; and has not involved the experimental validation of uncultivable microbial genes. Since 99% of the total microbial community consists of uncultivable microbes, it is necessary to take them into account while looking for potential genes or secondary bioactive compounds. Several organisms ranging from prokaryotes to eukaryotes produce reddish-brown to black pigments known as melanin. These are high molecular weight, polyphenolic heteropolymers, considered as efficient UV-screening compounds. Besides potent UV absorbing function, melanins have also been reported to play a significant role in enhancing cell-survival under various stress conditions such as oxidative-stress (Rita and Pombeiro-Sponchiado, 2005), heat-stress (Madhusudhan et al., 2015) and metal-stress (Gadd and de Rome, 1988; Eisenman and Casadevall, 2012). Furthermore, melanins are also reported to have anti-cancer (Pombeiro-Sponchiado et al., 2017; El-Naggar and El-Ewasy, 2017), anti-inflammatory (Avramidis et al., 1998), antioxidant (ElNaggar and El-Ewasy, 2017; Rageh and El-Gebaly, 2018) and antiaging (Lu et al., 2014) functions, and may also induce protective immune responses (Stappers et al., 2018). Recently, Fajuyigbe et al. (2018) have reported that melanin may protect against DNA photodamage and concur with skin cancer incidence. Moreover, melanin comprises a number of pharmacological properties with diverse biological functions (ElObeid et al., 2017). Consequently, the recombinant production of melanin becomes commercially important (Huang et al., 2009). Since the melanin production is facilitated through multiple enzymes (Coelho et al., 2015), the creation of melanin producing clone is difficult and requires huge efforts. As an alternative approach, the screening of metagenomic clones for melanin production has been demonstrated (Lagunas-Muñoz et al., 2006; Huang et al., 2009). This approach seems more attractive because of several reasons. It doesn't require cloning of multiple genes and the melanin, produced by this method does bear higher bioactivity, since this gene-cluster has been evolved under environmental pressure. Since microbial resources from highly polluted Amlakhadi canal have been little explored by metagenomics approach, the present study aimed to investigate the construction, screening and characterization of melanin producing clone from the Amlakhadi metagenomic library. The chemical characterization of produced melanin was also performed.

2.1. Chemicals and reagents E. coli EPI300™-T1R, E. coli BL21 as transformants were grown on Luria-Bertani (LB) broth (HiMedia, Mumbai, India). Restriction enzyme Bam HI was procured from Bangalore Genei, Bangalore, India. Chloramphenicol and other antibiotics were procured from HiMedia, Mumbai, India. The enzyme β-agarase and T4 DNA ligase were obtained from BioLab, USA and Bangalore Genei, Bangalore, India. Dimethyl sulfoxide (DMSO), methanol and Potassium bromide (KBr) were of HPLC grade and obtained from Merck, Germany. All other chemicals, reagents, solvents and media are of analytical and highest purity grades. 2.2. Strains and plasmids Large insert metagenomic library was constructed in Fosmid vector using ‘CopyControl™ HTP Fosmid Library Production Kit with pCC2FOS™ Vector (Epicenter, USA), containing E. coli EPI300™-T1R as host cells. The pET-28a(+) (New England Biolabs, USA) was used for sub-cloning and over expression vector in the host E. coli BL21 cells (New England Biolabs, USA). 2.3. Sampling Soil sediment samples from Amlakhadi canal (21° 36′ 0″ North, 73° 0′ 0″ East) polluted by industrial activities from Ankleshwar Industrial Estate, Ankleshwar, Gujarat, India were collected in February 2014. Amlakhadi canal, is flowing across the Ankleshwar Industrial Estate, receiving treated/partially treated/untreated industrial effluents (since last five decades) from thousands of industrial units manufacturing dye, dye-intermediates, pigments, pesticides, pharmaceuticals, specialty chemicals, petrochemicals, paints, textiles, engineering, plastics, rubbers and packaging, etc. Sub-surface (5–8 cm below the surface) soil samples about 1 kg each from three distinct sites were collected using standard soil sample collection sample kit, stored in sterile plastic bags and transported to laboratory under below ambient conditions and stored at 4 °C till the extraction of metagenomic DNA. 2.4. Metagenomic DNA extraction The metagenomic DNA from soil was extracted using a modified method of Desai and Madamwar (2007). In brief, the metagenomic DNA was extracted from 5 g of soil with 2–3 mm of glass beads (2 g) in 15 mL of extraction buffer (10% (w/v) sucrose, 100 mM Tris-Cl, 100 mM EDTA, 100 mM sodium phosphate, 0.5 M NaCl and 1% (w/v) Cetyltrimethylammonium bromide (CTAB), pH 8.0) under continuous shaking (75 rpm) at 37 °C for 1 h. Extracted cell mass was lysed using proteinase K (10 mg/mL) and sodium dodecyl sulfate (20%), which was followed by protein removal and DNA precipitation. The precipitated DNA pellet was washed with 70% ethanol for salt removal before dissolving the DNA into TE buffer for further use. Three different replicates from each sample were used for extraction of metagenomic DNA. The extracted DNA was pooled in equimillimolar ratio to obtain the composite metagenomic DNA. 2.5. Community genomic library construction From crude metagenome, the DNA of ~ 40 kb size was extracted from agarose gel using β-agarase (1 U/mL) gel elution method (BioLab, USA) as per the manufacturer's instructions. The extracted DNA (of ~ 40 kb) was end-repaired and phosphorylated at 5´- end. The 5-phosphorylated DNA was re-purified by β-agarase gel elution method and cloned into the pCC2 FOS™ vector, using the

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CopyControl Fosmid Library Production Kit (Epicenter®) as per manufacturer's instructions. The ligated vectors were then transformed into the E. coli EPI300™-T1R cells with the help of lambda phages extract (MaxPlax™) (Huang et al., 2009). The transformed cells were selected under selective pressure of chloramphenicol (12.5 μg/mL).

2.6. Screening and characterization of melanin-like pigment producing clone The prepared metagenomic library was screened for pigment production by spreading the library using various dilutions (10−1 to 10−8) on Luria-Bertani agar medium. Initially, clones producing brown colored zone were selected as melanin-like pigment producing clones. These clones were further characterized by sub-cloning and sequence analysis of sub-cloned plasmid. For sub-cloning, the plasmid was digested using BamHI restriction enzyme and then digested fragments were ligated into pET-28a(+) vector using T4 ligase (New England Biolabs, USA). The ligated products were further sub-cloned into E. coli (BL21) cells. The sub-clone was also checked for melanin production by visual observation of brown zone. The sequencing of sub-cloned insert plasmid (10 kb) was done using shotgun sequencing in an Ion Torrent sequencer using a 316 chip, with 200 bp chemistry (Thermo Scientific, USA).

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Table 1 Solubility analysis of melanin like pigment with different solvent and at acidic and alkaline pH. Solvents

Solubility

Deionized water 1 N HCl 0.1% NaOH Ethanol Ethyl acetate Chloroform Acetone Methanol Acetonitrile DMSO Dichloromethane Butanol Phenol

Insoluble Insoluble Soluble Insoluble Insoluble Insoluble Insoluble Insoluble Insoluble Soluble Insoluble Insoluble Soluble

2.7. Extraction and purification of melanin The transformed cells of E. coli (BL21) harboring 10 kb clone containing gene of melanin pigments were optimally cultured at 37 °C in a LB-broth under shaking condition. After removal of cell mass (by centrifugation), the melanin, present in the medium (supernatant) was precipitated by two step process. In the first step, the pH of the

23 kb 10 kb 3 kb 1 kb 0.5 kb

Fig. 1. (A) Agarose gel-electrophoretic profile of molecular weight marker (M), cloned 40 kb insert (1), BamHI digested 40 kb insert (2), sub-cloned 10 kb insert (3). (B) Pigment producing sub-clone grown on kanamycin (100 μg/mL) containing Luria-Bertani (LB) agar plate. (C-D) Appearance of melanin-like pigment at various concentrations (C) and in a dried precipitated form (D).

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medium was increased up to 12.0 using 5 N NaOH and continuously stirred for 1 h at room temperature. In the second step, the pH of the medium was decreased up to 2.0 using HCl (1 N), and incubated at room temperature for one week to precipitate out the melanin from the aqueous solution. Subsequently, the mixture was boiled for 1 h to avoid melanoidins formation prior to harvesting the melaninprecipitates. The obtained melanin powder (precipitates) was

repetitively washed with 0.1 N HCl, followed by deionized water. The melanin powder was further washed with ethanol to remove the impurities. The washed melanin powder was dried at room temperature for 2 h, powdered and stored at room temperature for further analysis. The whole process of precipitating out the melanin from the aqueous phase (medium) was performed twice from two separate sets of experiments.

AA

BB

C

D

E

F

Fig. 2. Scanning electron micrograph of control (A) and pigment producing sub-clone (B) of E. coli (BL21) cell. (C\ \F) Scanning electron microscopic images of melanin granules at 6.0 K х (C, D), 12.0 K х (E) and 16.0 K х (F) magnifications.

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2.8. Physiochemical characterization of melanin-like pigment 2.8.1. Biochemical and solubility analysis The pigment was primarily identified by few biochemical assays and solubility analysis. The pigment in powder form were assessed for its reactivity and solubility properties (separately) in deionized water, 1% KMnO4, 1% FeCl3, 1 N HCl, 0.1 N NaOH, 30% H2O2, chloroform, dichloromethane, ethanol (95%), acetone, ethyl acetate, methanol, acetonitrile, DMSO, butanol and phenol (saturated) as described earlier (Sajjan et al., 2010). 2.8.2. Electron microscopy (EM) The dried melanin powder (1–2 mg) and melanin-producing clone were observed under scanning (ESEM EDAX XL-30, Philips, Netherlands) and transmission electron microscope (Tecnai 20, Philips, Holland). 2.8.3. Spectroscopic analysis The melanin powder was dissolved in dimethyl sulfoxide (DMSO) and its light absoprtion property was analysed between 200 and 900 nm using a UV–Visible spectrophotometer (Specord 210, Analytik JenaAG, Germany). DMSO was used as a control blank. The raw data were transferred to a microcomputer, and the peaks were analyzed with the software (SPECORD® 210) provided by the manufacturer. 2.8.4. LC-MS/MS The melanin was further characterized by liquid chromatography (LC) - mass spectrometry (MS). LC was performed using methanol: water (60:40) as a mobile phase and C18 reverse phase column (5 μm, 250 × 4.6 mm) as a stationary in LCQ Fleet Ion Trap LC/MS (Thermo Scientific, USA) and HPLC system equipped with a photodiode array (PDA) detector. The DMSO-dissolved melanin (20 μL) was injected through an auto-sampler with a flow rate 1.0 mL/min. The PDA detection wavelength was operated at 270 nm, and PDA scan wavelength was from 200 to 900 nm. The column oven temperature was set at 40 °C. LC was followed by electron spray ionization (ESI) mass spectrometry (MS) using a LCQ Fleet Ion Trap LC/MSn as described by Wang et al. (2015).

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Therefore, to explore the functional potential of indigenous microbial community of the polluted canal and to devise the effective bioremediation strategies, functional metagenomic approach was applied and large insert library was constructed to study the genes and pathways involved in the degradation of xenobiotic compounds.

3.1. Construction of metagenomic library To get the highly diverse genes, we have isolated metagemone from the three different sites, which was mixed in equimolar ratio before cloning. Metagenomic library of 3.92 × 106 of the clones of average 40 kb DNA was constructed using fosmid vector. To check the redundancy of clones, fosmids from five randomly picked clones were checked by restriction digestion analysis. The agarose gelelectrophoretic profiles of digested fosmids clearly ruled out the presence of redundancy among library clones (Supplementary Fig. S1).

3.2. Screening and sub-cloning of pigment producing clones Pigment producing clone was identified by the visual brown colored zone around colony. The pigment-producing clone containing 40 kb insert was sub-cloned to over-express the pigment. The 40 kb insert was digested with different restriction enzymes and cloned into the E. coli (BL21) (Fig. 1A). Sub-clone was observed to contain 10 kb insert as shown in Fig. 1A. Fig. 1B shows the pigment-producing sub-clone, obtained with BamHI. Melanin-like pigment was extracted and the sub-clone was observed to produce 500 mg/L pigment. The appearance of pigment at various concentrations and as a precipitate in dry form is shown in Fig. 1C and Fig. 1D, respectively.

A

2.8.5. FTIR and NMR The melanin powder (5–10 mg) was mixed with potassium bromide (KBr) in 5:95 ratio and analyzed at mid IR region (400–4000 cm−1) using an infrared spectrophotometer (Spectrum GX, PerkinElmer, USA) against the blank of KBr (100%). The 1H and 13C NMR graph was obtained by loading DMSO dissolved melanin powder (5–10 mg) on Bruker-Avance II (500 MHz) (Bruker, USA) against the blank of DMSO (100%). All the spectra were collected in the replicates of three. 2.9. Homology-based structure and function prediction The sequence of 10 kb sub-cloned insert was analyzed to find melanin bio-synthesis enzymes associated genes. The sub-cloned insert was noticed to contain gene showing significant similarity with the gene of melanin bio-synthesis enzyme during NCBI BLAST analysis. The matched sequences were taken to predict the model for the possible 3D structure of the protein by homology modeling using SWISSMODEL (https://swissmodel.expasy.org). The possible function of proteins was also predicted by ‘molecular function ontology’ using the PredictProtein online tool (https://www.predictprotein.org).

B 42.17 nm 40.40 nm

3. Results and discussion Due to the continuous exposure of recalcitrant xenobiotic compounds, at Amlakhadi canal, the indigenous microbial community might have evolved and acquired the essential functional properties (genes/proteins) required to resist and to metabolise such compounds.

27.34 nm Fig. 3. Transmission electron microscopic images of melanin-like pigment granules (A) with different size (B).

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3.3. Characterization and structural identification of melanin-like pigment The pigment was not completely soluble in neither of the solvents including water, ethanol and methanol; however, it was completely soluble in Dimethyl sulfoxide (DMSO). The DMSO soluble pigment was extracted and characterized. First, several chemical assays were performed to identify the pigment. The solubility of pigment in various solvent is depicted in Table 1. The pigment was found to be readily soluble in 0.1 N NaOH. Furthermore, pigment's reaction with the 1% KMnO4 resulted in color change of pigment from brown to red (Supplementary Fig. S2A), which was observed to revert upon addition of 1% FeCl3 (Supplementary Fig. S2B). Moreover, the pigment color was bleached upon reaction with 30% H2O2 (Supplementary Fig. S2C). This type of chemical characteristics indicated that this compound may belong to the melanin (Ravishankar et al., 1995; Sajjan et al., 2010), which was further analyzed using various methods. 3.3.1. Scanning and transmission electron microscopy The SEM study revealed the altered surface properties of melaninproducing clone and pigment molecule itself. The surface of melaninproducing clone gives a brighter appearance than the control E. coli (BL21) due to the presence of melanin sheath (Fig. 2A,B). The morphological characterization and particle size distribution of melanin from E. coli (BL21) was done by Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). It has been observed that

A

the natural melanin appears as a small sphere in the form of granules (Nofsinger et al., 2000; Tarangini and Mishra, 2013; Mbonyiryivuze et al., 2015; El-Naggar and El-Ewasy, 2017). In the present study, the purified melanin pigment synthesized by E. coli (BL21) appears to have definite small granules under SEM imaging (Fig. 2C-F). Furthermore, SEM image of pigment molecules shown in Fig. 2 resembles to the surface structure of melanin pigment (Pitkänen et al., 2007; El-Naggar and El-Ewasy, 2017). Further, TEM analysis also suggested that the granules of melanin pigment are spherical to a certain extent (Fig. 3). The granule size measured from TEM micrograph was about 25–50 nm, which is correspondence with earlier reports of Liu and Simon (2003). Moreover, the electron microscopic image analysis showed that the obtained melanin is formed by aggregation of numerous spherical granules with unusual size distributions. 3.3.2. UV–visible spectrophotometeric analysis of the purified melanin-like pigment Fig. 4A shows the UV–visible absorbance spectrum of purified melanin pigment. The absorbance maximum was observed under UV-B region at 290 nm, which decreased gradually under visible region. This is a characteristic property of melanin, for example, this type of absorbance profile has also been observed for melanin like pigment isolated from a marine fungus (Ravishankar et al., 1995). Moreover, melanin pigment from different sources showed different UV–visible absorption maxima, for example, the melanin pigment synthesized by Streptomyces

0.42 0.35

Absorbance

0.28 0.21 0.14 0.07 0.00 280

390

500

610

720

830

940

1050

Wavelength [nm]

B

Fig. 4. (A) UV–visible absorbance spectrum of the purified melanin-like pigment. (B) Fourier transform infrared (FTIR) spectrum of the purified melanin-like pigment.

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bikiniensis M8 exhibited UV absorption maximum at 230 nm with decreased absorbance towards the visible region, which is the characteristic property of melanin (Deepthi and Rosamma, 2014). The absorption maxima of purified melanin pigment from Phyllosticta capitalensis (Suryanarayanan et al., 2004) and Streptomyces glaucescens strain NEAE-H (El-Naggar and El-Ewasy, 2017) was observed at 250 nm. In addition, the purified melanin from Actinoalloteichus sp. MA-32 and Chroogomphus rutilus had maximum absorption at 300 nm (Manivasagan et al., 2013) and 212 nm (Hu et al., 2015), respectively. 3.3.3. Fourier transform-infrared spectroscopy (FTIR) analysis Fig. 4B shows the FTIR profile of the purified melanin pigment. The appearance of broad absorbance around 3400 cm−1 indicates the presence of –OH and -NH2 groups, whereas the peak in the region 3000–2800 cm−1 signified the presence of the alkyl group. The peaks ~1640 cm−1 and ~1538 cm−1 are indicative of carboxylic group (-COOH) having the asymmetric and symmetric stretching. Thus, –COOH is most likely present in tautomeric form (Nakamoto, 1970). The absorbance at 1540 cm−1 is appeared due to the presence of -NH with bending mode. Further, the bending of phenolic –OH and –CN groups are confirmed by the peak ~1380 cm−1 and ~1454 cm−1, respectively. The presence of C_O stretching in pigment chemical structure is evidenced by peak at ~1640 cm−1. Overall, the general patterns of the FTIR profile of the melanin pigment extracted from E. coli (BL21) almost in agreement with previous melanin extracted from various organisms (Centeno and Shamir, 2008; Hewedy and Ashour, 2009; Magarelli et al., 2010; Sajjan et al., 2010; El-Naggar and El-Ewasy, 2017; Gustavsson et al., 2016). 3.3.4. LC-MS/MS analysis The Liquid chromatography (LC) profile of the purified melanin pigment (Fig. 5) showed two major peaks with retention time of 2.41 (peak a) and 2.98 (peak b) (Fig.5), with m/z value 475.5 (Fig. 5A) and

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701.5 (Fig. 5C), respectively. The MS/MS analysis of both peaks has been shown in Fig. 5B and D. The mass of peak b is nearly double than the mass of the single melanin molecule. Therefore, here, it is most likely that the melanin is present in dimeric form as shown in Fig. 5C inset. The presence of melanin dimer has also been reported previously (Stark et al., 2003; Pezzella et al., 2006). Peak a (475.5 m/z) (Fig. 5, Fig. 5A) might be aroused due to the asymmetric fragmentation of melanin dimer due to the loss of four N-C-COOH groups (2 from each melanin molecule) of the 284 Da mass. The possible chemical structure of ‘melanin dimer truncate’ corresponding to peak a i.e., 475.5 m/z is shown in Fig. 5A inset. The LC-MS results differ from the results of Wang et al. (2015), in which, the authors found the L-DOPA like molecule with retention time 3.2 min with m/z value 196. Moreover, melanin is generally found in the different polymer form (Sun et al., 2016) and their chemical structure may vary in different organisms.

3.3.5. 13C and 1H NMR Fig. 6A shows the 13C NMR spectrum of the purified melanin pigment. It shows the peaks at 171, 100, 120–140, 20–24, 39/56 ppm corresponding to the carboxylic acid moieties (R-COOH), carbonyl (C=O) group, aromatic rings, amine (-NH2) and methyl (-CH2) groups, and non-cyclic aliphatic chain, respectively. Peaks at 100 ppm could be due to the indole or pyrrole ring carbons. All functional groups corresponding to specific peak are listed in Table 2. The 1H NMR was performed to confirm the presence of such structural arrangement in pigment (Fig. 6B). The 1H NMR of the melanin pigment shows a resonance at 9.2 ppm and in the range of 7.2–8.0 ppm, which correspond to N\\H proton and aromatic proton, respectively. Further, the appearance of resonance at 6.6 and 7.1 ppm also confirmed the vinylic proton (C=C) in the pigment. Based on the above analysis, the chemical structure of the purified melanin-like pigment has been predicted as shown in Fig. 6B inset, which is quite close to the chemical structure of a

Fig. 5. Liquid chromatogram of the purified melanin-like pigment with absorption maximum at 2.41 min (a) and 2.98 min (b). MS spectra of LC peak a (RT: 2.41 min; A) and peak b (RT: 2.98 min; C). MS-MS spectra of the peak a (475.42 m/z; B) and peak b (701.50 m/z; D).

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Fig. 6. 13C (A) and 1H NMR (B) spectra and predicted chemical structure of the purified melanin-like pigment.

melanin pigment. Similar types of NMR results have also been noticed in previous studies (Song et al., 2016; Zong et al., 2017). 3.4. Structure-modeling and analysis From the shotgun sequencing of 10 kb sub-clone insert, partial gene sequence of the enzyme 4-Hydroxyphenylpyruvate-dioxygenase

(HPPD), which is involved in the melanin biosynthesis was obtained. This sequence showed 100% similarity and 56% query cover with HPPD partial gene sequence of Paenibacillus sp. DMB20 (Accession number KKO53146). The 3-D structure model for both present HPPD and Paenibacillus HPPD were predicted and validated (Fig. 7). The structure of Paenibacillus HPPD consists of five helixes and two β-sheets. In the available crystal structure of HPPD, the C-terminal domain contains

S. Amin et al. / Science of the Total Environment 635 (2018) 323–332 Table 2 13 C and 1H analysis for the functional group identification of pigment. Functional groups

Chemical shift (ppm) 13

\ \NH Aromatic proton Pyrrole ring \ \NH2 \ \OH proton \ \COOH carboxylic acid \ \CH2 or C\ \O group of molecules \ \C_C vinyl proton

C

– 130 – 40–42 – 171 20–24 –

1

H

9.25 7.22–7.5 1.0 9.1 3.4 – – 6.6

active site, where “iron” is surrounded by amino acids extending inwards from beta sheets. The structure of present HPPD only contains the C-terminal domain of HPPD as compared to Paenibacillus HPPD, which might have retained the active site. To confirm this, the function of present HPPD was predicted using PredictProtein molecular function ontogeny, which indicated that this HPPD have similar function to that of HPPD i.e. dioxygenase activity (Supplementary Fig. S3). Moreover, the structure and function prediction analysis confirmed that the 10 kb insert contains gene for HPPD-like enzyme, which might be playing an active role in production of melanin-like pigment. 4. Conclusion Melanin is one of the most common biopolymeric pigments produced by almost all biological systems ranging from prokaryotic as well as eukaryotic organisms. Herein, a metagenomic fosmid library of high-molecular-weight metagenomic DNA from the industrially polluted Amlakhadi canal, Ankleshwar, Gujarat, India was constructed.

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A clone DM1 producing brown colored pigment was screened and further sub-cloned to identify the genes involved in melanin synthesis. The purified melanin pigment extracted from recombinant E. coli (BL21) cells showed the physicochemical properties of a typical melanin compound. UV–visible spectroscopy, LC-MS/MS, 1H-/13C NMR and FTIR analysis revealed the similarity of present pigments with melanin. Furthermore, a protein 4-Hydroxyphenylpyruvate-dioxygenase (HPPD) was inferred from the gene sequence which is assumed to be involved in melanin biosynthesis. The sequence-homology based 3D model of HPPD was also explored. SEM and TEM analysis were performed to elucidate the morphology of extracted melanin pigment, and it was revealed that it is an aggregate of small granules of different size. Moreover, various types of melanin pigments with different properties have been reported from diverse organisms, but their accurate structure is still ambiguous and needs extensive research to establish their structural and functional properties. Overall, the construction of metagenomic libraries of microorganisms from unusual and extreme environments would be valuable for the identification of novel genes. The synthesis of various natural products including melanin should be of great importance in industrial processes towards biotechnological applications. Acknowledgement This work was supported by the Department of Biotechnology, Ministry of Science and Technology, New Delhi, India (BT/1/CEIB/09/V/05). The authors are also thankful to Sophisticated Instrumentation Centre for Applied Research and Training (SICART), Vallabh Vidyanagar, Gujarat, India, for providing FTIR, LC-MS and electron-microscopy facility. Central Salt and Marine Chemicals Research Institute (CSMCRI), Bhavnagar, is also duly acknowledged for FT-NMR facility. Conflict of interest Authors declare no conflict of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2018.04.107. References

Fig. 7. The cartoon diagram of predicted 3-D structure of 4-hydroxyphenylpyruvate dioxygenase (HPPD) of Paenibacillus sp. DMB20 (A) and HPPD of present 10 kb insert sub-clone (B). The figure was prepared in PyMOL 1.3.

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